| Background:Renal cell carcinoma(RCC) is the most common type of adult kidney tumor, accounting for about 85% of all primary renal tumors and about 3% of all cancers in adults. Operation can be taken for early local kidney cancer. However, there are still many patients would be confronted with the risk of tumor recurrence and metastasis and therefore more poor prognosis. The 5-year survival rate of RCC patients with metastases is only 9%. In order to treat these patients with advanced RCC, it is urgent need to find new biomarkers as therapeutic targets. MiRNAs are a class of regulatory molecules with highly conserved about 19-24 nucleotides in length, through gene targeting the 3’untranslated region (3’UTR) of non-complementary sequence of base pairing, miRNA could regulate the expression level of target gene by completed mRNA degradation or inhibiting its translation. Several studies have reported that miRNAs play the key role in developmental regulation; cell proliferation, differentiation, invasion and apoptosis. MiR-495 have abnormal expression which were found in a variety of tumors, such as non-small cell lung cancer, breast cancer, glioblastoma, stomach cancer and leukemia and which play an important role possibly as proto-oncogenes or tumor suppressor genes in the development of tumors. But miR-495 expression and function in renal cell carcinoma is unknown.Objective:Study on miR-495 expression in human RCC, and to investigate the effect of abnormal expression of miR-495 on RCC cell proliferation and migration capabilities. By predict and validate target genes of miR-495 to explore the molecular mechanism of miR-495 in RCC.Methods:Selected four human RCC cell lines and one normal human kidney cell line, the expression levels of miR-495 in RCC cell lines were detected by qRT-PCR. The clinical kidney cancer and adjacent normal tissues were collected at same time and the expression levels of miR-495 in RCC tissue specimens were also detected by qRT-PCR. One kind of 786-0 cell lines which have stable overexpression of miR-495 has been built by transient transfection technology and in which the expression of miR-495 was validated by qRT-PCR. Further experiments would be done to evaluate that the overexpression of miR-495 in 786-0 whether impacts on cell itself function including cell proliferative activity detected by CCK8, cell cycle tested by flow cytometry and cell migration capabilities by scratch experiment.SATB1 could be as the targeted gene of miR-495 predicted by searching TargetScan and other databases. Luciferase reporter gene analysis would be used to verify whether miR-495 can target SATB1 mRNA 3’UTR and qRT-PCR and WB would be done to detect the expression of mRNA and protein of SATB1 in 786-0 which had a stable expression of miR-495. In order to further clarify the molecular mechanism of miR-495 concerned with SATB1 gene in RCC in vitro,786-0 with stably expressing miR-495 would be transfected by SATB1 vector or negative control (NC) vector. The transfect outcome of SATB1 gene in miR-495 overexpressing 786-0 cells can be verified by WB. Then CCK8, flow cytometry and scratch experiment would be tested to evaluate the re-expression of SATB1 gene whether reversed the miR-459-induced inhibition of cell proliferation and migration.Results:Compared with normal human kidney cell line HK-2 and adjacent normal tissues, the expression of miR-495 in four human RCC cell line (769-P,786-0, A498 and SN12-PM6) and clinical RCC tissue samples were all significantly reduced tested by qRT-PCR and 786-0 had lowest level of miR-495 expression compared with other three RCC cell lines.The stably expressing miR-495 in 786-0 cells successful acquired and verified by qRT-PCR to detect the expression levels of miR-495. flow cytometry analysis showed that the cell cycle of 786-0 cells with stably expressing miR-495 were arrested in GO/G1 phase, CCK8 test showed that stable expression of miR-495 could inhibit the proliferation activity of 786-0 cells and scratches experiments show that the migration activity of 786-0 cells was also significantly inhibited because of the stable expression of miR-495.The data of TargetScan6.2 and other databases showed SATB1 has the putative target of miR-495. Luciferase reporter assay showed that the luciferase reporter activity decreased approximately 67% in the 786-0 cells which co-transfected the p-MIR-SATB1 (SATB1-3’UTR-WT) and miR-495 mimics campared with control group which transfected the p-MIR-SATB1 (SATB1-3’UTR-MUT) or miR scramble. Moreover, we found that the ectopic expression of miR-495 suppressed the SATB1 mRNA and protein level in 786-0 cells by using qRT-PCR and Western blot.In order to further clarify the molecular mechanism of miR-495 concerned with SATB1 gene in RCC in vitro, the re-expression of SATB1 was overexpression by using SATB1 vector. We rescued the expression of SATB1 in miR-495 overexpressing 786-0 cells. Compared with the NC vector, CCK8 assay showed that re-expression of SATB1 increased the miR-495 overexpressing 786-0 cells proliferation, cell cycle assays showed that miR-495 overexpressing 786-0 cells transfected with SATB1 vector had an obvious cell cycle at the S phase and the migration abilities of miR-495 overexpressing 786-0 cells were increased after SATB1 vector transfection. Rescue experiment to confirm that miR-495 acts as a tumor suppressor in renal cell carcinoma cells by regulating SATB1 expression.Conclusion:this study provided novel evidence that miR-495 functions as a tumor suppressor miRNA in RCC through inhibiting SATB1 expression. The ability of miR-495 to target SATB1 may provide one such mechanism of post-transcriptional control of SATB1. Our data on miR-495 suggested that this miRNA could be a potential target for the treatment of RCC in future. |
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