反义抑制miRNA-155对皮肤鳞状细胞癌A431细胞增殖、凋亡及侵袭力的影响

摘要

目的 观察转染反义微小核糖核酸(miRNA)-155对人皮肤鳞状细胞癌A431细胞增殖、凋亡、迁移和侵袭力的影响.方法 采用脂质体介导对皮肤鳞状细胞癌A431细胞转染miRNA-155无义序列(阴性对照组)、转染反义miRNA-155(转染组),空白对照组细胞不作处理.实时定量聚合酶链式反应(qRT-PCR)检测转染后A431细胞miRNA-155的相对表达水平;采用四甲基偶氨唑盐(MTT)检测转染后皮肤鳞状细胞癌A431细胞增殖活性;采用流式细胞术(FCM)检测转染后皮肤鳞状细胞癌A431细胞周期改变和凋亡变化;采用Transwell法检测侵袭力与细胞迁移能力.结果 转染组A431细胞中miRNA-155 mRNA的相对表达水平低于空白对照组和阴性对照组(F=634.57,P<0.01),但空白对照组和阴性对照组比较差异无统计学意义.转染72 h后,转染组较空白对照组和阴性对照组细胞存活率明显降低,转染120 h降低最为明显(P<0.05).转染组G0/G1期细胞增多,S期细胞减少,整体细胞增殖指数降低,A431细胞凋亡率、细胞迁移和侵袭力升高(P<0.05),但各组G2/M期细胞周期变化、后2组A431细胞凋亡率、细胞迁移和侵袭力差异无统计学意义.结论 皮肤鳞状细胞癌A431细胞转染反义miRNA-155可减少miRNA-155的表达,有效抑制A431细胞增殖,促进其凋亡.miRNA-155可能成为治疗皮肤鳞状细胞癌基因表达调控的新靶点.%Objective To investigate the effects of antisense oligonucleotide against miRNA-155 (AS-miRNA-155) on proliferation,apoptosis and invasion and migration abilities of human cutaneous squamous cell carcinoma cell line A431. Methods AS-miRNA-155 was transfected into human cutaneous squamous cell carcinoma A431 cells by using LipofectamineTM 2000. Blank control without transfection and transfected with non-sense sequence were used as non-sense sequence control. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miRNA-155 in A431 cells. Cell proliferation was analyzed using dimethyl thiazolyldiphenyl tetrazolium (MTT) assay. Cell cycle arrest and apoptosis were studied by flow cytometry (FCM). Invasion and migration were measured by Transwell chamber assays. Results The relative expression of miRNA-155 mRNA was lower in the transfection group than that in the blank control group and the negative control group (F=634.57, P<0.01), but there was no significant difference between the blank control group and the negative control group. After 72 h transfection, the survival rate was significantly lower in the transfection group than that of the blank control group and the negative control group, and the transfection rate decreased significantly by 120 h (P<0.05). Cells of G0/G1 phase increased, Cells of S phase reduced, the overall PI value decreased in transfection group, and the apoptosis rate of A431 cells, migration and invasion of cells increased (P<0.05). There was no significant difference in G2/M cycle between transfection group, blank control group and negative control group. There were no significant differences in A431 cell apoptosis rate, cell migration and invasive ability between blank control group and negative control group. Conclusion Antisense oligonucleotide against miRNA-155 can inhibit the expression of miRNA-155, the proliferation and promote the apoptosis of human cutaneous squamous cell carcinoma A431 cells, which indicates that miRNA-155 may become a new target for the regulation of gene expression in cutaneous squamous cell carcinoma.