miR-21-3p通过下调CCT4抑制食管鳞癌细胞的迁移及侵袭

miR-21-3p inhibits migration and invasion of esophageal squamous cell carcinoma cells through down-regulation of CCT4

目的:探讨miR-21-3p对食管鳞癌细胞迁移和侵袭的影响及其作用机制。方法:通过数据库检索miR-21-3p的下游靶基因,GEPIA2数据库预测CCT4在食管鳞癌中的表达和对食管鳞癌患者预后的影响,培养食管鳞癌细胞TE-1、KYSE150,将miR-21-3p inhibitor、SiCCT4、Plenti-CMV-CCT4-GFP/Puro转染至TE-1和KYSE150细胞中,分为miR-21-3p inhibitor组和inhibitor NC组、SiCCT4组和SiNC组、Plenti-CMV-CCT4组和inhibitor+Plenti-CMV-CCT4组,采用qRT-PCR检测各组细胞中miR-21-3p和CCT4 mRNA的表达水平,Transwell迁移侵袭实验检测各组细胞的迁移及侵袭能力,Western blot检测转染后各组细胞CCT4蛋白的表达水平。结果:数据库预测结果显示CCT4在食管鳞癌中过表达,与食管鳞癌患者总体生存期相关,与miR-21-3p呈正相关。qRT-PCR显示miR-21-3p和CCT4在食管癌细胞相较于正常上皮细胞表达显著升高,且在抑制miR-21-3p后CCT4表达水平显著降低(P<0.05)。Transwell实验显示,inhibitor组迁移率及侵袭率显著低于inhibitor NC组(P<0.05);SiCCT4组迁移率及侵袭率显著低于SiNC组(P<0.05);inhibitor+Plenti-CMV-CCT4组迁移率及侵袭率显著高于inhibitor组(P<0.05);Western blot检测CCT4蛋白发现,inhibitor组蛋白低于inhibitor NC组(P<0.05),inhibitor+Plenti-CMV-CCT4组蛋白显著高于inhibitor组(P<0.05)。结论:miR-21-3p和CCT4在食管癌中呈现过表达,miR-21-3p通过下调CCT4表达抑制食管癌细胞迁移和侵袭能力。

Objective:To explore the effects of miR-21-3p on migration and invasion of esophageal squamous cell carcinoma cells and its mechanism of action.Methods:Database searched downstream target genes of miR-21-3p and GEPIA2 database predicted the CCT4 expression in esophageal squamous cell carcinoma and prognostic impact on patients with esophageal squamous cell carcinoma.Esophageal squamous cell carcinoma cells TE-1 and KYSE150 were cultured,and miR-21-3p inhibitor,SiCCT4,and Plenti-CMV-CCT4-GFP/Puro were transfected into TE-1 and KYSE150 cells,which were divided into miR-21-3p inhibitor group and inhibitor NC group,SiCCT4 group and SiNC group,Plenti-CMV-CCT4 group and inhibitor+Plenti-CMV-CCT4 group.qRT-PCR was used to detect the expression levels of miR-21-3p and CCT4 mRNA in the cells of each group,and Transwell migration and invasion assay was used to detect the migration and invasion ability of the cells of each group.Western blot was used to detect the expression level of CCT4 protein in each group of cells after transfection.Results:Database prediction results showed that CCT4 was overexpressed in esophageal squamous cell carcinoma,correlated with overall survival of patients with esophageal squamous cell carcinoma,and positively correlated with miR-21-3p.qRT-PCR showed that the expression of miR-21-3p and CCT4 was significantly elevated in esophageal cancer cells compared with normal epithelial cells,and the expression level of CCT4 was significantly reduced after inhibiting miR-21-3p(P<0.05).Transwell assay showed that the migration and invasion rates of inhibitor group were significantly lower than those of inhibitor NC group(P<0.05).The migration and invasion rates of SiCCT4 group were significantly lower than those of SiNC group(P<0.05).The migration and invasion rates of inhibitor+Plenti-CMV-CCT4 group were significantly higher than those of inhibitor group(P<0.05).Western blot detection of CCT4 protein revealed that the protein of inhibitor group was significantly lower than that of inhibitor NC group(P<0.05),and the protein of inhibitor+Plenti-CMV-CCT4 group was significantly higher than that of inhibitor group(P<0.05).Conclusion:miR-21-3p and CCT4 showed overexpression in esophageal cancer.miR-21-3p inhibited the migration and invasion ability of esophageal cancer cells by down-regulating CCT4 expression.