The Regulating Of MicroRNA-210on Proliferation,Apoptosis And Invasion In Renal Cancer Cells And Its Molecular Mechanism

Part ⅠThe expression of microRNA-210and correlation with pathologic staging and grading in clear cell renal cell carcinomaObjective To investigate the expression of microRNA-210(miR-210) in clear cell renal cell carcinoma (ccRCC) with regard to the clinical significance.Methods Expressions of miR-210was detected by real-time PCR in15cases of ccRCC and matched adjacent non-tumor tissue. The relevance between the expressions of miR-210and the pathologic staging and grading were also studied.Results The relative expression of miR-210in ccRCC is6.10±8.14, which was significantly higher than that in the matched adjacent non-tumor tissue with2.27±0.54(P<0.01). Grouping according to pathologic staging, the relative expression of miR-210was7.24±0.66 in I phase, and7.60±0.64in Ⅱ-Ⅲ phase, which had no significant difference (P>0.05).Grouping according to pathologic grading, the rdlative expression of miR-210was7.06±0.69in well differentiated group, and7.21±0.63in low-moderately differentiated group, which had no significant difference (P>0.05)Conclusions1. Compared with matched adjacent non-tumor tissue, the expression of miR-210was up-regulated in ccRCC, suggesting that miR-210may participate in the pathogenesis of ccRCC and detecting the expression of miR-210may be useful for early diagnosis of renal cancer.2,The expression of miR-210had no significant correlation with pathologic staging and grading in ccRCC. Objective To explore the effects of miR-210on biological characteristics of renal cancer cell, including proliferation, apoptosis and invasion ability.Methods The expression of miR-210in ACHN and HK2cells incubated in normal oxygen and hypoxia condition was detected by real-time PCR. Then the miR-210expression in ACHN cell was knockdown by transfection of has-mir-210inhibitor using LipofectamineTM2000. The cell proliferative vitality, apoptosis and migration activity were determined by MTT assay, flow cytometry and transwell migration assay respectively.Results In normal oxygen condition, the relative expression of miR-210in HK2and ACHN cells was1.47and3.43respectively. Incubating in hypoxia condition for12h,24h and48h, the expression of miR-210in ACHN cells was4.59,5.30and7.20respectively. The further study was classified into three group:ACHN cells in normal oxygen group, hypoxia group and hypoxia inhibition group. Every time (12h,24h,48h) after transfection, compared with normal oxygen group, the survival rates of hypoxia group significantly upregulated (P<0.05); however, compared with normal oxygen group or hypoxia group, survival rates of hypoxia inhibition group significantly downregulated (P<0.05) The same results were seen with respect to invasion ability. Every time (12h,24h,48h) after transfection, compared with normal oxygen group, the apoptosis rates of hypoxia group significantly downregulated (P<0.05); however, compared with normal oxygen group or hypoxia group, apoptosis rates of hypoxia inhibition group significantly upregulated (P<0.05)Conclusions1,hypoxia induce upregulation expression of miR-210in renal cancer cells.2,hsa-mir-210inhibitor could be effectively transfected to renal cancer cells, resluting in downregulating the expression of miR-210.3,Hypoxia enhance proliferation and invasion ability and induce apoptosis of renal cancer cells.4,The down-regulated expression of miR-210can inhibit the proliferation, invasion and induce apoptosis in hypoxia renal cancer cells. Objective To explore the mechanism of miR-210regulating the renal cancer cellMethods After predicting target genes of miR-210by bioinformatics methods (miRanda, Pictar, mirbase and Targetscan), real-time PCR and Western blot were used to detect the expression of miR-210and target gene expressionResults VMP1, HOXA9, CBX7, VAV3and FGFRL1were predicted to be target genes of miR-210.48h after transfection, the relative expression of miR-210in hypoxia inhibition group was significantly downregulated than that in hypoxia group (P<0.05). Real-time PCR showed the VMP1expression in normal oxygen group, hypoxia group, hypoxia inhition group were3.3408±0.4391,1.3457±0.5235and2.2383±0.1785respectively, there were significant difference between the three groups (P<0.05); Compared with hypoxia group, FGFRL1expression in hypoxia inhibition group were significantly upregulated (P <0.05); The HOXA9, CBX7and VAV3expressions in the three groups had no significant difference. Western blot showed the FGFRL1expressions in normal oxygen group, hypoxia group, hypoxia inhition group were0.208±0.01,0.151±0.01and0.471±0.11respectively, Compared with normal oxygen group, FGFRL1expression in hypoxia group were significantly downregulated (P<0.05). Compared with hypoxia group, FGFRL1expression in hypoxia inhibition group were significantly upregulated (P<0.05); HOXA9protein expression ratio (HOXA9/GAPDH) were0.283±0.016,0.535±0.021和0.509±0.024respectively, which had significant difference between hypoxia group and normal oxygen group (P<0.05) and no significant difference between hypoxia inhibition group and hypoxia group(P>0.05); The VMP1, CBX7and VAV3expression in the three group had no significant difference.Conclusions1. In hypoxia renal cancer cells, the expression FGFRL1had negatively correlation with the expression miR-2102. In hypoxia renal cancer cells the VMP1gene expression had correlation with the expression of miR-210, however, the VMP1protein expression had no correlation with the expression of miR-210.3. HOXA9,CBX7and VAV3expression had no significant correlation with the expression of miR-210.4. FGFRL1may be a potential target gene of miR-210in renal cancer, while VMP1,HOXA9,CBX7and VAV3were not target genes of miR-210.