Regulation of proliferation and invasion of renal cancer cells by miRNA-4469 via targeting PDIA4 gene
黄耿 1桂定文 1袁琛 1张晓玲 1黄丽琼 1吕晶丽 郎华
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作者信息
1. 鄂东医疗集团黄石市中心医院泌尿外科，黄石 435000
折叠
摘要
目的 探讨miRNA-4469（miR-4469）体外调控肾癌细胞增殖和侵袭的机制。 方法 基于OncomiR数据库分析miR-4469不同表达水平患者生存差异。采用实时荧光定量聚合酶链反应（qRT-PCR）法检测miR-4469在肾癌细胞株ACHN、OS-RC-2、SK-RC-20、769-P、A498和正常肾小管上皮细胞株HK-2中的表达，将miR-4469表达水平最低的肾癌细胞分为miR-4469组和对照组，分别转染miR-4469模拟物和阴性对照序列。采用CCK-8法检测两组细胞增殖能力（以吸光度值表示），采用Transwell实验分析两组侵袭细胞数。应用TargetScan Release 8.0软件预测miR-4469与蛋白二硫化物异构酶A4（PDIA4）mRNA的结合位点，采用双荧光素酶报告基因实验验证miR-4469与PDIA4 mRNA的靶向关系。采用qRT-PCR法检测各组细胞PDIA4 mRNA的表达量，采用蛋白质印迹法检测各组细胞PDIA4蛋白和PI3K-AKT-m-TOR通路蛋白表达水平。 结果 分析OncomiR数据库相关数据显示，miR-4469高表达肾癌患者的总生存优于miR-4469低表达患者（P<0.001）。各肾癌细胞株中miR-4469的相对表达量均低于HK-2细胞（均P<0.05），其中769-P细胞miR-4469表达量最低，选取769-P细胞进行后续实验。miR-4469组769-P细胞增殖能力低于对照组（P<0.01）。miR-4469组769-P细胞侵袭数少于对照组[（19± 3）个比（64±7）个，t＝5.44，P＝0.002]。与共同转染野生型PDIA4和miR-4469阴性序列组相比，共同转染野生型PDIA4和miR-4469模拟序列组细胞相对荧光素酶活性低（0.42±0.07比1.01±0.08，t＝5.74，P＝0.001）；共同转染突变型PDIA4和miR-4469阴性序列组与共同转染突变型PDIA4和miR-4469模拟序列组间细胞相对荧光素酶活性差异无统计学意义（0.99±0.11比1.02±0.11，t＝0.19，P＝0.001）。miR-4469组769-P细胞PDIA4 mRNA相对表达量低于对照组[0.98±0.23比7.19±2.23，t＝2.77，P＝0.032]。与对照组相比，miR-4469组769-P细胞PDIA4蛋白和PI3K-AKT-m-TOR通路相关的p-PI3K、p-AKT、p-mTOR、p-SGK1蛋白表达量均低（均P<0.05）。 结论 miR-4469与肾癌患者生存可能有相关性，其在各肾癌细胞株中表达均下调。miR-4469可能通过PDIA4调节PI3K-AKT-m-TOR通路，从而抑制肾癌769-P细胞增殖和侵袭。 Objective To explore the mechanism by which miRNA-4469 (miR-4469) regulates the proliferation and invasion of renal cancer cells in vitro. Methods The survival differences of patients with different expression levels of miR-4469 were analyzed based on the OncomiR database. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression of miR-4469 in renal cancer cell lines ACHN, OS-RC-2, SK-RC-20, 769-P, A498 and normal renal tubular epithelial cell line HK-2, and the renal cancer cells with the lowest expression level of miR-4469 were divided into miR-4469 group and control group, and were transfected with miR-4469 mimic and negative control sequence, respectively. The CCK-8 assay was used to detect the cell proliferation ability (expressed as absorbance value) in the two groups, and Transwell assay was used to analyze the number of invasive cells in the two groups. TargetScan Release 8.0 software was used to predict the binding site between miR-4469 and protein disulfide isomerase A4 (PDIA4) mRNA, and dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-4469 and PDIA4 mRNA. qRT-PCR method was used to detect the expression of PDIA4 mRNA in cells of each group, and Western blotting method was used to detect the expression levels of PDIA4 protein and PI3K-AKT-m-TOR pathway proteins in cells of each group. Results Analysis of relevant data from the OncomiR database showed that compared with patients with low miR-4469 expression, the overall survival of renal cancer patients with high miR-4469 expression was better (P < 0.001). The relative expression of miR-4469 in each renal cancer cell line was lower than that in HK-2 cells (all P < 0.05), and the expression of miR-4469 in 769-P cells was the lowest, which were selected to perform the subsequent experiments. The proliferation ability of 769-P cells in the miR-4469 group was lower than that in the control group ( P < 0.01). The number of 769-P cell invasions in the miR-4469 group were less than that in the control group [(19±3) cells vs. (64±7) cells, t = 5.44, P = 0.002]. Compared with the co-transfection of wild-type PDIA4 and miR-4469 negative sequence group, the relative luciferase activity of cells in the co-transfection of wild-type PDIA4 and miR-4469 mimic sequence group was lower (0.42±0.07 vs. 1.01±0.08, t = 5.74, P = 0.001) there was no statistical difference in cell luciferase activity between the co-transfected mutant PDIA4 and miR-4469 negative sequence group and the co-transfected mutant PDIA4 and miR-4469 mimic sequence group (0.99±0.11 vs. 1.02±0.11, t = 0.19, P = 0.001). The