miR-769-3p在膀胱癌组织中的表达及下调其表达对膀胱癌J82细胞迁移和细胞周期的影响

Expression of miR-769-3p in bladder cancer tissues and the effect of its down-regulation on the migration and cell cycle of bladder cancer J82 cells

目的探究miR-769-3p在膀胱癌组织中的表达水平,通过下调膀胱癌J82细胞中miR-769-3p表达水平,观察沉默miR-769-3p对J82细胞迁移能力和细胞周期的影响。方法采用OncomiR数据库分析miR-769-3p在膀胱癌组织和癌旁组织中的表达差异。通过Lipofectamine 2000转染试剂转染J82细胞,分为si-miR-769-3p组(转染miR-769-3p小分子干扰片段)和对照组(转染无意义序列)。实时荧光定量聚合酶链反应(qRT-PCR)检测转染后miR-769-3p相对表达水平。通过细胞划痕实验和流式细胞仪检测比较两组J82细胞迁移能力和细胞周期的差异性。采用生物信息学软件MicroRNAdb预测miR-769-3p的靶基因。双荧光素酶报告基因实验验证miR-769-3p与靶基因的互补结合。qRT-PCR和Western blotting分别检测miR-769-3p的靶基因表达水平。计量资料以均数±标准差(±s)表示,两组间比较采用t检验。结果与癌旁组织相比,miR-769-3p在膀胱癌组织中表达显著升高,差异具有统计学意义(P<0.01)。si-miR-769-3p组miR-769-3p相对表达水平(1.02±0.16)明显低于对照组(4.50±0.60),差异具有统计学意义(P<0.01)。si-miR-769-3p组细胞迁移率[(26.67±3.98)%]明显低于对照组[(61.86±4.70)%],差异具有统计学意义(P<0.01)。si-miR-769-3p组处于G_(0)~G_(1)期的细胞比例[(57.66±5.74)%]显著多于对照组[(31.26±3.24)%],差异具有统计学意义(P<0.01)。双荧光素酶报告基因实验证实内皮素3(EDN3)是miR-769-3p的靶基因。对照组和si-miR-769-3p组J82细胞中EDN3 mRNA相对表达水平为1.99±0.66和6.98±0.76,与对照组比较,si-miR-769-3p组EDN3 mRNA相对表达水平明显高于对照组,差异具有统计学意义(P<0.01)。结论低表达miR-769-3p可以通过促进EDN3基因表达抑制膀胱癌J82细胞迁移能力,阻滞J82细胞周期。

Objective To explore the expression level of miR-769-3p in bladder cancer tissues,and observe the effect of silencing miR-769-3p on the migration ability and cell cycle of J82 cells by down-regulating the expression level of miR-769-3p in bladder cancer J82 cells.Methods The OncomiR database was used to analyze the expression differences of miR-769-3p in bladder cancer tissues and adjacent tissues.J82 cells were transfected with Lipofectamine 2000 transfection reagent and divided into si-miR-769-3p group(transfected with miR-769-3p small molecule interference fragments)and control group(transfected with meaningless sequences).quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the relative expression level of miR-769-3p after transfection.The cell scratch test and flow cytometry were used to compare the migration ability and cell cycle differences between the two groups of J82 cells.The bioinformatics software MicroRNAdb was used to predict the target gene of miR-769-3p.The dual-luciferase reporter gene assay was used to verify the complementary binding of miR-769-3p to the target gene.qRT-PCR and Western blotting were used to detect the expression levels of miR-769-3p target gene.Measurement data were expressed as mean±standard deviation(±s),and t-test was used for comparison between two groups.Results The expression of miR-769-3p was significantly increased in bladder cancer tissues compared with adjacent tissues,the difference was statistically significant(P<0.01).The relative expression of miR-769-3p in the si-miR-769-3p group(1.02±0.16)was significantly lower than that of the control group(4.50±0.60),the difference was statistically significant(P<0.01).The cell migration rate of the si-miR-769-3p group[(26.67±3.98)%]was significantly lower than that of the control group[(61.86±4.70)%],the difference was statistically significant(P<0.01).The proportion of cells in the G_(0)-G_(1)phase in the si-miR-769-3p group[(57.66±5.74)%]was significantly higher than that in the control group[(31.26±3.24)%],the difference was statistically significant(P<0.01).Dual-luciferase reporter gene assay confirmed that endothelin 3(EDN3)was the target gene of miR-769-3p.The relative expression of EDN3 mRNA in J82 cells in control group and si-miR-769-3p group was 1.99±0.66 and 6.98±0.76,compared with the control group,the EDN3 mRNA relative expression level of the si-miR-769-3p group was significantly higher than that of the control group,the difference was statistically significant(P<0.01).Conclusion Low expression of miR-769-3p can inhibit the migration of bladder cancer J82 cells and block the J82 cell cycle by promoting the expression of EDN3 gene.