Patent Application
20210190799
The present invention relates to a method for diagnosing liver diseases and a method for screening a therapeutic agent for liver diseases using the expression changes of TM4SF5 (transmembrane 4 L six family member 5) protein by confirming the expression changes of mRNAs and proteins of Srebp1 (Sterol regulatory element-binding protein 1), Srebp2 (Sterol regulatory element-binding protein 2), Fasn (Fatty acid synthase), CD36 (cluster of differentiation 36), Fabp1 (Fatty Acid-Binding Protein 1), Vldlr (very-low-density-lipoprotein receptor), Ldlr (low density lipoprotein receptor), ApoB100 (Apolipoprotein B 100), Pparα (Peroxisome proliferator-activated receptor alpha), Pparγ (Peroxisome Proliferator Activated Receptor Gamma), Leptin, Accα (acetyl-CoA carboxylase alpha), Accβ (acetyl-CoA carboxylase beta), collagen I, collagen type I alpha 1 chain, laminins, laminin α5, laminin γ2, laminin γ3, Socs1 (Suppressor of cytokine signalling 1), Socs3 (Suppressor of cytokine signalling 3), Sirt1 (Sirtuin 1), Sirt5 (Sirtuin 5), Sirt6 (Sirtuin 6), α-SMA (α-smooth muscle actin), MCP1 (monocyte chemoattractant protein 1), TGFβ1 (transforming growth factor beta 1), or F4/80 antigen (macrophage biomarker) and the changes in the phosphorylations of STAT3 (Signal transducer and activator of transcription 3), c-Src, FAK(focal adhesion kinase), mTOR, S6K, ULK (UNC-51-like kinase), 4EBP1(Eukaryotic translation initiation factor 4E-binding protein 1) and Akt proteins related to obesity due to fatty liver, hepatitis, fibrosis, cancer development and metabolic disorders, depending on the expression of TM4SF5 in the cells and the tissues obtained from cells and mice, by confirming the disease characteristics of the liver tissue by comparing cell damage, cell arrangement pattern disorder, collagen I or laminin synthesis/accumulation, and AFP (Alpha-fetoprotein), FUCA (AFU, Alpha-L-fucosidase), CD34 (human hematopoietic stem cell and endothelial cell marker), HIF1α (Hypoxia-inducible factor 1-alpha), Ki-67 (Antigen KI-67), or Cyclin D1 expression/accumulation, by confirming the level of triglyceride (TG), free fatty acid (FFA), cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDL (Low-density lipoprotein), glucose or insulin in plasma samples of animals, by measuring the increase in weight, and by measuring the increase in weight/liver weight.
The liver has many functions such as metabolism of lipids, detoxification, bile excretion, storage of various nutrients, hematopoiesis, blood clotting and regulation of circulating blood volume. Therefore, when the liver failure occurs, various functions are degraded, and in the worst case, life is difficult to maintain.
More particularly, the functions of the liver are as follows. First, the liver has a function of managing energy metabolism, so all nutrients such as carbohydrates, fats and proteins including amino acids absorbed from food are metabolized as substances capable of producing energy in the liver and are supplied to or stored in the body. Second, about 2,000 enzymes, albumin, and coagulation factors present in the liver synthesize, store, and distribute bile acids, phospholipids and fats such as cholesterol. Third, the liver has the functions of detoxification and decomposition. The liver detoxifies drugs, alcohol, and toxic substances, so it is easy to damage liver cells during this process. Therefore, liver diseases caused by drugs, poisons, or alcohol can often occur. In addition, the liver has the function of excreting various metabolites into the duodenum, and immune function, etc., so the liver is important for maintaining life.
Liver disease can be classified into viral liver disease, alcoholic liver disease, drug toxic liver disease, fatty liver, autoimmune liver disease, metabolic liver disease, and others depending on the cause. Liver disease is the first cause of death in the world as well as in Korea, as it is found only after considerable progress because there is no initial symptom. Therefore, research on an effective diagnosis method and a treatment method for liver disease is required.
When the liver is stimulated by alcohol, viruses, or harmful environmental factors, hepatic stellate cells are activated to secrete various cytokines including TGF (transforming growth factor (3). TGFβ is a cytokine known to play an important role in the development and carcinogenesis process. The TGF receptor phosphorylates and activates intracellular Smad2/3 proteins by the activated TGFβ, binds to Smad4, which moves into the nucleus, and promotes the transcription of several related genes.
Many of the proteins whose expression is regulated by TGFβ1 are associated with the induction of fatty liver and steatohepatitis. If metabolic function is abnormally regulated through the changes in expression of the proteins whose expression is regulated by TGFβ1, the expressions of fat biosynthesis-related enzymes, signal transduction proteins or enzymes and proteins involved in the absorption and accumulation of fat are regulated to increase as nutrients such as carbohydrates, fats, or proteins (including amino acids) are ingested excessively. So, fat is accumulated in the liver epithelial cells, fatty liver (steatosis) develops, and steatohepatitis (steatohepatitis) is induced if inflammation develops further.
The fat biosynthesis-related enzymes or signal transduction proteins or factors include Srebp1, Srebp2, Fasn, Pparα, Pparγ, Leptin, Accα, Accβ, Sirt1, Sirt5, Sirt6, insulin, or glucose, and the enzymes and proteins involved in the absorption and accumulation of fat include CD36, Fabp1, Vldlr, Ldlr, ApoB100 and the like. If the fatty liver becomes severe due to the above reasons, steatohepatitis accompanied by inflammation may occur, the amount of triglyceride or triacylglycerol in plasma, free fatty acid and cholesterol (VLDL and LDL) is increased, the symptoms of obesity or abdominal obesity may be induced, and the weight can be increased.
On the other hand, TGF promotes the synthesis of collagen to induce liver fibrosis, and affects not only hepatic stellate cells but also surrounding hepatocytes, causing EMT (epithelial to mesenchymal transition). If liver fibrosis persists, cirrhosis is eventually induced, so understanding the process of liver fibrosis is necessary to treat cirrhosis.
A lot of cytokines such as TGFβ1 are secreted by inflammation. Hepatic stellate cells and other hepatocytes are activated by the secreted cytokines, and many extracellular matrixes such as collagen I, fibronectin and laminin are synthesized and accumulated outside cells. In this case, the amount of mRNA and protein of MCP1 or F4/80 antigen, the inflammation-related factor, may be increased, and damage of cells in tissue, cell arrangement pattern disorder, or synthesis accumulation of collagen I or laminin may occur.
Alcoholic liver damage is caused by alcohol itself or by the compounds produced in the metabolic process of alcohol, which leads to lipid accumulation, hepatocellular damage and fibrosis. In addition, if hepatocytes are damaged by various causes such as chronic hepatitis B, chronic hepatitis C, chronic autoimmune disease, chronic biliary tract disease, chronic heart disease, parasites and drug intoxication, various cytokines and reactive oxygen species are produced by the interaction of various cells, such as hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells. Due to this, the extracellular matrix (ECM) is damaged, and abnormal proliferation of ECMs such as collagen I and III is induced, thereby leading to liver fibrosis.
In general, hepatic fibrosis is reversible, unlike cirrhosis, composed of thin fibrils, and nodules are not formed therein. In addition, liver fibrosis can be restored to normal if the cause of liver damage disappears, but if recurrence of liver fibrosis is repeated, cross-linking between ECMs increases to form thin microfibers and it progresses to irreversible cirrhosis with nodules. This cirrhosis is a chronic disease that pathologically involves necrosis, inflammation and fibrosis, and ultimately progresses to liver cancer if neglected.
In general, it is known that the liver tissue of liver cancer patients has increased mRNA or protein expression of AFP (Alpha-fetoprotein), FUCA (AFU, Alpha-L-fucosidase), CD34 (human hematopoietic stem cell and endothelial cell marker), HIF1α (Hypoxia-inducible factor 1-alpha), Ki-67 (Antigen KI-67) or Cyclin D1.
Meanwhile, TM4SF5 (transmembrane 4 L6 family member 5) protein is known as a type of tetraspanin. The TM4SF5 protein is a water-insoluble protein and includes four regions that pass through the cell membrane, two ring structures outside the cell, one ring structure present in the cytoplasm, and two terminal structures. These proteins form a giant tetraspanin-web or a tetraspanin-enriched microdomain (TERM) complex in the cell membrane with cell adhesion molecules such as integrin. This complex contributes to various biological functions such as cell adhesion, proliferation and migration. TM4SF5 protein is known to be overexpressed in human liver cancer cells.
In this regard, Korean Patent No. 10-0934706 discloses a method for screening anticancer substances using the cancer cells expressing TM4SF5 protein and an anticancer composition comprising a compound that inhibits the activity of TM4SF5 protein.
The present inventors tried to develop a method to diagnose liver diseases by using the expression changes of TM4SF5 protein. In the course of our study, the present inventors confirmed in the liver tissue or hepatocytes obtained from the TM4SF5 protein over-expressing transgenic mouse or the Tm4sf5 gene knockout transgenic mouse (KO mouse) that (1) the expression changes of Srebp1 (Sterol regulatory element-binding protein 1), Srebp2 (Sterol regulatory element-binding protein 2), Fasn (Fatty acid synthase), CD36 (cluster of differentiation 36), Fabp1 (Fatty Acid-Binding Protein 1), Vldlr (very-low-density-lipoprotein receptor), Ldlr (low density lipoprotein receptor), ApoB100 (Apolipoprotein B 100), Pparα (Peroxisome proliferator-activated receptor alpha), Pparγ (Peroxisome Proliferator Activated Receptor Gamma), Leptin, Accα (acetyl-CoA carboxylase alpha), Accβ (acetyl-CoA carboxylase beta), collagen I, collagen type I alpha 1 chain, laminins, laminin α5, laminin γ2, laminin γ3, Socs1 (Suppressor of cytokine signalling 1), Socs3 (Suppressor of cytokine signalling 3), Sirt1 (Sirtuin 1), Sirt5 (Sirtuin 5), Sirt6 (Sirtuin 6), α-SMA (α-smooth muscle actin), MCP1 (monocyte chemoattractant protein 1), TGFβ1 (transforming growth factor beta 1) or F4/80 antigen (macrophage biomarker) mRNA or protein, (2) the changes in the phosphorylation of STAT3 (Signal transducer and activator of transcription 3) protein, Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, FAK (focal adhesion kinase), mTOR, S6K, ULK, 4EBP1 and Akt protein, (3) the disease characteristics of the liver tissue by comparing cell damage, cell arrangement pattern disorder, collagen I or laminin synthesis/accumulation, and AFP (Alpha-fetoprotein), FUCA (AFU, Alpha-L-fucosidase), CD34 (human hematopoietic stem cell and endothelial cell marker), HIF1α (Hypoxia-inducible factor 1-alpha), Ki-67 (Antigen KI-67), or Cyclin D1 expression/accumulation, (4) the level of triglyceride (TG), free fatty acid (FFA), cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDL (Low-density lipoprotein), glucose or insulin in plasma samples of animals, and (5) TM4SF5 played a positive role in developing fatty liver, steatohepatitis and liver fibrosis by measuring weight gain and weight/liver weight increase. The present inventors further confirmed, while the transgenic mouse continued to be raised, that the expression pattern of the mRNA and protein above had been changed and the phosphorylation pattern of the protein above had been changed to express the symptoms of liver fibrosis, hepatitis, liver cirrhosis or liver cancer.
