PTEN慢病毒过表达肝癌稳转株的构建及对Huh-7肿瘤活性的影响

Construction of PTEN lentiviral stablization of cell line and its effect on tumor activity of Huh-7

目的构建过表达PTEN的人肝癌稳转细胞株,观察PTEN对肝癌细胞生物学行为的影响并探讨潜在机制。方法将编码PTEN的基因编码片段克隆到慢病毒载体pLV-puro-GFP,利用293T细胞得到慢病毒原液感染肝癌细胞Huh-7,嘌呤霉素筛选获得稳定过表达PTEN的Huh-7细胞。用RT-PCR、免疫荧光和Western blot法检测细胞内基因和蛋白的表达;Transwell检测细胞迁移活性;探讨AKT抑制剂MK2206对稳转株的药理作用。结果成功构建了PTEN过表达的肝癌稳转细胞株(Huh-7/hPTEN)。与对照株Huh-7/EGFP相比,Huh-7/hPTEN细胞PTEN的蛋白和mRNA表达升高(P<0.01),P53蛋白表达升高,Slug和AR的mRNA表达水平下降(P<0.01),HNF4α的mRNA表达升高(P<0.01),耐药蛋白ABCB1和ABCC2的表达下降,E-cadherin蛋白和mRNA表达升高(P<0.01),Vimentin的蛋白和mRNA表达下降(P<0.01),细胞迁移性能减少;Huh-7/hPTEN细胞对AKT抑制剂MK2206的利用率升高,并影响相关蛋白的表达。结论成功构建PTEN过表达肝癌稳转株,PTEN过表达可改善肝癌Huh-7细胞肿瘤生物学效应,致癌蛋白表达下降,迁移减弱。

Objective To establish stable human hepatocellular carcinoma cell lines overexpressing PTEN,investigate the effect of PTEN on the biological behavior of hepatocellular carcinoma cells,and explore probable mechanisms.Methods The PTEN gene was cloned into the lentiviral vector pLV-puro-GFP,and the lentiviral stock was obtained from 293T cells to infect liver cancer cells Huh-7.Puromycin was used to determine the stability of Huh-7 cells overexpressing PTEN.RT-PCR,immunofluorescence,and Western blotting were employed to detect gene and protein expression.Transwell was utilized to assess cell migration activity.MK2206 was designed to evaluate pharmacological action.Results A stable liver cancer cell(Huh-7/hPTEN)overexpressing PTEN and a control group(Huh-7/EGFP)were successfully generated.Huh-7/hPTEN cells showed higher PTEN protein and mRNA expression(P<0.01),higher P53 protein expression,lower Slug and AR mRNA expression levels(P<0.01),and higher HNF4ɑmRNA levels(P<0.01).Drug-resistant proteins ABCB1 and ABCC2 mRNA and protein expression decreased,whereas E-cadherin protein and mRNA expression increased(P<0.01)and Vimentin protein and mRNA expression decreased(P<0.01).Cell migration performance decreased.Huh-7/hPTEN increased the utilization rate of AKT inhibitor MK2206 in cells while weakening the expression of related proteins.Conclusion PTEN overexpression can alter the tumor biological effects of Huh-7 liver cancer cells,reduce the expression of oncogenic proteins,and decrease migration.