Patent Application
20190276895
The present invention provides a method and a kit for evaluation of the prognosis of breast cancer, by using snoRNA U50 as a biomarker.
Breast cancer is the most common cancer type among women with an increasing incidence rate worldwide and the second leading cause of cancer death. For clinical implications and treatment strategies, immunohistochemistry (IHC) markers, such as ER, PR and HER2, combined with clinicopathological variables, including tumor size, tumor grade and nodal involvement, have been utilized routinely. With the promotion of prevention medicine, breast cancer can be diagnosed in the early stages. However, there is no suitable prognostic marker for the early prediction of breast cancer survival. Furthermore, traditional pathological markers used to classify breast tissues into the TNM stages have limitations for discriminating individual variability. Hence, the need for establishing a molecular marker for early detection is urgent.
Small nucleolar RNAs (snoRNAs) are small noncoding RNAs ranging from 60-300 nucleotides. These snoRNAs are essential for numerous cellular processes and execute their function mainly in the nucleolus. SnoRNAs are classified according to their structures and function into C/D box and H/ACA box types. By binding different associated proteins and enzymes, C/D and H/ACA box snoRNAs become small nucleolar RNA-protein complexes (snoRNPs) and execute site-specific methylation or pseudouridylation of rRNAs guided by snoRNAs. Although they are considered to be housekeeping RNAs, aberrant expression of snoRNAs has been reported recently in human cancers. SNORA42 is elevated in nonsmall cell lung cancer (NSCLC) patients, which is correlated with poor prognosis. SNORD76 expression is lower in grade III/IV patients compared to grade II patients, which is considered as a tumor suppressor in glioblastoma. According to Siprashvili, et. Al, “The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer “Nature Genetics, volume 48, pages 53-58 (2016), SNORD50 (U50) is a putative tumor suppressor since the deletion of U50 is correlated with poor prognosis in breast cancer patients. Although 20% of breast cancers are heterozygous deletion of the U50 locus in the genome, no homozygous deletion has been observed. U50 snoRNA has a noncanonical function in inactivating KRAS, indicating its tumor-suppressive properties. However, the role and impact of U50 in breast cancer has not been elucidated.
In this study, we quantified the copy number of snoRNA U50 using absolute quantification real-time PCR in breast cancer patient tissues. To evaluate the potential RNA level of U50 as a prognostic marker, we validated and correlated U50 expression with clinical characteristics, including the stage, TNM stage and survival times.
The present invention therefore provides a method and a kit using snoRNA U50 as a biomarker, which can effectively diagnose the breast cancer.
According to one embodiment, a method for the detection of the presence of or the risk of breast cancer in a subject is provided. The RNA expression level of snoRNA U50 is detected in a sample isolated from a subject, wherein a decreased expression level of the snoRNA with reference to a normal control indicates the presence of or the risk of breast cancer in the patient from whom the sample was isolated.
According to another embodiment, a method for predicting the survival time of a patient suffering from a breast cancer is provided. First, the RNA expression level of snoRNA U50 is measured, in a sample isolated from a subject, After comparing said expression level with a predetermined reference value, a good prognosis is provided when the expression level of the selected snoRNA is higher than the predetermined reference value and a poor prognosis is provided when the expression level of the selected snoRNA is lower than the predetermined reference value.
According to another embodiment, a kit for the detection of the presence of or the risk of breast cancer in a subject is provided, which comprises nucleotides capable of specific binding to an snoRNA U50.
These and other objectives of the present invention will no doubt become obvious to those of ordinary skill in the art after reading the following detailed description of the preferred embodiment that is illustrated in the various figures and drawings.
To provide a better understanding of the presented invention, preferred embodiments will be made in detail. The preferred embodiments of the present invention are illustrated in the accompanying drawings with numbered elements.
Small nucleolus RNAs (snoRNAs) are small noncoding RNAs that are essential for numerous cellular processes and are generally considered housekeeping genes. In the present invention, the copy numbers of snoRNA U50 (5′-3′, SEQ ID: NO. 1) is quantified in breast cancer patient tissues and found that a higher level of U50 expression is correlated with better overall survival in breast cancer patients, suggesting that U50 is an independent prognostic marker. In addition, the level of U50 was lower in stage 2 and 3 than in stage 1 tumors, whereas no correlation was observed with lymph node metastasis. The present invention has shown the clinical relevance of snoRNA U50 in human breast cancer, which provides important clinical information for further research.
