Identification Of Differentially Expressed MicroRNAs In Clear Cell Carcinoma Of Kidney And The Study Of Biological Characteristics Of 786-O Cell Line By Expressing Or Inhibiting MiR-3133

Background and objectives:Kidney cancer (renal cell carcinoma, RCC),which originates from renal tubular epithelial cells, is a common malignant tumor of kidney accounting for 2%～3% of all malignant tumors in adults,85%～90% of primary malignant tumor of kidney. Clear cell renal cell carcinoma(ccRCC) is the most popular one of RCC the histological type of which still includes papillary renal cell carcinoma, chromophobe Carcinoma, collecting duct cancer and not elsewhere classified. And it accounts for about 70～80% of renal cell carcinoma. There is no effective treatment of Cc-RCC at present except surgery in early stage, which, in combination with its concealed onset and diagnostic difficulty in early stage, results in high mortality. Cc-RCC is not sensitive to both chemotherapy and hormone therapy, as in other types of RCC. Although bio-targeted therapy for advanced renal cell carcinoma has made some progress in recent years, the effect is very limited. Statistical data shows that metastasis or recurrence occurs in approximately one-third of patients after surgery who was diagnosed as limitative renal carcinoma before surgery,5-year survival rate less than 10% in patients with advanced renal cell carcinoma. So researches on early diagnosis and effective treatment of advanced renal cell carcinoma have been very popular recently for recurrence and metastasis remain major factors threatening life expectancies of patients with renal cell carcinoma. Current studies have shown that small RNA (microRNA, miRNA) is likely to be a new marker for early diagnosis of renal cell carcinoma and a new target for the treatment of patients at the advanced stage.Studies on the pathogenesis of renal cell carcinoma mainly focus on genomics, proteomics and microRNA genomics these years,of which the recently discovered miRNA is a tiny RNA which is very common in multi-cellular organisms. As a post-transcriptional regulation factor,composed of 20～22 nucleotides, miRNA plays an important role in many biological process including growth and development of individuals, proliferation and differentiation of cells,etc. And the mechanism is that RNA-induced silencing complex (RISC),induced by the combination of miRNA and non-translation area (3'UTR) of targeted gene, degrades mRNA of targeted gene or suppresses translation of proteins, miRNA does not code proteins itself. Evidence is increasing that miRNA is very involved in the development of cancer, mediated by its downstream targeted genes. And it plays a biological role as a gene that suppresses or precipitates the development of a tumor through involvement in proliferation, apoptosis, invasion of tumor cells, or angiogenesis of a tumor.There is a complex link between MiRNA genes and the body. An miRNA may have multiple targeted genes, and a targeted gene may be regulated by multiple miRNA. It was proved in recent years that more than 50% of miRNAs were located in chromosome areas which would change when tumor occurred. These areas include minimal regions of loss of heterozygosity (LOH), which usually contain tumor suppressor genes,and minimal regions of amplification, which may contain cancer genes. Changes of miRNA expression profiling in some diseases have distinctive manifestation,and there are significant discrepancies between of the normal adjacent tissue and that of the tumor,especially in cancers. It was found that the analysis of the different levels of tumor specific miRNA contributes to make an early diagnosis of tumors, which is expected to be molecular markers of the metastasis and prognosis of malignant tumors. Preliminary result shows that MiRNA expression spectrums of the renal cell carcinoma and adjacent normal renal tissue have characteristic changes, on which there is few reports at home and abroad. This study was to research the MiRNA expression spectrums of the renal clear cell carcinoma (ccRCC) and normal kidney tissues by high flux, high sensitivity and high specific miRNA microarray (Exiqon corporation),and to screen specifically expressed miRNA which was then validated using real-time quantitative PCR.The regulatory network between miRNA and gene within the body is very complex that a miRNA can have multiple targeted gene, and a gene can be combined with multiple miRNAs. We have completed gene expression spectrums of 4 case diagnosed as ccRCC by Agilent chip several years earlier. Now upon that we employed related miRNA targeted gene forecast software:TargetScan,miRanda and PietTar to forecast the differentially expressed miRNA genes which were screened in the last step of the experiment. Then we can find the probable targets of miRNA on targeted gene 3'UTR region, and analyze the relation between the differentially expressed miRNA and targeted genes to lay foundation for further research. Our destination is the role of miRNA in the development of renal tumor.MiR-3133 has a conspicuous down-regulated expression in ccRCC. And it probably acts as a tumor suppressor gene in the biology of renal cell