Patent Application
20100317840
The present invention relates to an RNA interfering technology, particularly to an hnRNP K expression-inhibiting compound and a siRNA sequence thereof.
Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the ribonucleoprotein family. HnRNP K can directly interact with DNA and RNA via the K homology domain and can regulate gene expression in different aspects, including transcription, translation and ligation. HnRNP K may induce canceration via regulating the expression of oncogenes c-myc and eIF4E. HnRNP K can respectively join with the internal ribosome entry site of c-myc and the polypyrimidine of the promoter of eIF4E to induce the expression of c-myc and eIF4E. As hnRNP K normally exists in the nucleus, it cannot undertake regulation and translation unless it transfers to cytoplasm. A recent paper pointed out that hnRNP K may cause the metastasis of fibrosarcoma cells. Therefore, hnRNP K is a potential target for metastasis therapy. Some clinical researches pointed out that hnRNP K is abnormally expressed in cells of some specified cancers, including colorectal cancer, esophagus cancer, lung cancer, oral squamous cell cancer, and prostate cancer. The reduced hnRNP K expression in nuclei also correlates with the shorter survival period of the patients of Dukes C colorectal cancer.
The Inventors found that hnRNP K is over-expressed in nasopharyngeal cancer and clinically correlates with the overall survival rate of a patient and the incidence rate of distant metastasis. Therefore, hnRNP K can be used as a biomarker for predicting the malignancy of cancer. In the current cancer therapy technology, there is still none molecular inhibiting method or medicine inhibiting the expression of hnRNP K—the over-expressed gene in colorectal cancer, lung cancer, oral squamous cell cancer, and nasopharyngeal cancer.
The primary objective of the present invention is to provide an hnRNP K (heterogeneous nuclear ribonucleoprotein K) expression-inhibiting compound and a siRNA (small interfering RNA) sequence thereof, wherein the siRNA corresponding to TP is used to effectively inhibit hnRNP K expression and suppress the growth, metastasis and invasion of cancer.
To achieve the abovementioned objective, the present invention proposes an hnRNP K expression-inhibiting compound and a siRNA sequence thereof, wherein a siRNA is used to inhibit hnRNP K expression, and wherein a small segment of RNA having about 18-24 pieces of nucleotides matches with the mRNA (messenger RNA) of hnRNP K and then the intracellular dicer recognizes the segment of RNA, whereby the mRNA of hnRNP K is cut off, and the hnRNP K expression is inhibited. The siRNA designed by the present invention matches with hnRNP K mRNA and has a transcribable sequence
which can inhibit hnRNP K expression and thus can reduce the survival rate of cancer cells in an anoxic environment.
Below, the present invention is described in detail in cooperation with the attached drawings to make easily understood the objective, characteristics and accomplishments of the present invention.
The hnRNP K (heterogeneous nuclear ribonucleoprotein K) expression-inhibiting compound and a siRNA (small interfering RNA) sequence thereof can effectively inhibit hnRNP K expression and thus can reduce the survival rate of cancer cells in an anoxic environment and can then suppress the growth of cancer cells, includes colorectal cancer, lung cancer, oral squamous cell cancer, prostate cancer, nasopharyngeal cancer. The nasopharyngeal cancer cells are used as the exemplification of the abovementioned cancer cells in the present invention.
In related experiments, the Inventors found that hnRNP K and the target thereof—TP are over-expressed in nasopharyngeal cancer. The abnormal hnRNP K expression and TP over-expression in cytoplasm correlates with the shorter overall survival period and the higher incidence rate of distant metastasis. A multivariate analysis shows that hnRNP K and TP in cytoplasm are the independent factors for prognosis. Further, TP over-expression in nasopharyngeal cancer cells makes the cancer cells more sensitive to the intermediate product of capecitabine—the precursor medicine 5-fluoro-5′-deoxyuridine (5′-DFUR), which can induce the apoptosis of cancer cells. Besides, the removal of serum will increase the stability of TP and cause TP over-expression.