To show the potential of snoRNA U50 as a prognostic marker, the following context will validated and correlated U50 expression with clinical characteristics, including stage, TNM stage and survival times.
We analyzed 142 samples of breast cancer tissues from individual patients. Specimens were collected from National Cheng Kung university hospital (Tainan, Taiwan) during 2003-2013 from patients 23 to 83 years of age. Frozen breast cancer tissues were used according to approved IRB protocols from NCKU Hospital. Tumor stage was classified according to the American Cancer Society.
Total RNA was extracted using TRIzol (Invitrogen) according to manufacturer's protocol. Briefly, chloroform was added and homogenized samples were centrifuged at 14000 g for 30 mins. The clear upper aqueous layer was isolated and precipitated with isopropanol at −80° C. for 1-2 hours. Samples were centrifuged at 14000 g for 30 mins, and the supernatant was discarded. The resulting pellets were washed with 75% ethanol and preserved in nuclease-free water at −80° C. Total RNA concentrations were determined by spectrophotometer (NanoDrop Lite Microlitre Spectrophotometer).
Reverse transcription was performed using the RevertAid RT Reverse Transcription Kit (Thermo Scientific™) according to the manufacturer's protocol. A reaction containing 200 ng template RNA, 1 μL random hexamer, and 2 μL 10 mM dNTP mix with nuclease-free water adjusted to 12 μL was incubated at 65° C. for 5 mins. A mixture of 4 μL 5×reaction buffer, 1 μL RNase inhibitor, and 1 μL RevertAid RT was added, the volume was brought 20 μL to with nuclease-free water, and the reactions were incubated at 25° C. for 5 mins, followed by 42° C. for 60 mins. Finally, the reactions were terminated at 70° C. for 5 mins. cDNAs were preserved at −20° C.
A standard curve was established using linearized U50 overexpressing plasmid. Ten-fold dilutions were used, and the initial concentration was adjusted to 108 copy numbers to achieve the final concentration within 40 cycles. Real-time PCR was performed in 48 well optical plates with four repetitions. Total volume consisted of 2.5 μL Fast SYBR™ Green PCR Master Mix, 0.5 μL forward primer, 0.5 μL reverse primer, 0.5 μL nuclease-free water and 1 μL template. The real-time PCR program was carried out according to the manufacturer's protocol, which included 95° C. for 20 sec to activate the polymerase, 40 cycles of 95° C. for 3 sec and 60° C. for 30 sec.
SnoRNA detection has been described previously (ref). Briefly, the total RNA was extracted and reverse transcription was performed. Samples were further applied to absolute real-time PCR to calculate the copy numbers of snoRNAs. The primers used for detecting U50 have been described previously (ref): forward primer: 5′-TATCTGTGATGATCTTATCCCGAACCTGAAC-3′ (SEQ ID: NO. 1) and reverse primer: 5′-ATCTCAGAAGCCAGATCCGTAA-3′(SEQ ID: NO. 2).
A total of 2000 or 4000 MCF-7/Control and MCF-7/U50 cells were seeded into 6-well plates. After 7-14 days, colonies were formed and fixed with 3.7% paraformaldehyde at room temperature for 30 mins. The fixed colonies were stained by 0.05% Coomassie blue for 15 mins at room temperature. The colonies in every well were counted using ImageJ software.
Cells were seeded a −80% confluency. Expression plasmids for snoRNA U50 (pSIREN-RetroQ-U50) were a kind gift from Dr. Dong. The plasmids were transfected into MCF-7 cells using the HyFect™ DNA transfection reagent according to the manufacturer's protocol (Leadgene Biomedical, Taiwan). Briefly, 6 μg of plasmid was mixed with the cells and incubated for 25 mins at room temperature. The cells were then added to dishes with fresh medium and incubated for 48 hours.
We used a log-rank test in Kaplan-Meier curves for overall and relapse-free survival. A t-test was used to compare U50 expression with ER, PR, HER2 and the tumor size. For multigroup analysis, one-way ANOVA with Tukey's multiple comparisons test was used.
Absolute Quantitative Real-Time PCR for snoRNA U50 in Breast Cancer Patient Tissues.
We used absolute quantitative real-time PCR to quantify U50 snoRNA copies numbers. A ten-fold serial dilution of linearized plasmid template was performed, and the initial concentration was adjusted so all the dilution points could be detected within 40 CT cycles. The result is shown in
We analyzed 142 samples of breast cancer tissues from individual patients. The clinical and pathological characteristics of patients are listed in Table 1.