In the Tm4sf5 gene knockout (KO) mouse, there were no significant changes in the expression pattern or the phosphorylation pattern of mRNAs and proteins of the confirmed factors; the degree of glucose resistance, insulin resistance or weight gain caused by the high-fat diet, high-carbohydrate diet, high-amino acid (arginine) diet, or high-sucrose diet which can cause obesity and metabolic disease was weak; and the levels of plasma triglyceride, cholesterol and AST/ALT were not much increased. Therefore, it was confirmed by the results above that the outbreak of liver diseases including fatty liver, hepatitis, liver fibrosis, liver cirrhosis and liver cancer can be induced by the expression of TM4SF5, leading to the completion of the present invention.
(Patent Reference 1) Korean Patent No. 10-0934706
It is an object of the present invention to provide a method for diagnosing liver diseases using the expression changes of TM4SF5 protein.
It is another object of the present invention to provide a method for screening a candidate substance for treating liver diseases or an anti-obesity candidate substance using the expression changes of TM4SF5 protein.
It is another object of the present invention to provide a method for preparing an animal model of portal hypertension using a TM4SF5 gene knock-out mouse and an animal model prepared by the method.
To achieve the above objects, the present invention provides a method of providing information for the diagnosis of liver diseases comprising the following steps:
1) selecting a sample, in which the expression level of TM4SF5 (transmembrane 4 L6 family member 5) protein is increased, obtained from a suspected liver disease patient compared to a sample obtained from a normal control group;
2) measuring the expression level of SREBP1 (sterol regulatory element-binding transcription factor 1) mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the sample selected in step 1); and
3) comparing the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the sample selected in step 1) measured in step 2) with the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the normal control group sample.
The present invention also provides a method for screening a candidate substance for treating fatty liver comprising the following steps:
1) treating a test substance to the cells expressing TM4SF5 and SREBP1 proteins;
2) measuring the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the cells of step 1); and
3) selecting a test substance that suppresses the expression level of SREBP1 mRNA or protein and increases the phosphorylation level of one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the cells of step 1), or suppresses the expression level of SREBP1 mRNA or protein and reduces the synthesis of monoacyl-, diacyl- or triacyl-glycerol in step 2) compared to the control group not treated with the test substance.
The present invention also provides a method for screening a candidate substance for treating obesity, fatty liver or liver cancer comprising the following steps:
1) treating a test substance to the cells or the animal model expressing TM4SF5 protein;
2) measuring the binding of TM4SF5 protein to any one or more selected from the group consisting of mTOR protein, SLC7A1 protein and arginine in the cells or the animal model of step 1);
3) measuring the phosphorylation level of mTOR protein, S6K protein, UNC-51-like kinase 1 (ULK1) protein or 4EBP1 protein in the cells or the animal model of step 1);
4) measuring the level of monoacyl-, diacyl- or triacyl-glycerol in the cells or the animal model of step 1);
5) measuring any one or more selected from the group consisting of weight gain, glucose resistance, insulin resistance and glycolysis reactivity in the cells or the animal model of step 1);
6) measuring the expression levels of the genes related to glycolysis in the cells or the animal model of step 1); and
7) selecting a test substance that suppresses the binding of TM4SF5 protein to any one or more selected from the group consisting of mTOR protein, SLC7A1 protein and arginine in step 2), inhibits the phosphorylation of mTOR protein, S6K protein, UNC-51-like kinase 1 protein or 4EBP1 protein in step 3), reduces the level of monoacyl-, diacyl- or triacyl-glycerol in step 4), and decreases the weight gain, glucose resistance, insulin resistance or glycolysis reactivity in step 5).
The present invention also provides a method for preparing a portal hypertension animal model comprising the step of mating a TM4SF5 knock-out (KO) mouse with a mouse having the genotype of APCmin/+ (adenomatous polyposis colimin/+).
In addition, the present invention provides a portal hypertension animal model prepared by the above method.
The present invention can be effectively used to diagnose obesity and liver disease or to screen a candidate substance for treating obesity or liver disease by measuring the expression changes of TM4SF5 protein by confirming that the metabolic function is reduced in the cells and transgenic mice over-expressing TM4SF5 protein; the weight is gained; the expression and accumulation of mRNAs and proteins of the factors involved in the biosynthesis of fat including TM4SF5 expression-dependent proteins such as SREBP1 protein are increased by high carbohydrate, fat and amino acid diet; the characteristics of obesity, fatty liver and hepatitis appear by reducing the phosphorylation of any one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR protein, S6K protein, ULK protein, 4EBP1 protein and Akt protein; and the expression of SREBP1 protein is decreased, the phosphorylation of STAT3 protein is increased, and the accumulation of extracellular matrix such as collagen and laminin is increased, indicating the characteristics of liver fibrosis or cirrhosis when the transgenic mice continues to be raised.
Hereinafter, the present invention is described in detail.
The present invention provides a method of providing information for the diagnosis of liver diseases comprising the following steps:
1) selecting a sample, in which the expression level of TM4SF5 (transmembrane 4 L6 family member 5) protein is increased, obtained from a suspected liver disease patient compared to a sample obtained from a normal control group;
2) measuring the expression level of SREBP1 (sterol regulatory element-binding transcription factor 1) mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the sample selected in step 1); and
3) comparing the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the sample selected in step 1) measured in step 2) with the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 (signal transducer and activator of transcription 3) protein, c-Src (cellular sarcoma) protein, FAK (focal adhesion kinase) protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the normal control group sample.
The term “TM4SF5 (transmembrane 4 L6 family member 5) protein” used in this specification is a protein included in tetraspanine, tetraspan or TM4SF (transmembrane 4 super family), the membrane receptor group that cross the cell membrane 4 times, and has a structure similar to each other that passes through the cell membrane four times. The TM4SF5 protein shares a structure including four hydrophobic sites that are biochemically estimated to be transmembrane domains.
The term “SREBP1 (sterol regulatory element-binding transcription factor 1) protein” used in this specification is a transcription factor that binds to the promoter of a gene and regulates the transcription thereof, and means a factor that regulates the expression of a gene involved in sterol biosynthesis. The SREBP1 protein is regulated by insulin and regulates the expression of a gene involved in glucose metabolism or fatty acid and fat production.
The term “STAT3 (signal transducer and activator of transcription 3) protein” used in this specification is a transcription factor belonging to the STAT protein family, and means a factor that transmits a signal to a lower level by being phosphorylated by cytokines and growth factors. The STAT3 protein is activated by phosphorylation at the 705th tyrosine residue by interferon, EGF (epidermal growth factor), IL-5 and IL-6, etc.
The terms “Srebp1 (Sterol regulatory element-binding protein 1), Srebp2 (Sterol regulatory element-binding protein 2), Fasn (Fatty acid synthase), CD36 (cluster of differentiation 36), Fabp1 (Fatty Acid-Binding Protein 1), Vldlr (very-low-density-lipoprotein receptor), Ldlr (low density lipoprotein receptor), ApoB100 (Apolipoprotein B 100), Pparα (Peroxisome proliferator-activated receptor alpha), Pparγ (Peroxisome Proliferator Activated Receptor Gamma), Leptin, Accα (acetyl-CoA carboxylase alpha) and Accβ (acetyl-CoA carboxylase beta)” used in this specification are enzymes or proteins involved in the accumulation or synthesis of fatty acids.
The terms “collagen I, collagen type I alpha 1 chain, laminin, laminin α5, laminin γ2 and laminin γ3” used in this specification are proteins corresponding to the extracellular matrix type. The terms “Socs1 (Suppressor of cytokine signalling 1), Socs3 (Suppressor of cytokine signalling 3), STAT3 (Signal transducer and activator of transcription 3), c-Src and FAK (focal adhesion kinase)” are proteins or signaling proteins involved in fibrosis.
The terms “Sirt1 (Sirtuin 1), Sirt5 (Sirtuin 5), Sirt6 (Sirtuin 6), α-SMA (α-smooth muscle actin), MCP1 (monocyte chemoattractant protein 1), TGFβ1 (transforming growth factor beta 1) and F4/80 antigen (macrophage biomarker)” used in this specification are factors involved in inflammation of the liver tissue.
The terms “AFP (Alpha-fetoprotein), FUCA (AFU, Alpha-L-fucosidase), CD34 (human hematopoietic stem cell and endothelial cell marker), HIF1α (Hypoxia-inducible factor 1-alpha), Ki-67 (Antigen KI-67) and Cyclin D1” used in this specification are cancer cell markers or related proteins. The terms “mTOR, S6K, ULK1, 4EBP1 and Akt” are signaling proteins involved in arginine metabolism in liver cancer cells.
The terms “triglyceride (TG), free fatty acid (FFA), cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDL (Low-density lipoprotein), glucose and insulin” used in this specification are factors related to liver tissue damage and fatty liver, hepatitis (or steatohepatitis) and liver fibrosis, and the levels can be confirmed in the animal plasma sample.
The terms “high fat diet, high carbohydrate diet, high amino acid (arginine) diet and high sucrose diet” used in this specification are diets related to obesity and metabolic disorders. It is possible to determine whether or not to induce liver disease including fatty liver, hepatitis, fibrosis and liver cancer by measuring the level of glucose resistance, insulin resistance, triglyceride, cholesterol or AST/ALT in plasma or by checking the degree of weight gain. In particular, since sucrose is decomposed into fructose and glucose in the body and used for cells, high concentration sucrose intake may have the effect of ingesting high concentration of fructose, which is included in carbonated beverages (sweetened beverages), juices, breakfast cereals, etc. for sweetness, causing metabolic diseases such as diabetes and obesity (Journal of Korean Oriental Association for Study of Obesity 2005:5(1): 121-131].
The term “liver disease” used in this specification may include obesity, metabolic disorders, glucose resistance, insulin resistance, weight gain, fatty liver, liver fibrosis, hepatitis, liver cirrhosis or liver cancer.
The TM4SF5, SREBP1, Srebp2, Fasn, CD36, Fabp1, ApoB100, Pparα, Pparγ, Leptin, Accα, Accβ STAT3, collagen type I alpha 1 chain, laminin and laminin γ2 used in the method of providing information of the present invention can be polypeptides composed of any amino acid sequence known in the art. The polypeptides can include variants or fragments of amino acids having different sequences by deletion, insertion, substitution of amino acid residues, or a combination thereof within a range that does not affect the function of the protein. The amino acid substitution in proteins or peptides that does not change the activity of the molecule as a whole is known in the art. In some cases, the polypeptide can be modified by phosphorylation, sulfation, acrylication, saccharification, methylation, farnesylation, etc.
In an embodiment of the present invention, the TM4SF5 protein can be a polypeptide composed of the amino acid sequence represented by SEQ. ID. NO: 1. The triglyceride (TG), Vldlr, Ldlr, and free fatty acid (FFA) are components of fatty acids and fats known in the art.
The method of providing information of the present invention may provide information for the diagnosis of liver disease by identifying the characteristics of TM4SF5-dependent factors, cells, tissues, or individuals, including the expression changes of SREBP1 protein and the phosphorylation level changes of STAT3 protein. The liver disease can be fatty liver, liver fibrosis, hepatitis, liver cirrhosis, or liver cancer.