carcinoma. As a newly discovered gene, it is difficult to determine the targeted genes and regulation mechanism of miR-3133, and there is no related international or domestic literature reported currently. For further research on the function of miR-3133, we synthesized its sequence, which was then transfected to human renal cancer 786-0 cell strains. RT-PCR and Westernblot were performed to detect downstream effect of silence miRNA gene; and scratch test was conducted to detect its migration efficacy, while line Transwell to detect its invasion efficacy. We also test the proliferation and apoptosis of the cancer cell strains by flow cytometry and MTT test.The objective of the study:1. Applied Exiqon's miRCURYTM LNA miRNA microarray detection cc-RCC of microRNA (miRNA) differential expression profiles, filter out differential gene expression in miRNA; real-time quantitative PCR validation results, with a view to establishing reliable cc-RCC miRNA expression profiles.2. The use of miRNA target gene prediction software, analyses of miRNA expression in renal clear cell carcinoma and gene expression profiling, aimed at finding differentially expressed miRNA target genes, revealing the relationship between regulation of target mRNA and miRNA, provides relevant basis for in-depth discussion on the pathogenesis of cc-RCC.3. Synthesis artificial sequences of miR-31333 was transfered to the 786-0 cell lines of renal cell carcinoma, through a series of in vitro cell biology experiment, observation of miR-3133 expression or suppression effects of 786-0 cells on Renal carcinoma biological properties, further confirmation of miRNA in the cc-RCC of an important role in the development process.Materials and methodsFirst, the establishment and screening of miRNA expression profiling of renal clear cell carcinoma.8 cases patients of cc-RCC were selected in June 2009-September 2010 in the Department of Urology, Nanfang Hospital.8 cases were in renal cell carcinoma after radical resection of tumor tissue were taken within 0.5h and the adjacent tissue (from cancer is greater than 2 cm) spare frozen in liquid nitrogen tanks. All specimens were confirmed by routine pathological examination. One specimen in accordance with the one-step extraction of total RNA, after passing through quality control, and labeled miRNA was isolated, made with the miRNA microarray hybridization solution, and then Jingxi film, scanning and data analysis, differentially expressed miRNA genes. Then results was verified by real time quantitative PCR, the renal clear cell carcinoma of miRNA expression profiling, differential expression analysis of possible clinical significance of miRNA and target genes, while classifying the newly discovered miRNA preliminary study.Second, the target genes prediction on differential expression miRNA of renal clear cell carcinoma.We based on thg miRNA expression profiles on the above and gene expression chip which we finished with Agilent in a few years ago,using bioinformatics methods, comprehensive analysis of mRNA and miRNA expression profiling to reveal the interaction between miRNA and mRNA regulation of relations. miRNA target gene prediction using three software, differential expression of miRNA genes predicted miRNA target genes to identify possible targets of 3'UTR region, analysis of differential expression of miRNA and target the complex relationship between mRNA.Third, the synthetic sequences of miR-3133, were transfected into 786-0 cells, through a series of in vitro cell biology experiments to observe the miR-3133 expression or inhibition of 786-0 cell biological traits impact.1. miR-3133/anti-miR-3133 Transfection and qRT-PCR test:①There were four groups:untransfected group (Mock), negative sequence transfection group (NC), miR-3133 mimic transfection group, miR-3133 inhibitor group;②Cell culture and transfection:The 786-O cells per well of about 1×106 cells seeded in 6-well culture plates, in 37℃,5% CO2 and saturated humidity transfected the plasmid-lipofectamine2000 complex to the corresponding cells.③The use of qRT-PCR detected 48 hours after transfection, each group's expression of miR-3133; with relative quantitative data analysis:2-ΔΔCt (Ct behalf of cycle threshold) that the gene expression, the average relative concentration=2-averageΔΔCT'100%, interference efficiency=(1-2-averageΔΔCT)'100%, the formula:ΔΔCt={Ct (ezrin)-Ct (β-actin)} interfere with cell-{Ct (ezrin)-Ct (β-actin)} control (untransfected cells).2. MTT cell proliferation test:After transfection, respectively,12h,24h,36h and 72h the cells were collected, each of 3 wells; detector in the enzyme-linked immunosorbent determination of the hole on the optical density (OD) were compared among groups cell proliferation.3. Scratches and pictures:After the success of transfection, each group has 6 duplicate wells, were cultured in 12-well plate, when the cell growth to the fusion of 95%, the vertical for tips to 1mL scratch test, calculated after 24 hours group of cells migrated to the number of scratch area and scratch area photographed calculated.4. Transwell invasion in vitro experiment:the cells in serum-free RPMI-1640 medium for overnight, the group of cells in