Expression Of MiR-205in Clear Cell Renal Cell Carcinoma And Its Associated Mechanism

Expression of miR-205in clear cell renal cell carcinoma and its associated mechanismBackgroundRenal cell carcinoma (RCC), as one of the common urological malignancies, accounts for3%of adult malignancies and its incidence is only secondary to bladder cancer in urological cancers. Clear cell renal cell carcinoma (ccRCC), the clear cell histology type, is the most frequent subtype of RCC and accounts for approximately80-85%of RCC. Currently, radical nephrectomy is effective for treating early RCC. However, for advanced RCC or recurrent and metastatic RCC after radical nephrectomy, effective treatment methods are very lacking. The5-year survival rate of those patients is less than9%. The studies have shown that carcinogenesis and progression of RCC are multistage process that involve lots of oncogenes, tumor suppressor genes and growth factors. Therefore, it is very important to clarify the molecular mechanism in RCC that will contribute to the early diagnosis and therapy of RCC.MicroRAN(miRNAs) are a class of naturally occurring, short non-coding, single stranded RNAs with22nucleotides in length, that regulate gene expression at the post-transcriptional level, by binding to the3’untranslated region (3’UTR) of mammalian target mRNAs and causing translational inhibition and/or mRNA degradation. By modulating physiological processes, such as metabolism, division, differentiation, development and apoptosis, miRNAs can simultaneously regulate many pathological processes, including human carcinogenesis. It has been repeatedly confirmed that miRNAs play an important role in the pathogenesis of RCC by targeting oncogenes or tumor suppressor genes to impacts on cell survival, proliferation, apoptosis and metastasis. Although the importance of miRNAs in RCC has attracted much attention in recent years, the function and its associated mechanism of the majority of the miRNAs in RCC remains unclear and further study is much needed.To find out the relationship between miR-205and carcinogenesis and progression of ccRCC, we first detected the expression of miR-205in the ccRCC and matched adjacent non-tumor tissue using real-time PCR. The relevance between the expression of miR-205and the pathologic staging and grading was also studied. The expression of miR-205in renal carcinoma cell line ACHN and Caki-1and normal renal tubular epithelial cell line HK-2was then detected by real-time PCR. Based on these results, we selected the suitable renal carcinoma cell line for the subsequent study in vitro. To explore the effects of miR-205on biological characteristics of renal carcinoma strain, the miR-205expression in ACHN cell was up-regulated by transfection of has-mir-205mimics using LipofectamineTM2000and the cell proliferative vitality, apoptosis, cell cycle, migration and invasion activity were determined by MTT assay, flow cytometry and transwell migration and invasion assay respectively. Last, using the bioinformatics methods (miRanda, Pictar and Targetscan) target genes of miR-205were predicted and real-time PCR and Western blot were used to detect the expression of miR-205and target gene expression in vitro. The study aimed to find out the possible mechanism of miR-205in ccRCC and provided a new viewpoint for diagnosis and therapy of RCC. Methods and Results1. MiR-205is significantly down-regulated in ccRCC tissue:The relative expression of miR-205by real-time PCR in ccRCC tissue was significantly lower than that in the matched adjacent non-tumor tissue (3.60±1.92vs6.39±3.39, P<0.05)2. The expression of miR-205has no significant correlation with clinical staging and pathologic grading in ccRCC:Twenty ccRCC tissue cases were respectively grouped according clinical staging and pathologic grading. The results showed that no significant difference was found between well differentiated group and low-moderately differentiated group or between I phase and Ⅱ-Ⅲ phase (3.66±2.24vs3.51±1.42and3.73±2.16vs3.36±1.48, P＞0.05)3. The up-regulated expression of miR-205inhibits the proliferation, invasion and induces apoptosis in ACHN cells:The expression of miR-205in Caki-1, ACHN and HK-2cell was detected by real-time PCR. The results showed that the relative expression of miR-205in Caki-1and ACHN was significantly lower than that in HK-2cell (1.32±0.13,1.06±0.13and3.95±0.37, respectively, P<0.05). ACHN was selected for the subsequent study in vitro. miR-205expression in ACHN cell was up-regulated by transfection of has-mir-205mimics using Lipofectamine2000. The further study was classified into three group:blank