The term “TM4SF5-dependent factors” used in this specification refer to factors that increase mRNA or protein in the tissues or cells by the expression of TM4SF5 protein (increase of TM4SF5 protein). In the case of fatty liver, the examples of such factors are SREBP1, SREBP2, Fasn, CD36, Fabp1, Vldlr, Ldlr, ApoB100, Pparα, Pparγ, Leptin, Accα, and Accβ. In the case of hepatitis, the examples of such factors are MCP1, TGFβ1, and F4/80 antigen. In the case of liver fibrosis, the examples of such factors are collagen I, collagen type I alpha 1 chain, laminins, laminin α5, laminin γ2, and laminin γ3. In the case of liver cancer, the examples of such factors are AFP, FUCA (AFU), CD34, HIF1a, Ki-67, and Cyclin D1.
In addition, the TM4SF5-dependent factors can include signaling proteins that increase phosphorylation in the tissues or cells according to the expression of TM4SF5 protein (increase of TM4SF5 protein), including STAT3, c-Src, FAK, mTOR, S6K, ULK1, 4EBP1, or Akt protein.
In addition, the TM4SF5-dependent factors may include factors that increase in plasma as fatty liver and hepatitis (or steatohepatitis) develop according to the expression of TM4SF5 protein (increase of TM4SF5 protein), including triglyceride (TG), free fatty acid (FFA), cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDL (low-density lipoprotein), glucose, or insulin.
The features that occur in the TM4SF5-dependent cells, tissues, or individuals include hepatocyte damage, cell arrangement pattern disorder, or increased collagen I or laminin synthesis accumulation as liver fibrosis develops according to the expression of TM4SF5 protein (increase of TM4SF5 protein).
In animal subjects, the expression of TM4SF5 protein (increase of TM4SF5 protein) can increase body weight; body weight/liver weight; weight gain according to high carbohydrate diet, high sucrose diet, high fat diet, low fat/high carbohydrate diet and high arginine diet; insulin resistance; glucose resistance; fatty liver and steatohepatitis; synthesis of extracellular matrix such as collagen and laminin; and accumulation of liver tissue.
In the method of providing information according to the present invention, when the level of the SREBP1, SREBP2, Fasn, CD36, Fabp1, Vldlr, Ldlr, ApoB100, Pparα, Pparγ, Leptin, Accα, or Accβ protein is increased, and the phosphorylation level of any one or more proteins selected from the group consisting of the STAT3 protein, c-Src protein, FAK protein, mTOR protein, S6K protein, ULK protein, 4EBP1 protein and Akt protein is decreased compared to the normal control group, it can be determined as fatty liver.
When the expression level of mRNA or protein of the SREBP1 is increased, and the level of monoacyl-, diacyl-, or triacyl-glycerol is reduced compared to the normal control group, it can be determined as fatty liver.
It was confirmed that the expression of TM4SF5, AFP, FUCA (AFU), CD34, HIF1a, Ki-67, and Cyclin D1 is increased in the sample of a patient with liver disease including liver cancer. The TM4SF5 protein was confirmed to bind to mTOR, SLC7A1 protein or arginine. It was also confirmed that the phosphorylation of mTOR protein, S6K protein, UNC-51-like kinase 1 (ULK1) protein or 4EBP1 is increased. The binding of the TM4SF5 protein to arginine can be mediated by the 124th to 129th residues from the N-terminus of the TM4SF5 protein.
In the method of providing information according to the present invention, when the expression level of the SREBP1, SREBP2, Fasn, CD36, Fabp1, Vldlr, Ldlr, ApoB100, Pparα, Pparγ, Leptin, Accα or Accβ protein is reduced, the phosphorylation level of STAT3 protein, c-Src protein, FAK protein or Akt protein is increased, and the expression of collagen I, laminin, laminin γ2 or α-SMA compared to the normal control group, it can be determined as liver fibrosis, hepatitis, liver cirrhosis or liver cancer.
In the method of providing information according to the present invention, the expression level of SREBP1 protein can be regulated by any one or more proteins selected from the group consisting of SIRT1, SIRT2, SIRT4, SIRT5, SIRT6 and SIRT7. Particularly, the increased expression of SREBP1 and SREBP2 proteins can be controlled by the decrease of the expression of SIRT1, SIRT5 and SIRT6 proteins, and the increase of the expression of SIRT2, SIRT4 and SIRT7 proteins.
The sample can be any sample as long as the expression of TM4SF5, SREBP1, SREBP2, Fasn, CD36, Fabp1, Vldlr, Ldlr, ApoB100, Pparα, Pparγ, Leptin, Accα or Accβ protein and the phosphorylation level of STAT3, c-Src, or FAK protein can be changed by liver disease.
The expression level or the phosphorylation level of the protein can be measured by any method known in the art. Particularly, the expression level of the protein can be measured by any one or more methods selected from the group consisting of Western blotting, enzyme-linked immunosorbent assay (ELISA), proteomic analysis, immunohistochemical staining, immunoprecipitation and immunofluorescence. Meanwhile, the expression level of mRNA can be measured by RT-PCR, real-time PCR or RNA-Seq.
In the method of providing information according to the present invention, the phosphorylation of STAT3 protein can be regulated by any one or more proteins selected from the group consisting of SOCS1 and SOCS3. Particularly, the decrease of the phosphorylation of STAT3 protein can be controlled by the increase of the expression of SOCS1 and SOCS3 proteins, and the increase of the phosphorylation of STAT3 protein can be controlled by the decrease of the expression of SOCS1 and SOCS3 proteins.
The method of providing information according to the present invention can further include a step of measuring the expression of any one or more mRNAs or proteins selected from the group consisting of SIRT1 (NAD-dependent deacetylase sirtuin-1), SIRT5, SIRT6, SREBP2, SREBP1c, CD36, FABP1 (fatty acid-binding protein 1), FASN (fatty Acid Synthase), LDLR (low density lipoprotein receptor), VLDLR (very Low Density Lipoprotein Receptor), PPARγ (peroxisome proliferator-activated receptors γ), TIMP1 (The tissue inhibitor of metalloproteinase-1), TGFβ1 (Transforming growth factor beta 1), TNFα (tumor necrosis factor α), vimentin, MCP1 [monocyte chemotactic protein 1 (CCL2)], laminin α2, laminin α3, laminin α5, laminin γ2, laminin γ3, SOCS1 (suppressor of cytokine signaling 1), SOCS3, ApoB100 (Apolipoprotein B), PPARα, Leptin, Acc (Acetyl-CoA carboxylase)α, Accβ, F4/80 antigen, collagen I, collagen type I alpha 1 chain, AFP (Alpha-fetoprotein), FUCA (AFU, alpha-L-fucosidase 1), CD34, HIF1α (Hypoxia-inducible factor), Ki-67 and Cyclin D1. When the expression level of mRNA or protein of SIRT1, SIRT5, SIRT6, laminin α5, laminin γ2 or laminin γ3 is decreased, the expression level of mRNA or protein of SREBP2, SREBP1c, CD36, FABP1, FASN, LDLR, VLDLR, PPARγ, TIMP1, TGFβ1, TNFα, vimentin, MCP1, SOCS1, SOCS3, ApoB100, PPARα, Leptin, Accα or Accβ is increased, the level of monoacyl-, diacyl-, and triacyl-glycerol is increased, and the phosphorylation level of any one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR protein, S6K protein, ULK protein, 4EBP1 protein and Akt protein is decreased or not changed compared to the normal control group, it can be determined as fatty liver. On the other hand, when the expression level of mRNA or protein of SREBP2, SREBP1c, CD36, FABP1, FASN, LDLR, VLDLR or PPARγ is decreased or not changed compared to the normal control group, the expression level of mRNA or protein of SIRT1, SIRT5, SIRT6, TGFβ1, TNFα, vimentin, laminin, laminin γ2, collagen SOCS1, SOCS3, F4/80 antigen, collagen I, collagen type I alpha 1 chain, AFP (Alpha-fetoprotein), FUCA (AFU, alpha-L-fucosidase 1), CD34, HIF1α (Hypoxia-inducible factor), Ki-67 or Cyclin D1 is increased, the cytokine/chemokine factors such as MCP1, TGFβ1 and F4/80 antigen is increased, or the phosphorylation level of any one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR protein, S6K protein, ULK protein, 4EBP1 protein and Akt protein is increased compared to the normal control group, it can be determined as liver fibrosis, hepatitis, liver cirrhosis or liver cancer.
When the expression level of mRNA or protein of SREBP2, SREBP1c, CD36, FABP1, FASN, LDLR, VLDLR or PPARγ is reduced, the expression level of mRNA or protein of SIRT1, SIRT5, SIRT6, TGFβ1, TNFα, vimentin, laminin, laminin γ2, collagen I, SOCS1, SOCS3, F4/80 antigen, collagen I, collagen type I alpha 1 chain, AFP (Alpha-fetoprotein), FUCA (AFU, alpha-L-fucosidase 1), CD34, HIF1α (Hypoxia-inducible factor), Ki-67 or Cyclin D1 is increased, the expression level of mRNA or protein of AFP, FUCA (AFU), CD34, HIF1α, Ki-67, Cyclin D1, laminin, collagen I or laminin γ2 is increased, the phosphorylation level of any one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR protein, S6K protein, ULK protein, 4EBP1 protein and Akt protein is increased compared to the normal control group, it can be determined as liver cancer.
As the expression of the TM4SF5 protein increases, the amount of any one or more selected from the group consisting of triglyceride (TG), free fatty acid (FFA), cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDL (Low-density lipoprotein), glucose and insulin in plasma can be increased as fatty liver and hepatitis develop. As the expression of the TM4SF5 protein increases, hepatocyte damage, cell arrangement pattern disorder or increased synthesis accumulation of collagen I or laminin may be seen in the tissue as liver fibrosis develops. The expression of TM4SF5 protein can increase body weight; body weight/liver weight; weight gain according to high carbohydrate diet, high sucrose diet, high fat diet, low fat/high carbohydrate diet and high arginine diet; insulin resistance; glucose resistance; fatty liver and steatohepatitis; or synthesis of extracellular matrix such as collagen and laminin in patients.