First, we used the Cutoff Finder (http://molpath.charite.de/cutoff/), which determines of cutoff point of molecular markers to determine the optimal cutoff point. We divided breast cancer patients into two groups using cutoff point: U50-high and U50-low by using RNA expression level of 227.5 ng as a cutoff number in the present embodiment. Next, we investigated the correlation between U50 snoRNA copy numbers and breast cancer patient survival and found that higher expression of U50 was statistically and significantly correlated with better overall survival in breast cancer patients (P=0.001, HR=0.17;
Associations of U50 Expression with ER, PR, and HER2
Since ER, PR and HER2 expression demonstrate distinct morphologies and clinical implications in breast cancer patients, we analyzed the correlation between U50 expression and breast cancer subtypes. In ER/PR/HER2-positive and ER/PR/HER2-negative breast cancer patients, U50 expression was not significantly different (P=0.42, 0.21 and 0.85 in ER, PR and HER2 groups, respectively;
Since U50 can be used to predict breast cancer patient survival (
We have shown that U50 is a prognostic marker for early breast cancer detection. Since U50 expression is decreased in patients with tumors >20 mm (
Since there is no study indicating the relationship between the snoRNA U50 and the breast cancer in an RNA level, the present invention has identified the prognostic value and molecular function of RNA expression of snoRNA U50 in breast cancer. Accordingly, the present invention provides a method for detection of the presence of or the risk of breast cancer in a subject. First, the RNA expression level of snoRNA U50 is detected, in a sample isolated from a subject. In one embodiment, the subject refers to patient or person suspected to have breast cancer and can be male or female. Sample is prepared from frozen breast tissue. After carrying out a total RNA extraction process, the expression level of snoRNA U50 is detected by calculating the number of snoRNA U50 a quantities RT-PCR. In one embodiment, the primer in RT-PCR is shown in SEQ ID: NO. 2 and SEQ ID: NO. 3. The result is compared to a normal control, wherein a decreased expression level of the snoRNA with reference to a normal control indicates the presence of or the risk of breast cancer in the patient from whom the sample was isolated. In one embodiment, the normal control value is between 350 ng and 100 ng. In another embodiment, the expression level of snoRNA U50 between 400 ng and 225 ng (preferably between 383.11 ng and 269.49 ng) indicates the stage 1 of breast cancer, the expression level of snoRNA U50 between 225 ng and 100 ng (preferably between 205.44 and 157.96 ng) indicates the stage 2 or stage 3 of breast cancer, the expression level of snoRNA U50 less than 100 ng (preferably between 64.39 ng and 19.97 ng) indicates the stage 4 of breast cancer.
The present invention further provides a method for predicting the survival time of a patient suffering from a breast cancer. First, measuring the expression level of snoRNA U50, in a sample isolated from a subject. Next, said expression level with a predetermined reference value is measured. According to the result, a good prognosis is provided when the expression level of the selected snoRNA is higher than the predetermined reference value and a poor prognosis when the expression level of the selected snoRNA is lower than the predetermined reference value.
The present invention also provides a kit for the detection of the presence of or the risk of breast cancer in a subject. The kit contains components required for detecting the RNA expression of snoRNA U50 in a sample, such as Northern blot analysis, RT-PCR, microarray analysis or sequencing analysis from an object. In one embodiment, the kit contains components for performing quantitative RT-PCR, including: nucleotides: a reverse transcriptase, a polymerase, a forward primer (SEQ ID: NO. further include assay containers (tubes), buffers, or enzymes necessary for carrying out the detection assay. In one embodiment, the kit can shows if the RNA expression level U50 of a subject is higher than a predetermined value, for example, by showing a bar, a signal or other readable forms to give an evaluation of prognosis of breast cancer.
In summary, U50 expression can be used to differentiate breast cancer patient survival. Higher expression of U50 is correlated with better overall survival and relapse-free survival, which is a prognostic marker in breast cancer. We also revealed that U50 is an independent molecular marker for discriminating early stage breast cancer. Along with tumor progression, we demonstrated the tumor suppression activity of U50 in inhibiting tumorigenesis, which is consistent with the clinical patient data. In this study, we established a new molecular marker, U50, for breast cancer early detection and prognosis prediction.
Those skilled in the art will readily observe that numerous modifications and alterations of the device and method may be made while retaining the teachings of the invention. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.
This application claims the benefit of priority of U.S. Provisional Application No. 62/641,385, filed on Mar. 11, 2018, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62641385 | Mar 2018 | US |
