肾癌组织中miRNA-4729的甲基化程度及其对肾癌细胞增殖和迁移能力的影响

Methylation degree of miRNA-4729 in renal cancer tissues and its effect on proliferation and migration abilities of renal cancer cells

目的探讨肾癌组织中miRNA-4729(miR-4729)的甲基化程度及其对肾癌细胞株增殖和迁移能力的影响。方法利用SurvivalMeth数据库中数据(更新时间2022年10月),分析178例肾癌组织中miR-4729的甲基化程度。通过miRNApath软件预测与miR-4729具有互补结合位点的靶基因。选择正常肾小管上皮细胞株HK-2和肾癌Caki-1、A-498、ACHN、786-O细胞株,采用实时荧光定量聚合酶链反应(qRT-PCR)检测各细胞株中miR-4729的相对表达量及甲基化抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对肾癌细胞中miR-4729表达的影响。将miR-4729相对表达量最低的A-498细胞分别转染miR-4729模拟物(miR-4729组)和miRNA-NC(NC组),采用集落形成实验和划痕实验检测过表达miR-4729对A-498细胞增殖、迁移能力的影响。采用双荧光素酶报告基因实验验证miR-4729和DEAD框肽5(DDX5)的靶向关系。采用蛋白质印迹法检测过表达miR-4729对A-498细胞中DDX5蛋白和AKT信号通路相关蛋白(p-AKT、p-IKKα、p-Tpl2、AS160)表达的影响。结果SurvivalMeth数据库中数据分析结果显示,肾癌组织中miR-4729的甲基化程度高于癌旁组织(P<0.01)。肾癌Caki-1、A-498、ACHN、786-O细胞和正常肾小管上皮HK-2细胞中miR-4729相对表达量分别为0.62±0.05、0.16±0.04、0.53±0.02、0.69±0.03和0.99±0.07,差异有统计学意义(F=47.39,P<0.01)。与加入二甲基亚砜(DMSO)培养的各组细胞分别比较,加入5-Aza-CdR培养的肾癌Caki-1、A-498、ACHN、786-O细胞中miR-4729相对表达量均高(均P<0.01)。集落形成实验结果显示,miR-4729组和NC组A-498细胞集落形成数分别为(53±6)个和(102±10)个,差异有统计学意义(t=4.25,P<0.01)。划痕实验结果显示,miR-4729组和NC组A-498细胞划痕愈合率分别为(42.3±2.7)%和(67.6±4.8)%,差异有统计学意义(t=4.58,P<0.01)。双荧光素酶报告基因实验结果显示miR-4729直接靶向结合DDX5。miR-4729组和NC组A-498细胞中DDX5 mRNA相对表达量分别为0.93±0.25和5.29±0.74,差异有统计学意义(t=5.60,P<0.01)。蛋白质印迹法检测结果显示,与NC组比较,miR-4729组A-498细胞中DDX5蛋白表达低,AKT信号通路相关蛋白p-AKT、p-IKKα、p-Tpl2、AS160表达亦均低。结论过表达miR-4729通过靶向抑制DDX5基因表达降低AKT信号通路活化水平,进而抑制肾癌细胞的增殖和迁移。

ObjectiveTo explore the methylation degree of miRNA-4729(miR-4729)in renal cancer tissues and its impact on the proliferation and migration abilities of renal cancer cell lines.MethodsData from the SurvivalMeth database(updated in October 2022)was used to analyze the methylation degree of miR-4729 in 178 renal cancer tissues.Target gene with complementary binding sites to miR-4729 was predicted by miRNApath software.The normal renal tubular epithelial cell line HK-2 and the renal cancer cell lines Caki-1,A-498,ACHN,and 786-O were selected.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-4729 in each cell line and the effect of methylation inhibitor 5-aza-2'-deoxycytidine(5-Aza-CdR)on the expression of miR-4729 in renal cancer cells.A-498 cells with the lowest relative expression of miR-4729 were transfected with miR-4729 mimic(miR-4729 group)and miRNA-NC(NC group),and colony formation assay and scratch assay were used to detect the effect of overexpression of miR-4729 on the proliferation and migration abilities of A-498 cells.Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-4729 and DEAD box peptide 5(DDX5).Western blotting was used to detect the effect of miR-4729 overexpression on the expression of DDX5 protein and AKT signaling pathway-related proteins(p-AKT,p-IKKα,p-Tpl2 and AS160)in A-498 cells.ResultsThe analysis results of data from the SurvivalMeth database showed that the methylation degree of miR-4729 in renal cancer tissues was higher than that in paracancerous tissues(P<0.01).The relative expressions of miR-4729 in renal cancer Caki-1,A-498,ACHN,786-O cells and normal renal tubular epithelial HK-2 cells were 0.62±0.05,0.16±0.04,0.53±0.02,0.69±0.03,and 0.99±0.07,respectively,and the difference was statistically significant(F=47.39,P<0.01).Compared with various cell groups cultured with dimethyl sulfoxide(DMSO),the relative expressions of miR-4729 in renal cancer Caki-1,A-498,ACHN and 786-O cells cultured with 5-Aza-CdR were higher(all P<0.01).The results of colony formation assay showed that the number of colonies formed in A-498 cells of the miR-4729 group and NC group were 53±6 and 102±10,respectively,and the difference was statistically significant(t=4.25,P<0.01).The results of scratch assay showed that the scratch healing rates of A-498 cells in the miR-4729 group and NC group were(42.3±2.7)%and(67.6±4.8)%,respectively,and the difference was statistically significant(t=4.58,P<0.01).The results of dual-luciferase reporter gene assay showed that miR-4729 directly targeted and bound to DDX5.The relative expressions of DDX5 mRNA in A-498 cells of the miR-4729 group and NC group were 0.93±0.25 and 5.29±0.74,respectively,and the difference was statistically significant(t=5.60,P<0.01).The results of Western blotting showed that compared with the NC group,the expression of DDX5 protein in A-498 cells of the miR-4729 group was lower,and the expressions of AKT signaling pathway-related proteins p-AKT,p-IKKα,p-Tpl2 and AS160 were also lower.ConclusionsOverexpression of miR-4729 decreases the activation level of AKT signaling pathway by targeting and inhibiting the expression of DDX5 gene,thereby inhibiting the proliferation and migration of renal cancer cells.