In a specific embodiment of the present invention, the present inventors prepared a mouse model (52 weeks old) transformed with a construct expressing TM4SF5 protein (see
In addition, hepatocytes were obtained from the liver tissue of the prepared transgenic mouse, and the expression changes of genes and proteins related to fatty liver were confirmed. As a result, it was confirmed that the expression of mRNAs or proteins of SREBP1, SREBP2, SREBP1c, CD36, Fabp1, Fasn, Accα, Accβ, Ldlr, SOCS1 and SOCS3 was increased; the phosphorylation of STAT3 protein was reduced; and the levels of triglyceride (TG), AST and ALT in the liver tissue were increased (see
On the other hand, it was confirmed that the increase of ApoB100, LdlR, Srebp2, Pparγ and leptin was weak in the liver tissue of the animal in which Tm4sf5 gene was removed as a heterozygote compared to the normal animal not over-expressing TM4SF5 (see
When TM4SF5 was over-expressed in a cell model or free fatty acid was treated to a TM4SF5 non-expressing cell line, it was confirmed that the increase of the expression of SREBP1 or Pparγ protein is inversely correlated with the phosphorylation of STAT3 protein (see
In adipocytes (3T3-L1), it was confirmed that fat was accumulated depending on the expression of TM4SF5, and the levels of mRNA and protein of Pparγ, CD36, Fasn, Srebp1 or Fabp1 were maintained (see
It was confirmed that the increase of the expression of mRNA or protein of SREBP1, SREBP2 or SREBP1c was increased by the decrease of the expression of SIRT1, SIRT5 or SIRT6 gene, and the increase of the expression of SIRT2, SIRT4 or SIRT7 gene. It was also confirmed that the increase of the phosphorylation of STAT3 protein was regulated by the expression of SOCS1 and SOCS3 genes and proteins (see
In a specific embodiment of the present invention, when TM4SF5 was expressed in the primary liver epithelial cells isolated from 52-week-old C57BL/6 normal animals or free fatty acid (FFA) was treated thereto, it was confirmed that the expression of SOCS1 and SOCS3 had positive feedback (or correlation) with the expression of TM4SF5. The expression of SREBP1 and the expression of SOCS3 were confirmed to have positive feedback, and the expressions of the proteins (Srebp1, Socs1 and Socs3) associated with the expression of TM4SF5 were negatively correlated with the phosphorylation of STAT3 protein (negative feedback) (see
In addition, it was confirmed in the TM4SF5 gene knockout mouse (TM4SF5 gene KO mouse) that the ratio of liver weight/weight of both males and females was lower than that of the normal animals at 3 or 6 months of age (see
When the high fat diet was fed freely for 10 weeks, the normal animal showed a significant increase in weight compared to the normal diet, but the TM4SF5 gene knockout mouse showed a low level of weight gain and low levels of cholesterol and FFA in the liver tissue (see
In the 52-week-old TM4SF5 knockout C57BL/6 mouse (Tm4sf5−/+, the levels of Socs1 and Socs3 mRNAs and proteins were reduced compared to those in the normal mouse. When the high fat diet was fed, the normal animal showed the symptoms of steatohepatitis, but the knockout animal showed the weak symptoms, and at this time, Srebp1c mRNA and Srebp1 protein were reduced (see
Furthermore, it was confirmed that the TM4SF5 protein was involved in arginine transport and induced S6K activity by binding to mTOR, SCL7A1 and arginine (see
In the TM4SF5 gene KO mouse, weight gain, fat accumulation, glucose resistance, insulin resistance or liver tissue damage was suppressed even when the high carbohydrate or high arginine diet unlike in the normal mouse (see
In the TM4SF5 gene KO mouse, unlike in the normal mouse, it was confirmed that the function of glycolysis for energy production was reduced by measuring the extracellular acidification rate (ECAR) by applying pharmacological stress to mitochondria. Through RNA-Seq analysis, a group of genes that depended on the expression of TM4SF5 was identified (see
When the TM4SF5 gene KO mouse was fed a high sucrose diet, unlike the normal animal, the symptoms of fatty liver were weak, and the levels of AST, ALT and total cholesterol in plasma were low. When the lipid component was analyzed, it was confirmed that the contents of monoacyl-, diacyl- and triacyl-glycerol were low in the Tm4sf5 gene KO mouse compared to those in the normal mouse (see
The expression of SREBP1, SREBP2, SREBP1c, SOCS1 or SOCS3 mRNA or protein in the liver tissue of the mouse (78 weeks old) transformed with a construct expressing TM4SF5 protein was not decreased or increased compared to that of the normal control group not expressing TM4SF5, the phosphorylation of STAT3 protein was increased, the levels of various factors related to fatty liver were similar to the levels present in the normal animal (without increasing), the mRNA levels of genes related to liver fibrosis and inflammation were increased, and the liver tissue exhibited the phenotype of liver fibrosis, liver cirrhosis or hepatitis (see
In addition, the present inventors constructed a liver disease animal model of liver fibrosis/liver cirrhosis by administering CCl4 for 4 weeks or 16 weeks, according to the conventional method for preparing a liver disease animal model, and confirmed the liver tissue damage and the expression accumulation in the animal model (see
Through the liver tissue staining of the animals, it was confirmed that the expression of TM4SF5 and the expression of α-SMA, collagen I, laminin or laminin γ2 were increased, and the phosphorylation of STAT3, c-Src, FAK or Akt protein was correlatively increased in the liver disease animal model of liver fibrosis/liver cirrhosis and primary epithelial cells (see
The present inventors confirmed that the expression of collagen in hepatic stellate cells and the expression of laminin in hepatic epithelial cells were regulated by the phosphorylation of STAT3 protein by binding to the promoters of collagen type I alpha 1 chain and laminin γ2 (see
When the expression of laminin γ2 or collagen type I alpha 1 chain was suppressed in the normal animal and CCl4 was treated to the animal, it was confirmed that the liver tissue damage was inhibited, the expression of TGFβ1, α-SMA, laminin or collagen was suppressed, the phosphorylation of STAT3 protein was inhibited, and the expression of laminin γ2 or collagen type I alpha 1 chain was important for liver fibrosis (see
When Tm4sf5 gene was over-expressed in FVB/N animals, the nodules suggesting a tumor were confirmed in the liver tissue, the expression of CD34, α-SMA, AFP, FUCA, laminin, laminin γ2, collagen, MCP-1, F4/80 antigen, Hif1a, Ki67 or Cyclin D1 mRNA or protein was increased, and the level of AST, ALT, LDL or triglyceride (TG) in plasma was increased (see
In the liver cancer model treated with DEN, it was confirmed that the nodule formation and the liver tissue damage were observed in the liver tissue, and the expression of TM4SF5, laminin, collagen or laminin γ2 was increased, and the phosphorylation of STAT3 protein was increased (see
Therefore, when TM4SF5 protein is increased in the cancer region or the surrounding area of the liver tissue sample of a patient with suspected liver disease, the expression of SREBP1, SREBP2, SREBP1c, laminin or collagen mRNA or protein and the phosphorylation level of STAT3, c-Src, FAK or Akt protein are measured (see
The present invention also provides a method for screening a candidate substance for treating fatty liver:
1) treating a test substance to the cells expressing TM4SF5 and SREBP1 proteins;
2) measuring the expression level of SREBP1 mRNA or protein, and the phosphorylation level of one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the cells of step 1); and
3) selecting a test substance that suppresses the expression level of SREBP1 mRNA or protein and increases the phosphorylation level of one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK protein, mTOR, S6K, ULK, 4EBP1 and Akt proteins in the cells of step 1), or suppresses the expression level of SREBP1 mRNA or protein and reduces the synthesis of monoacyl-, diacyl- or triacyl-glycerol in step 2) compared to the control group not treated with the test substance.
The TM4SF5, SREBP1, SREBP2, Fasn, CD36, Fabp1, ApoB100, Pparα, Pparγ, Leptin, Accα, Accβ STAT3, collagen type I, laminin and laminin γ2 proteins have the characteristics as described above. For example, the TM4SF5, SREBP1 and STAT3 proteins may be any sequence well known in the art, and can include variants or fragments of the sequence. Specifically, the TM4SF5, SREBP1 and STAT3 proteins may be the polypeptides composed of the amino acid sequences represented by SEQ. ID. NO: 1, NO: 2 and NO: 3, respectively. In addition, The triglyceride, Vldlr, Ldlr and free fatty acid are the components of fatty acid and fat known in the art.
In the method for screening a candidate substance for treating fatty liver according to the present invention, the candidate substance capable of treating fatty liver can be screened by using the expression changes of TM4SF5, SREBP1, Srebp2, Fasn, CD36, Fabp1, ApoB100, Pparα, Pparγ, Leptin, Accα or Accβ protein, and the changes of the phosphorylation level of STAT3, c-Src, FAK (focal adhesion kinase), mTOR, S6K, ULK1, 4EBP1 or Akt protein in the cells expressing the proteins.
The method for screening a candidate substance for treating liver cancer according to the present invention can further include a step of confirming the increase of the expression of any one or more proteins selected from the group consisting of CD34, AFU, FUCA, laminin γ2, HIF1α and cyclin D1 together with the expression of TM4SF5 protein, or confirming the binding of TM4SF5 protein to mTOR, SLC7A1 or arginine. The candidate substance for treating liver disease including liver cancer selected by the screening method according to the present invention can inhibit the binding of the TM4SF5 protein to mTOR, SLC7A1 or arginine.
In a specific embodiment of the present invention, the present inventors prepared a transgenic mouse model expressing TM4SF5 protein, and confirmed that the formation of fat in the liver tissue of the mouse model was promoted to display the phenotype of fatty liver (see
In addition, it was confirmed that the expression of SREBP1, SREBP2, SREBP1c, CD36, Fabp1, Fasn, Accα, Accβ, Ldlr, SOCS1 or SOCS3 mRNA or protein was increased in the liver tissue of the transgenic mouse or the hepatocytes obtained from the liver tissue, the phosphorylation of STAT3 protein was decreased, and the levels of triglyceride (TG), AST and ALT were increased in the liver tissue (see
Therefore, it was confirmed that a candidate substance for treating fatty liver can be screened by measuring the expression level of SREBP1, SREBP2, SREBP1c, CD36, Fabp1, Fasn, Accα, Accβ, Ldlr, SOCS1 or SOCS3 protein and the phosphorylation of STAT3, c-Src or FAK protein in the cells expressing TM4SF5 protein.
In a specific embodiment of the present invention, the present inventors prepared a transgenic mouse over-expressing TM4SF5 protein, and confirmed that the formation of fat was promoted in the transgenic mouse (see
The present invention also provides a method for screening a candidate substance for treating liver fibrosis, hepatitis or liver cirrhosis comprising the steps of treating a test substance to the cells expressing TM4SF5 protein and having phosphorylated STAT3 protein; measuring the expression level of SREBP1 protein and the phosphorylation level of any one or more proteins selected from the group consisting of STAT3, c-Src, FAK, mTOR, S6K, ULK, 4EBP1 and Akt in the cells; and selecting a test substance that increases the expression level of SREBP1 protein and suppresses the phosphorylation level of STAT3 protein compared to the control group not treated with the test substance.
The TM4SF5, SREBP1 and STAT3 proteins have the characteristics as described above. For example, the TM4SF5, SREBP1 and STAT3 proteins may be any sequence well known in the art, and can include variants or fragments of the sequence. Specifically, the TM4SF5 protein may be the polypeptide composed of the amino acid sequence represented by SEQ. ID. NO: 1.
In the method for screening a candidate substance for treating fatty liver according to the present invention, the candidate substance capable of treating liver fibrosis, hepatitis, liver cirrhosis or liver cancer can be screened by using the expression changes of SREBP1 protein, and the changes of the phosphorylation level of any one or more proteins selected from the group consisting of STAT3 protein, c-Src protein, FAK, mTOR, S6K, ULK, 4EBP1 or Akt protein in the cells expressing TM4SF5 and SREBP1 proteins.
The present invention also provides a method for screening a candidate substance for treating obesity, fatty liver or liver cancer comprising the following steps:
1) treating a test substance to the cells or the animal model expressing TM4SF5 protein;
2) measuring the binding of TM4SF5 protein to any one or more selected from the group consisting of mTOR protein, SLC7A1 protein and arginine in the cells or the animal model of step 1);
3) measuring the phosphorylation level of mTOR protein, S6K protein, UNC-51-like kinase 1 (ULK1) protein or 4EBP1 protein in the cells or the animal model of step 1);
4) measuring the level of monoacyl-, diacyl- or triacyl-glycerol in the cells or the animal model of step 1);
5) measuring any one or more selected from the group consisting of weight gain, glucose resistance, insulin resistance and glycolysis reactivity in the cells or the animal model of step 1);
6) measuring the expression levels of the genes related to glycolysis in the cells or the animal model of step 1); and
7) selecting a test substance that suppresses the binding of TM4SF5 protein to any one or more selected from the group consisting of mTOR protein, SLC7A1 protein and arginine in step 2), inhibits the phosphorylation of mTOR protein, S6K protein, UNC-51-like kinase 1 protein or 4EBP1 protein in step 3), reduces the level of monoacyl-, diacyl- or triacyl-glycerol in step 4), and decreases the weight gain, glucose resistance, insulin resistance or glycolysis reactivity in step 5).
