MicroRNA-122Expression In Human Renal Cell Carcinoma And Its Molecular Mechanisms On Invasion And Migration Of Renal Cell Carcinoma Cells

Renal cell carcinoma (RCC) is one of the most common kindney malignancies,which is the secondary cancer in the urinary system carcinoma. Renal Clear cellcarcinoma (CCC) accounts for approximately70%of the cases. Surgery is the mainmethod for treating RCC, because of the complicated and still unclear mechanisms.About50%members of those patients are in their late stage when firstly diagnosed ofthis caner and lose their best opportunities for treatment. Therefore, increasedunderstanding of the molecular mechanisms of RCC progression (recurrence,metastasis or drug resistance) is needed to provide a rationale for the effectivetherapeutic methods of RCC.MicroRNAs (miRNAs) are short noncoding oligonucleotides with imperfectcomplementarity predominantly to the3’ untranslated region (UTR) of target mRNAsand cause translational repression or mRNA cleavage. miRNAs predominantly act byinhibiting mRNA translation although mRNA degradation and mRNA cleavage mayalso contribute to downregulation of protein levels and play a role in the pathogenesisof cancer with function as tumor suppressors or oncogenes. miRNA-122-5p (miR-122)is one of the most frequent miRNA isolated in the liver and plays important roles inmany aspects of liver physiology, such as stress response and lipid metabolism.Recently numerous studies have shown aberrant expression of miR-122in humancancer tissues, including RCC, suggesting that it is a candidate tumor activator inRCC. However, up to date, there are no studies of miR-122in renal cell carcinomacells. Methods:In order to explore the relationship between miR-122and clear cell renal cellcarcinoma (ccRCC), we detected the expression of miR-122in human ccRCC andadjacent non-tumor tissues through real time PCR, and to find out the relationshipsbetween its expression and clinical stage, histological grade of the patients.Furthermore, we examined the expression of miR-122in different RCC cell lines, andassessed the impact of miR-122on cell proliferation, colony formation, invasion andmigration after transfection. Last, we predicted miR-122target genes and explored itspossible meachnisms for mediating the biological effects.Results:Real time PCR results demonstrated that the expression of miR-122wasup-regulated in all samples (4.04±2.23), compared to the matched adjacentnon-tumor tissues. According to the pathological grades, the relative expression ofmiR-122was3.24±1.32in well differentiated group, and5.05±2.76inlow-moderately differentiated groups, which had no significant difference (P>0.05).For the clinical stages, the relative expression of miR-122was3.49±1.49in thephage I, and5.69±3.35in the phage II-III, which had no difference between twogroups (P>0.05).Real time PCR results showed that A498and786-O cells have very lowexpression levels of miR-122, but high levels of miR-122in other cell lines. MTTassay showed that a significant increase in cell proliferation of A498and786-O in adose-dependent manner was observed following miR-122transfection, compared tocells transfected with miR-Con. We also observed that the effect of miR-122andanti-miR-122on the growth rate of both cell lines by counting cell numbers intriplicate wells every day for3days. The growth of A498and786-O was significantlyincreased by miR-122and decreased by anti-miR-122in a time dependent manner.For colony formation assay, our results showed that the colony numbers weresignificantly increased following miR-122mimics transfection and decreasedfollowing anti-miR-122transfection compared with the control cells. These results indicated that renal cell carcinoma cell proliferation could be significantly increasedby miR-122and decreased by anti-miR-122. We further detected the influence ofmiR-122on cell invasion and migration, A498and786-O cells transfected withmiR-122were applied to the Matrigel pre-coated invasion and collagen IV pre-coatedmigration assays. The results showed that invasion of A498and786-O cells wereincreased by miR-122. And the similar effect of migration was also observed in A498and786-O cells.To analyze the molecular mechanisms of miR-122on proliferation, invasion andmigration was examined in renal cell carcinoma cells, we transfected miR-122mimicsto investigate the activation of Akt and mTOR by Western blot analysis. Our resultsindicated that the phosphorylation levels of Akt (p-Akt Ser473) and mTOR (p-mTORSer2448) and mTOR downstream targets (p-p70S6K (Ser389) and p-4E-BP1(Ser65))were effectively increased after miR-122mimics transfection in A498and786-Ocells.Conlusion:1.Up-regulation of miR-122may be associated with the pathogenesis ofccRCC and have no significant correlation with pathological grade and clinical stagein ccRCC.2.A498and786-O cells have very low expression levels of miR-122, but highlevels of miR-122in other cell lines (ACHN, Caki-1, Caki-2).3.miR-122could promote cell proliferation and colony formation of RCC cells.4.miR-122could promote cell invasion and migration of RCC cells.5.Akt/mTOR signal pathways may be the potential target genes of miR-122.