miR-1-3p抑制肝癌细胞SMMC-7721增殖、迁移并诱导其凋亡

MiR-1-3p inhibits proliferation and migration of liver cancer SMMC-7721 cells and induces their apoptosis

目的探讨在肝癌细胞SMMC-7721中微小RNA(miR)-1-3p的表达变化对其增殖、迁移和凋亡的影响及miR-1-3p的作用机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-1-3p和CAAP1的相对表达量;SMMC-7721细胞按miR-1-3p过表达载体(miR-1-3p组)、pcDNA3组、miR-1-3p inhibitor组和NC inhibitor组(对照组)分别转染,采用qRT-PCR检测转染效果,CCK-8实验检测细胞增殖能力,克隆形成实验观察并比较组间克隆形成率,流式细胞术和Western blot检测细胞凋亡及CAAP1蛋白表达,划痕和Transwell实验观察SMMC-7721迁移和侵袭。利用miRDB、TargetScan、miRanda数据库预测miR-1-3p靶基因,双荧光素酶报告验证miR-1-3和CAAP1靶向性。结果miR-1-3p在SMMC-7721细胞中的表达明显低于正常肝细胞LO2,差异有统计学意义(P<0.05);miR-1-3p上调,SMMC-7721增殖、迁移和侵袭能力显著下降,凋亡显著增强,而下调miR-1-3p的表达则作用相反;miR-1-3p和CAAP1具有靶向性;miR-1-3p过表达明显抑制CAAP1表达。结论miR-1-3p上调可能通过与CAAP1的3′-UTR结合抑制SMMC-7721细胞增殖、侵袭、迁移。

Objective To investigate the effect of miR-1-3p expression change in hepatocellular carcinoma(HCC)SMMC-7721 cells on their proliferation,migration and apoptosis and the action mechanism of miR-1-3p.Methods The relative expression levels of miR-1-3p and CAAP1 were detected by the real-time fluorescence quantitative PCR(qRT-PCR).SMMC-7721 cells were transfected according to miR-1-3p overexpression vector(miR-1-3p group),pcDNA3 group,miR-1-3p inhibitor group and NC inhibitor group(control group),respectively.The qRT-PCR assay was used to detect the transfection effect.The CCK-8 assay was used to detect the proliferation ability of cells.The clone formation rates were observed and compared among the groups by the clone formation experiment.The cell apoptosis and CAAP1 protein expression were detected by flow cytometry and Western blot.The migration and invasion of SMMC-7721 were observed by the scratches and Transwell experiment.The MiRDB,TargetScan and miRanda databases were used to predict miR-1-3p target genes,and the dual-luciferin reports were used to verify the targeting of miR-1-3 and CAAP1.Results The expression of miR-1-3p in SMMC-7721 cells was significantly lower than that of normal liver cells LO2,and the difference was statistically significant(P<0.05);when miR-1-3p was up-regulated,the proliferation,migration and invasion abilities of SMMC-7721 were significantly decreased,and the apoptosis was significantly enhanced,while the down-regulating the expression of miR-1-3p had an opposite effect.MiR-1-3p and CAAP1 were targeted.The overexpression of miR-1-3p significantly inhibited the expression of CAAP1.Conclusion The up-regulation of miR-1-3p may inhibit the proliferation,invasion and migration of SMMC-7721 cells by binding to CAAP1′s 3′-UTR.