The term “mTOR (mammalian target of rapamycin)” used in this specification means a hub signal transduction for the regulation of cellular metabolic functions (GenBank accession number: NM_004958.3). The term “SLC7A1 (solute carrier family 7 member 1) protein” means an arginine transporter present in the cell membrane and lysosomal membrane (GenBank accession number: NM_003045.4).
The TM4SF5 and SLC7A1 proteins have the characteristics as described above. For example, the TM4SF5 and SLC7A1 proteins may be any sequence well known in the art, and can include variants or fragments of the sequence. Specifically, the TM4SF5 and SLC7A1 proteins may be the polypeptides composed of the amino acid sequences represented by SEQ. ID. NO: 1 and NO: 2, respectively.
In the method for screening an anti-obesity candidate substance according to the present invention, the anti-obesity candidate substance and the liver cancer cell survival inhibitor candidate can be screened by selecting a test substance that inhibits the binding of the TM4SF5 protein to mTOR, SLC7A1 or arginine. The binding of the TM4SF5 protein to arginine can be mediated by the 124th to 129th residues from the N-terminus of the TM4SF5 protein.
In a specific embodiment of the present invention, the present inventors prepared a transgenic mouse over-expressing TM4SF5 protein, and confirmed that the formation of fat was promoted in the transgenic mouse (see
Therefore, it was confirmed that the anti-obesity and anti-cancer candidates can be screened by measuring the inhibition of the binding of TM4SF5 protein to mTOR, SLC7A1 or arginine.
The present invention also provides a method for preparing a portal hypertension animal model comprising the step of mating a TM4SF5 knock-out (KO) mouse with a mouse having the genotype of APCmin/+ (see
As used herein, the “APC (adenomatous polyposis coli) gene” is a causative gene for familial colorectal adenomatosis, and the product synthesized from the said APC gene forms a complex with β-catenin to promote its degradation.
The TM4SF5 (GenBank Accession NO. NM_003963) and APC (GenBank Accession NO. M74088) genes can be the polynucleotides composed of any nucleotide sequences known in the art. The polynucleotide can be a polynucleotide composed of any nucleotide sequence encoding TM4SF5 protein. The TM4SF5 gene of the present invention can be a polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 3. The TM4SF5 gene may have 70%, 80%, 90%, 95% or 99% homology with the nucleotide sequence represented by SEQ. ID. NO: 3.
In a specific embodiment of the present invention, the inventors prepared a TM4SF5 gene knockout (KO) mouse, and then crossed the mouse with a mouse having the genotype of APCmin/+ to obtain offspring (see
Therefore, it was confirmed that an animal model of portal hypertension can be prepared by mating a TM4SF5 gene KO mouse and a mouse having the genotype of APCmin/+.
In addition, the present invention provides a portal hypertension animal model prepared by the above method.
The animal model can be prepared by the preparation method as described above. In one example, the preparation method can include a step of mating a TM4SF5 gene KO mouse with a mouse having the genotype of APCmin/+. At this time, the TM4SF5 and APC genes can have the characteristics as described above, and can include variants and fragments thereof. The TM4SF5 and APC genes can be the polynucleotides composed of the nucleotide sequences represented by SEQ. ID. NO: 3 and NO: 4, respectively.
In a specific embodiment of the present invention, the inventors prepared a portal hypertension animal model by mating a TM4SF5 knock-out (KO) mouse with a mouse having the genotype of APCmin/+ (see
Hereinafter, the present invention will be described in detail by the following examples.
However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.
In order to confirm the liver disease phenotype of the mouse over-expressing TM4SF5 protein, a transgenic mouse model was prepared in the following ways.
First, a construct wherein the Flag-labeled TM4SF5 protein (GenBank Accession NO. CAG33206) and BGH (bovine growth hormone) poly-A region (Macrogen, Korea) were expressed under the control of CMV promoter was constructed (J Cell Sci. 2012, 125(Pt 24):5960-73).
The prepared construct was injected into the fertilized egg of a C57BL/6 mouse using a microinjection method.
Two weeks after the injection, the liver tissue was obtained from the mouse and PCR was performed by the conventional method using the primers listed in Table 1 below (
As shown in
The mice prepared in Example <1-1> were raised for 52 weeks, and then sacrificed to obtain the liver tissues. The appearance of the obtained liver tissue was observed, and the results are shown in
As shown in
H&E staining was performed using the liver tissue of the transgenic mouse over-expressing TM4SF5 protein obtained in Example <1-1>.
First, the dissected liver tissue was fixed to paraffin, and then slides were made. For H&E staining, the obtained liver tissue was left in a 60° C. oven for about 20 minutes to remove paraffin. The paraffin-removed liver tissue was immersed in xylene for 5 minutes, and this process was repeated 3 times. Next, the liver tissue was sequentially placed in 100%, 90%, 80% and 70% ethanol, and distilled water for 3 minutes each, and then taken out, followed by reaction in a hematoxylin solution for 5 minutes. Upon completion of the reaction, the liver tissue was washed with tap water, followed by reaction in an eosin solution for 20 minutes. The liver tissue was washed again with tap water, and then sequentially placed in 70%, 80%, 90% and 100% ethanol, and a xylene solution for 3 minutes each, and then placed on a slide and mounted. The slide glass was observed using a microscope and the results are shown in
As shown in
Oil red 0 staining was performed using the liver tissue of the transgenic mouse over-expressing TM4SF5 protein obtained in Example <1-1> in the following ways.
First, blood of the transgenic mouse prepared in Example <1-1> was removed by adding a perfusate, and hepatocytes were separated using type 2 collagen. The isolated hepatocytes were filtered using a cell filter having a pore size of 40 μm, and centrifugation was performed to obtain pellets. The obtained pellets were cultured using the William's E medium supplemented with 1% penicillin/streptomycin and 10% FBS. At this time, the culture was performed using a plate coated with collagen.
The cultured hepatocytes were put in 10% formalin, fixed for 15 minutes, and washed with PBS. Meanwhile, the oil red 0 dye (Sigma, Germany) was mixed with sterile distilled water to prepare a mixed solution, and the prepared mixed solution was filtered. The filtered oil red 0 solution was added to the washed cells, which were stained for 30 minutes, followed by washing with distilled water. The stained cells were observed using a microscope, and the results are shown in
As shown in
The levels of triglyceride (TG), albumin and ALT in the blood of the transgenic mouse over-expressing TM4SF5 protein obtained in Example <1-1> were measured in the following ways.
First, blood was obtained before sacrificing the transgenic mouse. The obtained blood was placed in a 1.5 ml tube coated with 1 M EDTA, and 8 μl of 1 M EDTA was added thereto. Serum was separated by centrifuging the tube at 1,500×g and 4° C. for 15 minutes. The levels of triglycerides, albumin and ALT were confirmed from the separated serum using a blood analyzer (Drichem 4000, Fuji, Japan).
As a result, as shown in
The expression changes of the fatty liver-related genes in the liver tissue of the transgenic mouse prepared in Example <1-1> were confirmed by the following method.
First, cells were lysed by adding Qiazol (Qiagen, USA) to the obtained liver tissue, and chloroform was added thereto, followed by centrifugation at 12,000×g and at 4° C. for 15 minutes. After the centrifugation, RNA was precipitated by adding isopropanol to the obtained supernatant. The precipitated RNA was washed with 70% ethanol, and centrifuged for 5 minutes under the conditions of 7,500×g and 4° C. to obtain RNA pellets. The RNA pellets were dried at room temperature for 10 minutes. RNA was obtained by adding 30 μl of DEPC-distilled water to the dried pellets.
From the obtained RNA, gDNA was removed and cDNA was obtained using a reverse transcription kit (Toyobo, Japan) according to the manufacturer's protocol. Real-time PCR was performed by adding 2× evergreen master mix (Labopass, Korea) and 0.4 μM of forward and reverse primers listed in Table 2 below to the obtained cDNA. From the PCR, the expression level of each gene was obtained using the modified Pfaffl delta-delta Ct method.
As a result, as shown in
The expression changes of the fatty liver-related proteins in the liver tissue of the transgenic mouse prepared in Example <1-1> were confirmed by Western blotting.
Particularly, a lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium dioxycholate, 150 mM NaCl, 1 mM EDTA], SDS (sodium dodecyl sulfate), Na3O4V and protease inhibitor cocktail (GenDepot) were added to the obtained liver tissue, which was left at 4° C. for 15 minutes to lyse the tissue. The lysate was centrifuged for 30 minutes under the conditions of 13,000 rpm and 4° C. to obtain a supernatant. The proteins present in the supernatant were quantified using BCA reagent (Thermo Scientifics). 4× sample buffer [4 ml of 100% glycerol, 2.4 ml of Tris-HCl (pH 6.8), 0.8 g of SDS, 4 mg of brominated phenol blue, 0.4 ml of β-mercaptoethanol and 3.1 ml of H2O, final volume: 10 ml] was added thereto, which was boiled at 100° C. for 5 minutes. SDS-PAGE was performed, and the proteins were transferred to a nitrocellulose membrane (Whatman). The membrane was pretreated in a solution containing 5% skim milk for 1 hour, and reacted with the antibodies against laminin (Abcam, UK), ACC1 (Cell Signalling, USA), SREBP1 precursor (Santa cruz, USA), mature SREBP1 (Santa cruz, USA), MTP (Santa cruz, USA), PPARα (Santa cruz, USA), pY706STAT3 (Millipore, USA), STAT3 (Santa cruz, USA), α-tubulin (Sigma, USA) and TM4SF5 (J Clin Invest. 2008 April; 118(4): 1354-66) as the primary antibodies at 4° C. for 15 hours. Then, the membrane was reacted with the secondary antibody, and developed on an X-ray film using an ECL solution (Pierce, USA). The results are shown in
As shown in
TM4SF5 Protein The suppression of the STAT3 protein phosphorylation in the transgenic mouse over-expressing TM4SF5 protein, confirmed in Example <2-2>, was confirmed again by using histostaining.
The obtained liver tissue was left in a 60° C. oven for about 20 minutes to remove paraffin. The paraffin-removed liver tissue was immersed in xylene for 5 minutes, and this process was repeated 3 times. Next, the liver tissue was sequentially placed in 100%, 90%, 80% and 70% ethanol, and distilled water for 3 minutes each, and in tap water for 10 minutes. The liver tissue was put in 10 mM citric acid buffer (pH 6.0), and covered with foil, which was autoclaved. Upon completion of the autoclave, the tissue was sufficiently cooled, reacted in PBS for 10 minutes twice, and 3% hydrogen peroxide was made using methanol to undergo a step of quenching for 15 minutes. This was put in PBS again and reacted three times for 5 minutes each. Then, blocking was performed in PBS containing 5% horse or goat serum at 4° C. for one day. On the next day, the tissue was reacted three times with PBS for 5 minutes each, and reacted with biotin-conjugated IgG rabbit or mouse for 1 hour using the serum used for the primary reaction. The tissue was washed again with PBS, and reacted with an avidin-biotin-peroxidase complex prepared in advance for 30 minutes. The tissue was washed 3 times with PBS for 5 minutes each and stained with DAB. At this time, the reaction time was different according to the antibody used, so the time point was determined in comparison with the control group. The tissue stained with DAB was placed in distilled water and reacted with hematoxylin for more than 5 minutes. The liver tissue was washed with tap water, and then sequentially placed in 70%, 80%, 90% and 100% ethanol, and a xylene solution for 3 minutes each, and then placed on a slide and mounted.