Furthermore, RT-PCR-based immunoprecipitation and the transfer of hnRNP K from nucleus to cytoplasm shows that a UMP- and CMP-rich segment of TP can directly interact with hnRNP K. Therefore, inhibiting hnRNP K expression can reduce TP expression. It means that hnRNP K should be the upstream of TP. In the reaction mechanism, both the MEK inhibitor (Mitogen-activated protein/Extracellular signal-regulated Kinase) and the amino mutation of p-ERK (phosphorylated Extracellular signal-Regulated kinases) of hnRNP K can reduce hnRNP K expression in cytoplasm. Therefore, the phosphorylation of hnRNP K by ERK maybe plays an important role in inducing TP.
Besides, the TP expression activated by hnRNP K can inhibit the anoxic apoptosis of nasopharyngeal cancer cells. In conclusion, our experiments show that ERK can induce increasing hnRNP K expression. Thus, hnRNP K is the upstream of TP, and TP is the downstream target of hnRNP K. Both hnRNP K and TP are effective indicators to prognose nasopharyngeal cancer and deserves designing new targeted-therapy medicine thereof, which should benefit cancer therapy.
Therefore, the present invention suppresses the growth, metastasis and invasion of nasopharyngeal cancer via inhibiting hnRNP K expression. The hnRNP K expression-inhibiting compound may be the nucleotide-based molecules of RNA or DNA, which has a sense region and an antisense region jointly forming a duplex region. The sense region and the antisense region respectively have a length of 18-30 nucleotides. The antisense region has a sequence completely or partially matching the sequence of the mRNA of hnRNP K, whereby hnRNP K expression is inhibited, and the growth, metastasis, and invasion of cancer cells is suppressed.
In other words, the hnRNP K-inhibiting siRNA contains the following sequence:
Below, experimental data is used to prove the efficacies of the present invention. The cell lines of nasopharyngeal cancer NPC-TW01, NPC-TW02 and NPC-TW04 were cultivated in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at a temperature of 37° C. in a humidified 5% CO2 atmosphere. The cells are also cultivated in a serum-free DMEM, i.e. treated with a serum deprivation process. The cell lines NPC-TW02 is cultivated in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37° C. in a humidified 5% CO2 atmosphere. The expression of endogenous TP can be induced by the serum deprivation processing, as shown in
Refer to
Firstly, 2×105 cells are taken out and washed with PBS. Next, a buffer solution (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) is used to adjust the solution to have a cell concentration of 2×106/mL. Next, 5 μl Alexa Fluor® 488 annexin V is added into 100 μl cell suspension, and then the cell suspension reacts at an ambient temperature for 15 minutes. Next, the cell specimens (10000 events) are analyzed with a flow cytometer (a product of Becton Dickinson), as shown in
According to the operation manual, 50 μg transfection agent TransIT-TKO (a product of Mirus Bio Corporation) is used to transfect 50 nmol/l dsRNA duplexes to the cell line NPC-TW02, wherein a 21-bp hnRNP K and TP-addressing RNA duplex (SMARTpool reagents, Dharmacon, Lafayette, Colo.) and a 21-bp none-addressing RNA duplex (Research Biolabs Ayer Rajah Industrial Estate) are transfected to the cell line NPC-TW02. 24 hours later from transfection, the siRNA-containing culture solution is replaced with a serum-containing culture solution and a serum-free culture solution. Refer to
The mRNA of hnRNP K may have a sequence shown in Table. 1.
In conclusion, the present invention proposes an hnRNP K expression-inhibiting compound and a siRNA sequence thereof, which uses an RNA interfering technology to inhibit hnRNP K expression inside cancer cells and suppress canceration, including the growth, metastasis and invasion of cancer cells.
The present invention has been demonstrated with the embodiments described above. However, they are only to exemplify the present invention but not to limit the scope of the present invention. Any equivalent modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention, which is based on the claims stated below.