the Transwell plate from each of three duplicate wells, adding the full lower chamber medium 500pL, the room contains an added×106 cells in serum-free medium 200μL,24 hours later with a cotton swab to remove the upper surface of the cell membrane and gel, and then fixed, stained, selected under the microscope at 400 times the view count of 5 different cells under the surface of the diaphragm, calculated cell invasion.ResultsFirst, the establishment and selection of renal miRNA expression profile of renal clear cell carcinoma1. The results of total RNA extraction and quality testing of 8 cases of carcinoma and 4 cases of adjacent tissue is ideal, whose A260/A280 value is between 1.8～2.1, and electrophoresis with 18s and 28s of total RNA were clear, showing that nucleic acid ingredient are full, free of degradation and pollution.2. Chip sample scatter chart shows the relationship between tumor samples and the hybridization signal correlation of control groups is lower, reflecting there is a significant difference between the biological properties of experimental group and that of control group sample.3. There exists a difference in expression of miRNA between renal clear cell carcinoma and paracancerous tissue, in which raised miRNA genes consists 106, cut miRNA genes 184, with 111 new miRNA. by t test raised miRNA genes is only 3,and cut miRNA genes 38, which is more than cut miRNA.4. QRT-PCR texts shows cancer expression in the miR-142-5p and miR-375 is higher than their surrounding tissue and expression of miR-3133, miR-3907 and miR-1236 are lower than the adjacent tissues, consistent with the chip results.Second, the target genes prediction of differentially expressed miRNA of Renal clear cell carcinomaThought the computing method of biological information, it is shown that of the differentially expressed genes of 30 renal clear cell carcinoma,nearly half of cancer-related genes are regulated by miRNA,in which SEMA3A, BTG2 and HIF1A gene are respectively regulated by more than 10 miRNA lines. In addition, there exists the negetive correlation between genes and miRNA regulation thought analysis,meaning that raise genome miRNA of Regulation genome expression most appears in the cut genes of miRNA expression profiles.Third, the research of biological influence between miR-3133 and 786-0 cell line in human Renal carcinoma.1. Determination of transfection efficiency:at 48h after transfection to detect the expression of miR-3133.2. MTT assay results showed that:single factor analysis of variance showed that differences between groups was significant (F=11.835, P<0.05), different time levels were statistically significant (F=11.573, P<0.000), group Interaction between the other and time (F=79.245, P<0.000). Further analysis of individual effects, the results displayed in a fixed group of conditions, the time difference was statistically significant (P all P<0.001), factor conditions in a fixed time, from among the groups after 24h significantly (P all P<0.01), suggest miR-3133 was inhibited and 786-0 cell proliferation has been enhanced.3. Experiments of the cell migration showed:24 and 36 hours after the scratches were observed scratches to the size, contrast found cells completely fused in the miR-3133 inhibitor group after 36 hours; and scratch area was still the largest in miR-3133 mimic group. That show miR-3133 was inhibited and 786-0 cell migration has been increased.4. Invasion assay results showed that the cell numbers through matrigel film were 43±8/HP,17±4/HP,108±13/HP and 40±7/HP in 786-0 group, miR-3133 mimic group, miR-3133 inhibitor group and negative control group respectively. ANOVA analysis, the cell numbers through matrigel film were more than the other three groups in miR-3133 inhibitor group, with statistical significance (P<O 05).Conclusion1.The miRNA expression profile of renal clear cell carcinoma showed tissue specificity, up-regulated miRNA genes in renal clear cell carcinoma are 106, with 184 down-regulated miRNA, in which passed t-test up-regulated miRNA are 3, with down-regulated miRNA 38. 111 miRNA which were released in 16.0 miRBase firstly were detected in our experiment.2.1t is further validated that miR-155, miR-210, miR-141, miR-16, miR-200c and let-7 family miRNAs are closely associated with pathogenesis of renal clear cell carcinoma, which plays an important role in the disease process.3. Bioinformatics methods can comprehensively analysis the funtional relation of genomes and miRNA expression profiles, and establish a relationship between genes and miRNA regulation,also can better predict target genes of miRNA.There exists the negetive correlation between genes and miRNA regulation thought analysis,meaning that raise genome miRNA of Regulation genome expression most appears in the cut genes of miRNA expression profiles.4. MiR-3133 expression is significantly lower in renal clear cell carcinoma. In vitro experiment found that biological activity of 786-0 cell decreased after enhancing the expression of miR-3133 in renal clear cell carcinoma, while it increased after restraining,which showed miR-3133 may play a role of tumor suppressor gene in the biological process of renal clear cell carcinoma.