As shown in
The above results were reconfirmed using the hepatocytes over-expressing TM4SF5 protein.
First, hepatocytes were obtained under the same conditions and methods as described in Example <1-4>, except that the C57BL/6 normal mouse was used instead of the transgenic mouse over-expressing TM4SF5 protein. The obtained hepatocytes were transformed with the construct containing TM4SF5 gene prepared in Example <1-1>. Oil red 0 staining was performed using the cells transformed with the construct expressing TM4SF5 under the same conditions and methods as described in Example <1-4>. At this time, the hepatocytes obtained from the normal mouse and treated with fatty acids (FFA) were used as the positive control. The stained cells were observed using a microscope, and the results are shown in
As shown in
The expression changes of the fat-related genes were confirmed using the hepatocytes expressing TM4SF5 protein prepared in Example <3-1>. At this time, the hepatocytes over-expressing or not-expressing TM4SF5 protein treated with free fatty acid, or the hepatocytes expressing TM4SF5 protein, the normal hepatocytes treated with IL-6, a cytokine associated with fatty liver, and the hepatocytes expressing TM4SF5 protein treated with IL-6 were used for the comparison. The experiment was performed under the same conditions and methods as described in Example <2-1>, except that the primers listed in Table 3 were used.
As a result, as shown in
The expression changes of the fatty liver related proteins in the cells over-expressing TM4SF5 protein were confirmed by Western blotting. The experiment was performed under the same conditions and methods as described in Example <2-2>, except that the antibodies against laminin, SREBP1 precursor, mature SREBP1, PPARγ, pY705STAT3, STAT3, β-actin and Flag were used as the primary antibodies.
As a result, as shown in
To confirm whether the increase of the SREBP1 protein expression and the decrease of the STAT3 protein phosphorylation have a competitive relationship, Western blotting was performed in the same manner as described above using the hepatocytes over-expressing STAT3 protein treated with fatty acid. As a result, as shown in
Meanwhile, after over-expressing SREBP1 protein in the normal hepatocytes, the phosphorylation of STAT3 protein was confirmed by the same method as above. As a result, as shown in
Therefore, it was confirmed from the above results that the expression level of SREBP1 protein and the phosphorylation of STAT3 protein played opposite roles.
<4-1> Confirmation of Fat Production Inhibition in Adipocytes with Suppressed TM4SF5 Protein Expression
To confirm whether the fat production was suppressed when the expression of TM4SF5 protein was suppressed in adipocytes, oil red O staining was performed.
First, mouse 3T3-L1 adipocyte progenitor cells were prepared by culturing in DMEM supplemented with 10% NBCS (Gibco, 16010159) and 1% penicillin/streptomycin. The prepared cells were distributed in a 6-well plate at the density of 1×105/well.
After 4 days of the distribution, when the progenitor cells were full in the wells, the cells were further cultured for 48 hours. Then, the medium was replaced with the adipocyte differentiation medium (MDI medium containing 10% FBS) supplemented with 1 μM dexamethasone, 0.5 mM IBMX (3-Isobutyl-1-methylxanthine) and 10 μg/m of insulin (Sigma, USA). After culturing the cells for 2 days, the medium was replaced with DMEM supplemented with 10% FBS and 10 μg/m
of insulin. After replacing with the medium, the cells were cultured for 10 days, and then cultured using DMEM supplemented with 10% FBS and 1% penicillin/streptomycin to obtain differentiated adipocytes. The adipocytes were transfected with TM4SF5 shRNA (shTM4SF5, 5′-CCTGGAATGTGACGCTCTTCTCGCTGCTG-3′, SEQ. ID. NO: 35) using lipofectamine 3000.
As a result, as shown in
<4-2> Confirmation of Expression Change of Fat-Related Gene in Adipocytes with Suppressed TM4SF5 Protein Expression
Whether the expression of the fat-related gene changed when the expression of TM4SF5 gene was suppressed was confirmed by the following method. The experiment was performed under the same conditions and methods as described in Example <2-1>, except that the differentiated adipocytes obtained in Example <4-1> were treated with shRNA against TM4SF5, and then the primers listed in Table 4 were used.
As a result, as shown in
<4-3> Confirmation of Expression Change of Fat-Related Gene in Differentiation Process of Adipocytes with Increased TM4SF5 Protein Expression
Mouse 313-L1 preadipocytes were cultured in DMEM supplemented with 10% NBCS (Gibco, 16010159) and 1% penicillin/streptomycin. On the 4th day of culture, if the preadipocytes were filled with 100% in the culture vessel, the cells were further cultured for hours, and then treated with the adipocyte differentiation medium (MDI medium) supplemented with 1 μM dexamethasone, 0.5 mM IBMX (3-Isobutyl-1-methylxanthine), 10 μg/ml of insulin (Sigma, USA) and 10% FBS for 2 days. Thereafter, the medium was replaced with DMEM supplemented with 10% FBS and insulin (10 μg/ml) for 2 days. On the 10th day of culture, the cells were cultured with DMEM supplemented with 10% NBCS and 1% penicillin/streptomycin to differentiate into adipocytes.
At this time, whether the expression of the fat-related gene was changed in addition to the expression of TM4SF5 gene during the process of accumulating fat was confirmed by performing the experiment under the same conditions and methods as described in Example <2-2> using the primary antibodies against SREBP1 precursor, mature SREBP1, PPARγ, pY705STAT3, STAT3, β-actin (Cell Signaling Technology, USA), ERK (Cell Signaling Technology, USA), p-ERK (Cell Signaling Technology, USA), Akt (Cell Signaling Technology, USA) and TM4SF5.
As a result, as shown in
The effect of the increased expression of SREBP1 protein by the over-expression of TM4SF5 protein on the expression patterns of SIRT genes, the factors that regulate the expression of SREBP1 protein, was investigated. The experiment was performed under the same conditions and methods as described in Example <2-1>, except that the primers listed in Table 5 below were used.
As a result, as shown in
The effect of the STAT3 protein phosphorylation inhibition by the over-expression of TM4SF5 protein on the expression patterns of SOCS genes, the factors that suppress STAT3 protein, was investigated. The experiment was performed under the same conditions and methods as described in Example <2-1>, except that the primers listed in Table 6 below were used.
As a result, as shown in
The effect of the increased expression of SREBP1 protein and the STAT3 protein phosphorylation inhibition by the over-expression of TM4SF5 protein on the expression patterns of SIRT and SOCS proteins was investigated. The experiment was performed under the same conditions and methods as described in Example <2-2>, except that SCOS1 (Cell Signaling, USA), SOCS3 (Santa cruz, USA), SIRT1 (Santa cruz, USA) and β-tubulin were used as the primary antibodies.
As a result, as shown in
In addition, a culture medium in which AML12 cells, the normal hepatocytes transformed with a construct expressing TM4SF5 protein, were cultured was obtained on the 4th, 8th, and 12th days of culture, and 3T3-L1 cells were cultured in the obtained culture medium. The expression changes of SOCS3 protein in the cultured 313-L1 cells were confirmed by Western blotting in the same manner as above.
As a result, as shown in
The effect of the increased expression of SREBP1 protein and the STAT3 protein phosphorylation inhibition by the over-expression of TM4SF5 protein on the expression patterns of SIRT and SOCS proteins in the hepatocytes separated from the normal mouse was investigated.
First, the hepatocytes over-expressing TM4SF5 protein were prepared under the same conditions and methods as described in Example <3-1>. The expression changes of SOCS1 and SOCS3 genes were confirmed using the prepared hepatocytes under the same conditions and methods as described in Example <2-1>, except that the primers listed in Table 3 were used. As a result, as shown in
In addition, the expression changes of SOCS1 and SOCS3 proteins in the hepatocytes were confirmed by Western blotting. As a result, as shown in
The expression changes of SOCS1 and SOCS3 proteins in the hepatocytes were also confirmed by immunostaining. As a result, as shown in
Meanwhile, the hepatocytes over-expressing SREBP1 protein isolated from the normal mouse, and the expression changes of SOCS1 and SOCS3 proteins were confirmed by Western blotting using the prepared hepatocytes. As a result, as shown in
The primary hepatocytes isolated from the normal mouse (52 weeks old) were transfected with SOCS3 (NM_174466) shRNA (shSOCS3, sense 5′ CAACAUCUCUGUCGGAAGAUU-3′ SEQ. ID. NO: 111; antisense 5′ UCUUCCGACAGAGAUGUUGUU-3′ SEQ. ID. NO: 112) under the same conditions and methods as described in Example <4-1> to prepare hepatocytes wherein the expression of SOCS3 gene was suppressed, and the expression changes of SREBP1 and SOCS3 proteins and the phosphorylation changes of STAT3 were confirmed by Western blotting.
As a result, as shown in
First, the cas9/RGEN KO mouse in which exon 3 of the Tm4sf5 mouse gene (GenBank accession number: NM_029360.3) composed of 5 exons was removed was prepared using C57BL/6 mouse (Macrogen, Seoul). At this time, the mouse in which 522 bp of DNA containing TM4SF5 gene was deleted was obtained using the RGEN site shown in Table 7. In addition, the mouse in which TM4SF5 gene was deleted was prepared from the mouse obtained above using the mouse TM4SF5 primers shown in Table 7 below.
The mutant mouse was selected by observing the heterologous double-strand formation between the wild-type (normal type) and mutant PCR products through T7E1 analysis.
In addition, using C57BL/6 mouse, the cas9/RGEN KO mouse in which exon 1 of the Tm4sf5 mouse gene (GenBank accession number: NM_029360.3) was removed was prepared. At this time, the mouse in which 29 bp of DNA containing TM4SF5 gene was deleted was obtained using the RGEN site shown in Table 8. In addition, the Tm4sf5-Exon 1-KO mouse in which TM4SF5 gene was deleted was prepared from the mouse obtained above using the mouse TM4SF5 primers shown in Table 7 below. In other examples except
The mutant mouse was selected by observing the heterologous double-strand formation between the wild-type (normal type) and mutant PCR products through T7E1 analysis.
<7-2> Confirmation of Expression Changes of Factors that Regulate Phosphorylation of STAT3 Protein in TM4SF5 Gene KO Mouse
The expression changes of SOCS1 and SOCS3 genes that regulate the phosphorylation of STAT3 protein in the TM4SF5 gene KO mouse prepared in Example <7-1> were confirmed. The expression changes of SOCS1 and SOCS3 genes were confirmed under the same conditions and methods as described in Example <2-1> using the hepatocytes obtained from the mouse prepared above, except that the primers listed in Table 3 were used. As a result, as shown in
In addition, Western blotting was performed to confirm whether the suppression of the expressions of SOCS1 and SOCS3 genes was the same in proteins. The experiment was performed under the same conditions and methods as described in Example <2-2>, except that the hepatocytes obtained from the mouse prepared above were used and SOCS1, SOCS3 and β-tubulin were used as the primary antibodies. As a result, as shown in
Fat accumulation in the liver of the TM4SF5 gene KO mouse fed a high-fat diet was confirmed by H&E staining.
