下调lncRNA OIP5-AS1可通过miR-217/USP7轴抑制肝癌细胞EMT及侵袭迁移

Down-regulation of lncRNA OIP5-AS1 can Inhibit EMT,invasion and migration of hepatoma cells through miR-217/USP7 axis

目的探究lncRNA OIP5-AS1/miR-217/USP7分子轴对肝癌细胞EMT和侵袭迁移的调控作用。方法选取正常肝细胞HL-7702和肝癌细胞系(MHCC97H,Hep3B,HepG2和HCCLM3),实时荧光定量PCR法检测正常肝细胞和肝癌细胞系中lncRNA OIP5-AS1的表达水平,Transwell实验检测肝癌细胞在下调lncRNA OIP5-AS1的表达后其侵袭和迁移能力的变化,蛋白质印迹实验检测上皮-间质转化(epithelial-mesenchymal transition,EMT)相关蛋白E-cadherin,N-cadherin和Vimentin的表达情况。通过生物信息学方法和双荧光素酶报告基因实验预测并验证lncRNA OIP5-AS1、miR-217和USP7的靶向关系,蛋白质印迹和Transwell实验检测LncRNA OIP5-AS1/miR-217/USP7分子轴对肝癌细胞EMT和侵袭迁移能力的影响。结果lncRNA OIP5-AS1在肝癌细胞系中均明显高表达(P<0.05或P<0.01)。下调肝癌细胞中lncRNA OIP5-AS1的表达可明显抑制肝癌细胞EMT及侵袭迁移(P<0.05);lncRNA OIP5-AS1靶向结合miR-217,且USP7是miR-217的靶基因(P<0.05);进一步研究证实,IncRNA OIP5-AS1通过miR-217上调USP7的表达水平,从而促进肝癌细胞的EMT和侵袭迁移(P<0.05或P<0.01)。结论IncRNA OIP5-AS1通过靶向miR-217/USP7分子轴促进肝癌细胞的EMT和侵袭迁移。

Objective To explore the effects of the molecular axis of lncRNA OIP5-AS1/miR-217/USP7 on epithelial-mesenchymal transition(EMT), invasion, and migration of hepatocellular carcinoma(HCC) cells. Methods Normal hepatocyte HL-7702 and HCC cell lines(MHCC97 H, Hep3 B, HepG2, and HCCLM3) were selected for real-time quantitative PCR assay to detect the expression level of lncRNA OIP5-AS1, and Transwell experiment to detect the changes of invasion and migration ability of HCC cells after down-regulating the expression of lncRNA OIP5-AS1. The expression levels of E-cadherin, N-cadherin, and Vimentin proteins related to EMT, were detected by Western blotting. The targeting relationships of lncRNA OIP5-AS1, miR-217, and USP7 were predicted and verified by bioinformatics methods and double luciferase reporter gene experiments. Western blotting and Transwell experiments were used to detect the effects of the molecular axis of lncRNA OIP5-AS1/miR-217/USP7 on the EMT, invasion, and migration ability of HCC cells. Results lncRNA OIP5-AS1 was highly expressed in HCC cell lines(P<0.05 or P<0.01). Down-regulating the expression of lncRNA OIP5-AS1 in HCC cells significantly inhibited the EMT, invasion, and migration of HCC cells(P<0.05). LncRNA OIP5-AS1 targeted miR-217, and USP7 was the target gene of miR-217(P<0.05);Further experiment confirmed that lncRNA OIP5-AS1 up-regulated the expression level of USP7 via miR-217, thereby promoting EMT, invasion, and migration of HCC cells(P<0.05 or P<0.01). Conclusion lncRNA OIP5-AS1 promotes EMT, invasion, and migration of HCC cells via miR-217/USP7 molecular axis.