control group (B.C)、negative control (N.C)、miR-205mimics transfection group (miR-205). The expression of miR-205in the three groups was detected and the cell proliferation, apoptosis, cell cycle, migration and invasion activity were determined by MTT assay, flow cytometry and transwell migration and invasion assay respectively. The results showed that the relative expression of miR-205in miR-205group was significantly higher than that in B.C and N.C group (3.95±1.15,1.35±0.51and1.37±0.41, respectively, P<0.05);The proliferation of miR-205group was significantly slower than B.C and N.C group (P<0.05); The apoptosis rates of miR-205group significantly down-regulated compared with B.C and N.C group (P<0.05); The cell number in sub-G1phase significantly up-regulated while cell number in sub-G1phase significantly down-regulated in miR-205group compared with B.C and N.C group (P<0.05); The migration and invasive capacities of ACHN cell in miR-205group was significantly inhibited compared with B.C and N.C group (P<0.05).4. E2F1and ZEB2may be the downstream targets of miR-205: Using the bioinformatics methods (miRanda, Pictar and Targetscan), we predicted that E2F1and ZEB2may be the potential target genes of miR-205. The expression of E2F1and ZEB2in Caki-1, ACHN and HK-2cell was detected by real-time PCR and Western blot. The expression of E2F1and ZEB2in miR-205-overexpressed ACHN cell was also detected. The results showed that the expression of E2F1and ZEB2in ACHN and Caki-1cell was significantly higher than that in HK-2cell both at the level of mRNA and protein(P<0.05). The expression of ZEB2in miR-205group was significantly lower than that in B.C and N.C group both at the level of mRNA and protein(P<0.05). The expression of E2F1protein in miR-205group was significantly lower than that in B.C and N.C group (P<0.05), but no significant difference of the expression of E2F1mRNA was found in these three groups (P>0.05).5. MiR-205may regulate E2F1-mediated AKT signal pathway:The expression of AKT, pAKT, BAD and pBAD protein in miR-205, B.C and N.C group were detected by Western blot. The results showed that The expression of pAKT, pBAD protein in miR-205group was significantly lower than that in B.C and N.C group (P<0.05), but no significant difference of the expression of AKT and BAD protein was found in these three groups (P>0.05).6. MiR-205may regulate ZEB2-mediated EMT:The expression of E-cadherin and Vimentin protein in ccRCC tissue and matched adjacent non-tumor tissue was detected by immunohistochemisty. The expression of E-cadherin and Vimentin in Caki-1, ACHN and HK-2cell was detected by real-time PCR and Western blot. The expression of E-cadherin and Vimentin in miR-205-overexpressed ACHN cell was also detected. The results showed that the positive ratio of E-cadherin in ccRCC tissue was significantly lower than that in matched adjacent non-tumor tissue (35%vs75%, P<0.05). The positive ratio of Vimentin in ccRCC tissue was significantly higher than that in matched adjacent non-tumor tissue (80%vs25%, P<0.05). The expression of E-cadherin in ACHN and Caki-1cell was significantly lower than that in HK-2cell both at the level of mRNA and protein(P<0.05), while Vimentin in ACHN and Caki-1cell was significantly higher than that in HK-2cell (P＜0.05). The expression of E-cadherin in miR-205group was significantly higher than that in B.C and N.C group both at the level of mRNA and protein(P<0.05), while Vimentin in miR-205group was lower than that in B.C and N.C group (P＜0.05).Conclusion1. The expression of miR-205in ccRCC tissue was significantly down-regulated, suggesting that miR-205may participate in the pathogenesis of ccRCC and detection of miR-210may be useful for diagnosis of ccRCC. The expression of miR-205had no significant correlation with clinical staging and pathologic grading in ccRCC.2. The expression of miR-205in Caki-1and ACHN cell lines was significantly lower than that in HK-2cell line. Has-miR-205mimics can be effectively transfected to ACHN cell line, resluting in down-regulation of the expression of miR-205.3. Up-regulation of miR-205can effectively inhibited proliferation and migration and invasion activity, and induced apoptosis in ACHN cell line, suggesting that miR-205may play a role as tumor suppressor gene and up-regulation of miR-205may be an effective treatment for ccRCC.4. E2F1and ZEB2may be the downstream targets of miR-205in ACHN cell. MiR-205may regulate E2F1-mediated AKT signal pathway to induce apoptosis.5. EMT mechanism may exist in ccRCC. MiR-205may regulate ZEB2-mediated EMT to inhibit migration and invasion activity in ACHN cell.