First, the TM4SF5 gene KO mouse prepared in Example <7-1> was fed a 60% kcal high fat diet (Harlan, USA) for 10 weeks. The weight changes were measured weekly during the 10 weeks. Ten weeks later, H&E staining was performed under the same conditions and methods as described in Example <1-3>, except that the liver tissue was obtained from the mouse.
As a result, as shown in
The liver tissue was obtained from the TM4SF5 gene KO mouse fed a high fat diet, and the expression changes of the fat-related genes and proteins in the liver tissue were confirmed.
The experiment was performed to confirm the expression changes of the genes under the same conditions and methods as described in Example <2-1> using the hepatocytes obtained from the mouse prepared above, except that the primers listed in Table 9 below were used.
On the other hand, Western blotting was performed under the same conditions and methods as described in Example <2-2>, except that antibodies against SREBP1 precursor, mature SREBP1, CD36 (Santa cruz, USA) and α-tubulin (Cell Signaling Technology, USA) were used as the primary antibodies.
As a result, as shown in
For the measurement of fat in the liver tissue of the TM4SF5 gene KO mouse fed a high-fat diet, the tissue fixed to RNAlater was cut into pieces of ˜10 mg, and cholesterol (Abcam, ab65390), free fatty acid (Abcam, ab65341) and Triglyceride (Cell biolabs, STA-396) were measured.
As a result, as shown in
The phenotype of the offspring obtained by mating the TM4SF5 gene KO mouse prepared in Example <7-1> with the APCmin/+ mouse mutated to develop colon disease more likely (Central Lab. Animal Inc., Seoul, Korea) was confirmed.
The expressions of TM4SF5 and APC genes were confirmed under the same conditions and methods as described in Example <2-1> using the liver tissue of the obtained offspring, except that the primers listed in Table 10 below were used.
In addition, the obtained offspring were sacrificed and each organ was observed. The results are shown in
H&E and Mason's trichrome stainings were performed using the liver tissue of the obtained offspring. At this time, H&E staining was performed as described in Example <1-3>.
On the other hand, for Mason's trichrome staining, the liver tissue fixed to paraffin was left in a 60° C. oven for about 20 minutes to remove paraffin. The paraffin-removed tissue was placed in a heated Bouin's solution, followed by reaction for 1 hour. Upon completion of the reaction, the liver tissue was washed with tap water, placed in a hematoxylin solution, and reacted for 10 minutes. The liver tissue was washed again with tap water, placed in a biebrich scarlet-acid fushsin solution, and reacted for 5 minutes. Upon completion of the reaction, the liver tissue was placed in distilled water, and then placed in a phosphotungstic acid/phosphomolybdic acid solution, followed by reaction for 15 minutes. Thereafter, the liver tissue was reacted in an aniline blue solution for 10 minutes and 1% acetic acid for 1 minute, respectively, and then the tissue was dehydrated. The dehydrated tissue was placed in xylene, taken out, placed on a slide and mounted. The cells stained with the said two staining methods were observed using a microscope, and the results are shown in
As shown in
Immunostaining was performed to confirm the expression changes of TM4SF5, β-catenin and HIF1α proteins in the offspring obtained in Example <10-1>. The experiment was performed under the same conditions and methods as described in Example <2-3>, except that the antibodies against TM4SF5, β-catenin and HIF1α proteins were used as the primary antibodies.
As a result, as shown in
The fat-related signal transduction mechanism was confirmed in the hepatocytes of the offspring obtained by mating TM4SF5 gene KO mouse and APCmin/+ mouse by Western blotting. The experiment was performed under the same conditions and methods as described in Example <2-3>, except that the hepatocytes of the offspring obtained in Example <10-1> were used and the antibodies against laminin, fibronectin, pY142 β-catenin, pY705 STAT3, STAT3, pS9-GSK3β, GSK3β and TM4SF5 proteins were used as the primary antibodies.
As a result, as shown in
Therefore, it was confirmed that the expression of TM4SF5 protein caused disorders in the blood vessels and the portal vein of the liver, and induced fibrosis symptoms in the liver by promoting the expression of the fibrosis-related extracellular matrix.
It was confirmed that the characteristics of fatty liver were appeared by the over-expression of TM4SF5 protein. Immunoprecipitation was performed to confirm whether the TM4SF5 protein was bound to SLC7A1 or SLC38A9, the mTOR and arginine transporter.
First, HEK293T cells (KCLB, Korea) were prepared by culturing in 5% CO2 at 37° C. using DMEM containing 10% FBS and antibiotics. The prepared cells were distributed in 100 mm plates and cultured to the density of 60%, which were transfected with the construct expressing SLC7A1 or SLC38A9 protein labeled with HA tag or the construct labeled with STERP tag. The cells cultured for 2 days after the transfection were washed once with PBS and incubated in 5% CO2 at 37° C. for 50 minutes in the amino acid- or arginine-free medium. After the incubation, the cells were washed twice with PBS, and 500 μl of lysis buffer was added thereto, followed by reaction at 4° C. for 15 minutes. The cell lysate was centrifuged for 15 minutes at 4° C., 12,000×g to obtain supernatant. The protein included in the supernatant was quantified using BCA reagent (Thermo Scientifics, USA), and the beads coated with streptavidin were added thereto in proportion to the protein amount. The mixture was reacted at 4° C. for 4 hours while rotating, and then centrifuged at 4° C., 7,000×g for 5 minutes. After the centrifugation, lysis buffer was added to the obtained pellets, which were lightly mixed and then centrifuged for 5 minutes at 4° C., 7,000×g to obtain pellets. This washing process was repeated twice using lysis buffer and twice using PBS, and then 2× sample buffer was added to the washed pellets, which were boiled for 5 minutes to prepare a sample. Western blotting was performed under the same conditions and methods as described in Example <2-3>, except that the prepared sample was used, and HA (Covanvce, USA) and streptavidin-HRP (IBA, USA) were used as the primary antibodies.
As a result, as shown in
The TM4SF5 gene KO mouse was starved for 6 hours, and the content of arginase present in the liver was confirmed by measuring the expression of arginase 1 gene.
Particularly, the TM4SF5 gene KO mouse prepared in Example <7-1> was starved for 6 weeks, and then sacrificed to obtain the liver tissue. Real-time PCR was performed under the same conditions and methods as described in Example <2-1>, except that the obtained liver tissue was used and the primers known for the arginase gene were used.
As shown in
The following experiment was performed to confirm whether TM4SF5 protein directly affects the transport of arginine.
First, HEK293FT cells (Thermo, USA) were prepared by culturing in 5% CO2 at 37° C. using DMEM containing 10% FBS and antibiotics. The prepared cells were distributed in 150 mm plates and cultured to the density of 60%, which were transfected with the construct expressing TM4SF5, MetaP2, Castrol, TM4SF1, TM4SF4 and TM4SF5 proteins constructed in Example 11 using PEI. Two days after the transfection, the desired protein was precipitated under the same conditions and methods as described in Example 11 using the beads coated with streptavidin. The precipitate was added with 10 μM [3H]-arginine (American radiolabeled chemicals, USA), followed by reaction at 4° C. for 1 hour. At this time, a sample in which 10 mM of L-arginine was added to the same amount of beads was used as the control. Upon completion of the reaction, the beads were washed 3 times with lysis buffer, to which 2 m of scintillation cocktail (Ultima gold, Perkin Elmer, USA) was added. The mixture was vortexed and analyzed using a liquid scintillation counter (Tri-Carb, Perkin Elmer, USA).
As a result, as shown in
An experiment was performed to confirm whether the binding of TM4SF5 protein and arginine confirmed in Example <12-2> was concentration-dependent. The experiment was performed under the same conditions and methods as described in Example <12-2>, except that HEK293FT cells transformed with TM4SF5 protein were used and 0, 0.01, 0.05. 0.1 or 0.5 mM L-arginine was added.
As a result, as shown in
It was confirmed that TM4SF5 protein was directly bound to arginine, so the following experiment was performed to confirm which residue of TM4SF5 protein played an important role in binding to arginine.
First, a short extracellular loop (SEL) fragment mutant comprising the 31st to 42nd amino acid residues from the N-terminus of the amino acid sequence constituting TM4SF5 protein (SEQ. ID. NO: 1) and a long extracellular loop (LEL) fragment mutant comprising the 113th to 157th amino acid residues from the N-terminus were prepared. Or mutants of TM4SF5 protein were prepared by substituting the 124th to 129th amino acid residues and the 153th to 157th amino acid residues from the N-terminus, respectively, were prepared. As a result, SEL, LEL, W124A, G125A, Y126S, H127A, F128S, E129A, P153A, W154A, N155Q, V156A or T157A mutant was obtained in addition to the wild type of TM4SF5 protein (WT, full length). The binding of TM4SF5 protein and arginine was confirmed under the same conditions and methods as described in Example <12-2>, except that the construct expressing the obtained mutant protein was used.
As a result, as shown in
As shown in
On the other hand, as shown in
The body weight changes in the TM4SF5 gene KO mouse by the high arginine diet (High Arg Diet) were confirmed by the following method.
Particularly, the TM4SF5 gene KO mouse prepared in Example <7-1> was fed L-arginine (40 g/kg of mouse body weight) for 10 weeks. The weight changes were measured weekly during the 10 weeks, and the results are shown in
As shown in
The liver tissue was extracted from the TM4SF5 gene KO mouse fed a high arginine diet in Example <13-1>, and H&E staining was performed using the method described above.
As a result, as shown in
The proteins binding to TM4SF5 protein were analyzed by mass spectrometry, and GLUT1 (SLC2A1) protein was selected. The said GLUT1 protein is a glucose transporter, which is involved in the production of energy by moving into the cell membrane by insulin and supplying glucose inside the cell. Thus, the phosphorylation of S6Kinase was confirmed as follows using the cells transformed with a construct expressing TM4SF5 protein.
First, HEK293FT cells (Thermo, USA) were prepared by culturing in 5% CO2 at 37° C. using DMEM containing 10% FBS and antibiotics. The survival responsiveness of the cells was confirmed by investigating the survival of the prepared cells under the stress such as re-supply after deficiency of glucose.
As a result, as shown in
Glycolytic stress in the cells in which the expression of TM4SF5 protein was suppressed was measured using an XF analyzer (Sea Horse). To construct a TM4SF5 expression-suppressing cell line, the HEK293FT cell line was transfected with the pLKO.1 (addgene) lenti-viral plasmid, psPAX2 and pDM2.G constructs in which shRNA sequences (shTM4SF5 #2: 5′-accauguguacgggaaaaugugc-3′, SEQ. ID. NO: 95; shTM4SF5 #4, 5′-ccaucucagcuugcaaguc-3′, SEQ. ID. NO: 96) targeting TM4SF5 was inserted using PEI. After 5 hours, the culture medium was replaced and cultured for 24 hours to obtain shTM4SF5 lenti-virus. Hep3B cells were infected with the obtained virus and 4 ug/ml of polybrene for 24 hours, followed by selection with puromycin for 48 hours.
