沉默EFNA3基因表达抑制缺氧诱导的肝细胞癌细胞的迁移与侵袭  

EFNA3 gene silencing inhibits on the migration and invasion of hepatocellular carcinoma cells induce by hypoxia

作　　者：陈静[1] 余君铭 刘腾飞[1] 金文姣 苗春晓 李红[1] CHEN Jing;YU Junming;LIU Tengfei;JIN Wenjiao;MIAO Chunxiao;LI Hong(State Key Laboratory of Oncogenes and Related Genes,Shanghai Cancer Institute,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200032)

机构地区：[1]上海交通大学医学院附属仁济医院,上海市肿瘤研究所癌基因与相关基因国家重点实验室,上海200032

出　　处：《肿瘤》2020年第5期305-316,共12页Tumor

基　　金：国家自然科学基金资助项目(编号:81672832);上海市卫生计生系统优秀学科带头人培养计划(编号:2018BR20)。

摘　　要：目的 :探讨肝细胞癌中缺氧对肝配蛋白A3(ephrin A3,EFNA3)的调控作用,以及沉默EFNA3基因表达对缺氧诱导的肝细胞癌细胞迁移和侵袭的影响。方法 :肝细胞癌HCC-LY10和MHCC-97L细胞缺氧条件下培养0、12、24和48h,采用实时荧光定量PCR法检测2株细胞中EFNA3m RNA和长链非编码RNA(long non-coding RNA,Lnc RNA)EFNA3的表达水平,蛋白质印迹法检测EFNA3和缺氧诱导因子1α(hypoxiainduciblefactor-1α,HIF-1α)蛋白的表达水平。采用脂质体法将mi R-210-3p-mimic和阴性对照(NC-mimic)转入HCC-LY10和MHCC-97L细胞,实时荧光定量PCR检测mi R-210-3p的转染效率,以及mi R-210-3p过表达后常氧和缺氧条件下EFNA3 m RNA和Lnc RNA EFNA3表达水平的变化,同时采用蛋白质印迹法检测HIF-1α和EFNA3蛋白表达水平的变化。采用慢病毒感染法将靶向HIF-1α和HIF-2α基因的sh RNA慢病毒重组载体GV248-sh HIF-1α-1、GV248-sh HIF-1α-2、GV248-sh HIF-2α-1和GV248-sh HIF-2α-2及阴性对照GV248-NC转入HCC-LY10和MHCC-97L细胞,实时荧光定量PCR检测HIF-1α和HIF-2α的干扰效率,以及沉默HIF-1α和HIF-2α表达后缺氧条件下EFNA3m RNA和Lnc RNAEFNA3表达水平的变化;采用慢病毒感染法将靶向EFNA3基因的sh RNA重组慢病毒载体LVRU6GP-sh EFNA3-1和LVRU6GP-sh EFNA3-2及阴性对照LVRU6GPNC转入HCC-LY10和MHCC-97L细胞,采用蛋白质印迹法检测EFNA 3基因的干扰效率,Transwell小室法检测沉默EFNA3表达后缺氧条件下对肝细胞癌细胞HCC-LY10和MHCC-97L迁移和侵袭的影响。结果:在缺氧培养条件下,肝细胞癌HCC-LY10和MHCC-97L细胞中EFNA3 m RNA的表达水平无明显变化(P值均> 0.05),Lnc RNA EFNA3和EFNA3蛋白的表达水平均明显上调(P值均<0.01);HCCLY10和MHCC-97L细胞中稳定转染mi R-210-3pmimic后,mi R-210-3p的表达水平显著增加(P值均<0.001),mi R-210-3p对EFNA3 m RNA和Lnc RNAEFNA3的表达无明显影响(P值均> 0.05),但能够下调缺氧诱导的EFNA3蛋白表达水平(P值均<0.05);缺氧条件下Objective: To investigate the effect of hypoxia on the expression of ephrin A3(EFNA3) in hepatocellular carcinoma, and the effects of silencing EFNA 3 gene expression on migration and invasion abilities of hepatocellular carcinoma cells induced by hypoxia. Methods: The hepatocellular carcinoma HCC-LY10 and MHCC-97 L cells were cultured in hypoxic condition for 0, 12, 24 and 48 h, the expression level of EFNA3 mRNA and long non-coding RNA(LncRNA) EFNA3 were detected by real-time fluorescent quantitative PCR, the expression level of EFNA3 and hypoxia inducible factor-1α(HIF-1α) proteins were detected by Western blotting. The HCC-LY10 and MHCC-97 L cells were transfected with microRNA(miR)-210-3 pmimic and negative control(NC)-mimic by liposome, when cultured in normoxic and hypoxic condition for 48 h, the expression level of miR-210-3 p, EFNA3 mRNA and LncRNA EFNA3 were detected by real-time fluorescent quantitative PCR, the expression level of EFNA3 and HIF-1α proteins were detected by Western blotting. The HCC-LY10 and MHCC-97 L cells were infected with recombinant lentivirus vector carrying shHIF-1α and shHIF-2α targeting HIF-1α and HIF-2α genes(GV248-shHIF-1α-1, GV248-shHIF-1α-2, GV248-shHIF-2α-1 and GV248-shHIF-2α-2) and negative control plasmid(GV248-NC), when cultured in hypoxic condition for 48 h, the interference efficiency of HIF-1α and HIF-2α were detected by real-time fluorescent quantitative PCR, then the expression level of EFNA3 mRNA and LncRNA EFNA3 in HCC-LY10 and MHCC-97 L cells HIF-1α and HIF-2α silencing were detected by real-time fluorescent quantitative PCR. The HCC-LY10 and MHCC-97 L cells were infected with recombinant lentivirus vector carrying shEFNA3 targeting EFNA3 genes(LVRU6 GP-shEFNA3-1 and LVRU6 GP-shEFNA3-2) and negative control plasmid(LVRU6 GP-NC), the interference efficiency was analyzed by Western blotting. The effects of EFNA3 gene silencing on migration and invasion abilities of HCC-LY10 and MHCC-97 L cells under hypoxic condition were evaluated by Transwell ass

关 键 词：癌 肝细胞 低氧 竞争内源性RNA 细胞运动 

分 类 号：R735.7[医药卫生—肿瘤]