relative expression levels of PDIA4 mRNA in 769-P cells in the miR-4469 group were lower than that in the control group (0.98±0.23 vs. 7.19±2.23, t = 2.77, P = 0.032). Compared with the control group, the expression of PDIA4 protein and PI3K-AKT-m-TOR pathway-related p-PI3K, p-AKT, p-mTOR, and p-SGK1 proteins in 769-P cells in the miR-4469 group were all lower (all P < 0.05). Conclusions miR-4469 may be related to the survival of renal cancer patients, and its expression is down-regulated in various renal cancer cell lines. miR-4469 may inhibit the proliferation and invasion of renal cancer 769-P cells by regulating the PI3K-AKT-m-TOR pathway through PDIA4.
Abstract
Objective To explore the mechanism by which miRNA-4469 (miR-4469) regulates the proliferation and invasion of renal cancer cells in vitro. Methods The survival differences of patients with different expression levels of miR-4469 were analyzed based on the OncomiR database. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression of miR-4469 in renal cancer cell lines ACHN, OS-RC-2, SK-RC-20, 769-P, A498 and normal renal tubular epithelial cell line HK-2, and the renal cancer cells with the lowest expression level of miR-4469 were divided into miR-4469 group and control group, and were transfected with miR-4469 mimic and negative control sequence, respectively. The CCK-8 assay was used to detect the cell proliferation ability (expressed as absorbance value) in the two groups, and Transwell assay was used to analyze the number of invasive cells in the two groups. TargetScan Release 8.0 software was used to predict the binding site between miR-4469 and protein disulfide isomerase A4 (PDIA4) mRNA, and dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-4469 and PDIA4 mRNA. qRT-PCR method was used to detect the expression of PDIA4 mRNA in cells of each group, and Western blotting method was used to detect the expression levels of PDIA4 protein and PI3K-AKT-m-TOR pathway proteins in cells of each group. Results Analysis of relevant data from the OncomiR database showed that compared with patients with low miR-4469 expression, the overall survival of renal cancer patients with high miR-4469 expression was better (P < 0.001). The relative expression of miR-4469 in each renal cancer cell line was lower than that in HK-2 cells (all P < 0.05), and the expression of miR-4469 in 769-P cells was the lowest, which were selected to perform the subsequent experiments. The proliferation ability of 769-P cells in the miR-4469 group was lower than that in the control group ( P < 0.01). The number of 769-P cell invasions in the miR-4469 group were less than that in the control group [(19±3) cells vs. (64±7) cells, t = 5.44, P = 0.002]. Compared with the co-transfection of wild-type PDIA4 and miR-4469 negative sequence group, the relative luciferase activity of cells in the co-transfection of wild-type PDIA4 and miR-4469 mimic sequence group was lower (0.42±0.07 vs. 1.01±0.08, t = 5.74, P = 0.001) there was no statistical difference in cell luciferase activity between the co-transfected mutant PDIA4 and miR-4469 negative sequence group and the co-transfected mutant PDIA4 and miR-4469 mimic sequence group (0.99±0.11 vs. 1.02±0.11, t = 0.19, P = 0.001). The relative expression levels of PDIA4 mRNA in 769-P cells in the miR-4469 group were lower than that in the control group (0.98±0.23 vs. 7.19±2.23, t = 2.77, P = 0.032). Compared with the control group, the expression of PDIA4 protein and PI3K-AKT-m-TOR pathway-related p-PI3K, p-AKT, p-mTOR, and p-SGK1 proteins in 769-P cells in the miR-4469 group were all lower (all P < 0.05). Conclusions miR-4469 may be related to the survival of renal cancer patients, and its expression is down-regulated in various renal cancer cell lines. miR-4469 may inhibit the proliferation and invasion of renal cancer 769-P cells by regulating the PI3K-AKT-m-TOR pathway through PDIA4.
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