【摘要】 目的探讨miRNA-4429靶向结合髓样细胞白血病-1(MCL1)对食管鳞状细胞癌细胞增殖、凋亡、迁移与侵袭的影响。方法采用q RT-PCR法检测食管鳞状细胞癌细胞系和食管正常上皮细胞中miRNA-4429的相对表达量。采用脂质体转染法分别将miRNA-4429模拟物和模拟物对照转染至食管鳞状细胞癌EC9706细胞,分为实验组和对照组。应用CCK-8法、克隆形成实验、流式细胞术和Transwell小室实验等探究miRNA-4429对食管鳞状细胞癌细胞增殖、凋亡、迁移与侵袭的影响。构建MCL1的3’非编码区(3’UTR)野生型和突变型荧光素酶报告基因载体,应用荧光素酶报告基因实验验证MCL1是否为miRNA-4429的靶基因。采用Western blot法检测实验组和对照组MCL1蛋白的表达水平。结果 4株食管鳞状细胞癌细胞中miRNA-4429相对表达量均明显低于食管正常上皮细胞(均P<0.01);而与对照组比较,实验组细胞中miRNA-4429相对表达量显著上调(P<0.01)。实验组细胞OD值在72、96 h均明显低于对照组(均P<0.01);与对照组比较,实验组细胞集落形成数明显降低(P<0.01)。与对照组比较,实验组细胞凋亡率明显升高(P<0.01)。实验组细胞迁移数及侵袭数均明显低于对照组(均P<0.01)。mi RWalk、mi RDB和mi RTarBase预测结果表明,MCL1是miRNA-4429的作用靶基因。荧光素酶报告基因实验的结果表明,共转染MCL1 3’UTR-野生型荧光素酶报告载体后,与对照组比较,实验组荧光素酶相对活性降低(P<0.05);而共转染MCL1 3’UTR-突变型荧光素酶报告载体后,实验组与对照组荧光素酶相对活性比较差异无统计学意义(P>0.05)。实验组MCL1蛋白表达水平明显低于对照组(P<0.01)。结论食管鳞状细胞癌细胞株中miRNA-4429相对表达量降低,上调miRNA-4429表达可减弱食管鳞状细胞癌EC9706细胞的增殖、迁移和侵袭能力,促进细胞凋亡,其作用机制可能与靶向结合调控MCL1的表达有关。更多还原

【Abstract】 Objective To investigate the effects of miRNA-4429 on proliferation, migration and invasion in esophageal squamous cell carcinoma cells and its mechanism. Methods Quantitative real-time polymerase chain reaction(q RT-PCR) was used to detect the relative expression of miRNA-4429 in esophageal squamous cell carcinoma cell lines and human esophageal epithelial cells. Human esophageal cancer EC9706 cells were transfected with mimics NC and miRNA-4429 mimics by lipofectamine, which were divided into experimental group and control group. The proliferation, migration and invasion of EC9706 cells were detected by CCK-8, colony formation test, flow cytometry and transwell chamber assay, respectively. Luciferase reporter assay was used to confirm whether MCL1 was the target gene of miRNA-4429 by constructing the 3’UTR of MCL1 wild-type and mutant reporter vectors. The effects of miRNA-4429 on MCL1 protein levels were detected by Western blot. Results The relative expression of miRNA-4429 in esophageal squamous cell carcinoma cell lines was significantly lower than that in normal esophageal epithelial cells(P<0.01). Compared with the control group, the relative expression of miRNA-4429 in miRNA-4429 mimics-transfected EC9706 cells was significantly up-regulated(P<0.01). The proliferation of transfected EC9706 cells was significantly lower than that of the control group at 72 and 96 h after transfection(P<0.01). Compared with the control group, in transfected EC9706 cells the number of clone formation cells, the number of cell migration and cell invasion were significantly lower(P<0.01), the apoptosis rate was significantly increased(P<0.01). The prediction results of mi RWalk, Mi RDB and mi RTarBase showed that MCL1 was the target gene of miRNA-4429. The results of the luciferase reporter assay showed that the luciferase activity of EC9706 cells was significantly lower than that of the control group after co-transfection with MCL1 3’UTR-wild-type luciferase reporter vector(P<0.05), while the luciferase activity of transfected EC9706 cells did not change significantly after co-transfection with MCL1 3’UTR-mutant luciferase reporter vector(P<0.05).There was a statistically significant difference in the expression level of MCL1 protein between miRNA-4429-transfected EC9706 cells and the control cells(P<0.01). Conclusion The expression of miRNA-4429 is down-regulated in esophageal squamous cell carcinoma cell lines. Up-regulation of miRNA-4429 expression inhibits the proliferation, migration and invasion and promotes the cell apoptosis in EC9706 cells, which may be related to regulating MCL1 gene expression.更多还原