Particularly, Hep3B cells were distributed in XFp cell culture plates (Sea Horse bioscience, USA) at the density of 5×103 cells/well. The cells were cultured in 5% CO2 at 37° C. for 16 hours and then the medium was replaced with Sea Horse XF basal medium (Sea Horse bioscience, USA). The cells in the replaced medium were cultured for 1 hour in a 37° C. incubator without CO2 supply. The XFp cell culture plate containing the cultured cells was bound to a hydrated and calibrated sensor cartridge (Sea Horse bioscience, USA) at 37° C. and analyzed using an XFp analyzer. 100 mM glucose (A), 50 μM oligomycin (B), and 500 mM 2-deoxy-D-glucose (C) were loaded through the drug inlet.
As a result, as shown in
The following experiment was performed to confirm the expression changes of the glycolysis-related genes in the cells over-expressing TM4SF5 protein.
First, a SNU449 liver cancer cell line was transformed with a construct expressing TM4SF5 protein. The cells were crushed by adding liquid nitrogen, and RNA was extracted using an RNAeasy kit (Qiagen, USA) according to the manufacturer's protocol. DNAse was added to the extracted RNA to remove DNA, and cDNA was synthesized by the conventional method. An adapter was attached to the synthesized cDNA, which was amplified by PCR, and the PCR products having the size of 200 to 400 bp were selected. The sequence of the selected cDNA was analyzed using a HiSeq 4000 sequencer (Illumina, USA). Artifacts were removed through pre-processing of the sequencing results and mapped to the genome using a HISTA2 program. The expression levels were obtained through transcript assembly using StringTie from the mapped data.
As a result, as shown in
The weight changes of the TM4SF5 gene KO mouse fed a high-carbohydrate diet (70% kCal high-carbohydrate) or a high-sucrose diet (AIN-93G diet; It has a sucrose content of 10%, which is 3 times higher than that of a chow diet with a sucrose content of 3.15%.) under the same conditions and methods as described in Example <8-1> were confirmed, and the results are shown in
As shown in
The glucose resistance of the TM4SF5 gene KO mouse fed a high-carbohydrate diet or a high-sucrose diet under the same conditions and methods as described in Example <8-1> was measured by the following method.
Particularly, the mice fed a high-carbohydrate diet or a high-sucrose diet for 3 weeks and 10 weeks were starved for 16 hours, and blood was collected from the tail. Blood glucose in the collected blood was measured using a blood glucose meter (One touch ultra, Johnsons and Johnsons, USA). After measuring the blood glucose, 2 g/kg of glucose was injected into the mouse intraperitoneally, and blood was collected from the tail 30 minutes, 60 minutes, 90 minutes and 120 minutes after the injection. Then, blood glucose was measured.
As a result, as shown in
The insulin resistance of the TM4SF5 gene KO mouse fed a high-carbohydrate diet or a high-sucrose diet under the same conditions and methods as described in Example <8-1> was measured by the following method.
Particularly, the mice fed a high-carbohydrate diet or a high-sucrose diet for 10 weeks were starved for 6 hours, and blood was collected from the tail. Blood glucose in the collected blood was measured using a blood glucose meter (One touch ultra, Johnsons and Johnsons, USA). After measuring the blood glucose, 0.5 U/kg of insulin was injected into the mouse intraperitoneally, and blood was collected from the tail 30 minutes, 60 minutes, 90 minutes and 120 minutes after the injection. Then, blood glucose was measured.
As a result, as shown in
The levels of blood AST, ALT and cholesterol in the TM4SF5 gene KO mouse fed a high-carbohydrate diet or a high-sucrose diet under the same conditions and methods as described in Example <8-1> was measured using Fuji Dri-Chem 3500i.
As a result, as shown in
H&E staining was performed using the liver tissue of the TM4SF5 gene KO mouse fed a high-carbohydrate diet or high-sucrose diet in Example <16-1> using the method described above.
As a result, as shown in
The liver tissue was extracted from the TM4SF5 gene KO mouse fed a high-carbohydrate diet or high-sucrose diet in Example <16-1>. After lysophilization of the liver tissue and pulverizing thereof using a mortar, lipids were extracted with 0.3 ml of methanol and 0.1% butylated hydroxytoluene solution per 10 mg of the liver tissue. After adding methyl-tert-butyl ether containing 0.1% butylated hydroxytoluene to the extract, the mixture was shaken for 1 hour at room temperature. The mixture was diluted with 0.25 ml of H2O, vortexed at room temperature for 10 minutes, and centrifuged at 14,000 g at 4° C. for 15 minutes. The supernatant and the lower solution were separated, followed by drying. After treating 40 μl of CHCl3:MeOH (1:9) to 0.16 ml of the solution, lipid analysis was performed using LC-MS/MS (8040, Shimadzu, Japan).
As a result, as shown in
The mice over-expressing TM4SF5 protein were prepared under the same conditions and methods as described in Example <1-1>, which were bred for 78 weeks. The mice were sacrificed as described above, and the liver tissue was obtained therefrom. The phenotype of the liver tissue was confirmed by H&E and Mason's trichrome staining. As a result, as shown in
In addition, the expression changes of the fat-related proteins were confirmed by Western blotting as described above using the liver tissue, and the results are shown in
As shown in
In addition, immunostaining was performed as described above using the liver tissue, and the results are shown in
On the other hand, the expression changes of the genes related to fat metabolism, liver cirrhosis and hepatitis were confirmed using the liver tissue as described above. As a result, as shown in
Therefore, it was confirmed that fatty liver developed in liver cirrhosis or hepatitis after a certain period of time in the transgenic mouse over-expressing TM4SF5 protein, thereby the phosphorylation of STAT3 protein or the ECM level was increased.
It was confirmed that the fatty liver produced in the mouse over-expressing TM4SF5 protein showed the symptoms of liver cirrhosis and hepatitis over time. In general, it has been reported that the mouse administered with carbon tetrachloride for 4 weeks showed the symptoms of liver fibrosis, and the mouse administered for 16 weeks shows the symptoms of cirrhosis. Thus, the expression changes of TM4SF5 protein were confirmed in the model mouse induced with liver cirrhosis by a drug.
First, a mouse model in which liver disease was induced by intraperitoneally injecting carbon tetrachloride (CCl4) once a week for 1, 4 or 16 weeks at the concentration of 1 mg/kg to 4-week-old BALB/C mice (Orient Bio, Korea) was prepared. H&E and Mason's trichrome stainings were performed using the prepared model mouse, and the results are shown in
As shown in
The expression levels of proteins and mRNAs were confirmed as described above using the liver tissue of the model mouse, and the results are shown in
In addition, immunostaining was performed as described above using the liver tissue of the model mouse, and the results are shown in
On the other hand, after administering CCl4 to the TM4SF5 gene KO mouse prepared in Example <7-1> as described above, the liver tissue was obtained and Mason's trichrome staining was performed, and the results are shown in
The mechanism of regulating the expression of laminin protein was confirmed by the following method using the liver tissue of the liver disease model mouse prepared by drug administration in Example 18.
First, hepatocytes were obtained from the isolated liver tissue as described above. The expressions of TM4SF5 and STAT3 proteins in the obtained hepatocytes were suppressed by transfecting the cells with shTM4SF5 or silencing STAT3 [On-Target plus SMART pool siRNA (Thermo)], and then the expression changes of laminin were confirmed by Western blotting as described above.
As a result, as shown in
In addition, the separated liver tissue was treated with IL-6 and Western blotting was performed as described above to confirm whether the increased STAT3 phosphorylation and laminin protein expression were dependent on IL-6. As a result, as shown in
In order to confirm the position of laminin in the signal transduction mechanism as described above, Western blotting was performed as described above by treating laminin to the separated liver tissue. As a result, as shown in
The c-Src protein inhibitor PP2 (LC Laboratories, USA) or the control compound PP3 (LC Laboratories, USA) was added to the separated liver tissue, and the expression changes of the protein were confirmed by Western blotting as described above. As a result, as shown in
In addition, the phosphorylation of STAT3 protein and the expression changes of laminin protein in the HepG2 (Korean Cell Line Bank, Seoul) liver cancer cells in which TM4SF5 protein was suppressed were confirmed by Western blotting as described above. As a result, as shown in
Whether the phosphorylation of STAT3 protein confirmed to regulate the expression changes of laminin protein controls the expression through the promoter of laminin was investigated by luciferase assay.
First, the regions corresponding to −1871 to +388 (1 kb) and −592 to +388 (2.3 kb) of LAMC2 promoter and −2865 to +85 (0.9 kb), −2047 to +89 (2.1 kb) and −845 to +89 (2.9 kb) of COL1A1 promoter were amplified by PCR using the primers listed in Table 11 below.
A construct was prepared by inserting the amplified PCR product into pGL3 vector (Promega, Cat #.E1751, USA) (
As a result, the luciferase activity showing the promoter activity of laminin γ2 (Lamc2,
It is generally known that the disease is exacerbated by the accumulation of collagen activated by hepatic stellate cells. In addition, since the luciferase activity level of collagen I and laminin γ2 was different, confirmed by the above experiment, it was expected that different types of ECM would be expressed according to cell types, so the following experiment was performed.
First, fluorescent staining was performed as described above using the liver cirrhosis tissue. As a result, it was confirmed that laminin protein was expressed around the damaged liver tissue as the expression of TM4SF5 protein increased (
In addition, the hepatocyte marker albumin and the hepatic stellate cell marker α-SMA were stained along with collagen I and laminin in the same manner as described above, in order to more clearly identify the kind of the cells. As a result, as shown in
On the other hand, after suppressing the expression of TM4SF5 protein in the same manner as described in Example <4-1> in HepG2 cells, the expression changes of the protein was confirmed in the same manner as described above. As a result, as shown in
It was confirmed that the expression of laminin protein was regulated by STAT3 protein. First, siRNA for laminin γ2 (LAMC2) or collagen I (COL1A1) gene was injected into the tail vein of the mouse, and CCl4 was administered. The liver tissue was obtained from the mouse, which was stained by H&E staining. As a result, the liver damage caused by CCl4 was suppressed ( mRNA was decreased (
In the liver cancer animal model induced through fatty liver, liver cirrhosis, steatohepatitis and cirrhosis, it was confirmed whether the above-mentioned signaling was applied by the following method.
Particularly, the 52-week-old FVB/N animal model over-expressing TM4SF5 protein was bred for 1 year, and then sacrificed to extract the liver tissue. It was confirmed that TM4SF5 protein was over-expressed and nodules were formed in the extracted liver tissue (
The exacerbation process of liver disease was confirmed using the transgenic mouse as follows. Particularly, the transgenic mouse was induced with liver cancer by injecting diethylnitrosamine (DEN). The liver tissue was extracted from the mouse, and H&E staining was performed. As a result, it was confirmed that liver cancer was induced (
In addition, immunostaining was performed using the obtained liver tissue. As a result, it was confirmed that the expressions of TM4SF5, phosphorylated STAT3, laminins, laminin γ2 and collagen I proteins were increased (
The cancer tissue and the cancer surrounding tissue were obtained from liver cancer patients, and the expression changes of phosphorylated STAT3, laminin and collagen I were confirmed in the same manner as described above. At this time, the cancer surrounding tissue was a tissue at the stage before the onset of cancer, and it was expected to show the pathological symptoms of hepatitis, fibrosis and liver cirrhosis. As a result, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0140514 | Oct 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/012860 | 10/26/2018 | WO | 00 |
