自噬基因WIPI2在人肝细胞癌索拉非尼耐药细胞株Huh7-SR中的表达及意义  

Expression and significance of autophagy gene WIPI2 in sorafenib-resistant human hepatocellular carcinoma cell strain Huh7-SR

作　　者：陈灿金 叶在秉 阮煜 童洪飞[1] 倪仲琳[1] 赵亚新[1] CHEN Can-jin;YE Zai-bing;RUAN Yu;TONG Hong-fei;NI Zhong-lin;ZHAO Ya-xin(Department of Surgery,The Second Affiliated Hospital of Wenzhou Medical University,Yuying Children’s Hospital,Zhejiang 325027,China)

机构地区：[1]温州医科大学附属第二医院/育英儿童医院外科,浙江温州325027

出　　处：《肝胆胰外科杂志》2020年第3期164-168,174,共6页Journal of Hepatopancreatobiliary Surgery

基　　金：浙江省公益技术应用项目(LGD19H160003);浙江省自然基金项目(LY19H190003);浙江省自然科学家基金项目(LY19H290009)。

摘　　要：目的探讨自噬基因WIPI2在人肝细胞癌索拉非尼耐药细胞株Huh7-SR中的表达及意义。方法以人肝细胞癌Huh7细胞为亲本细胞,采用药物浓度梯度法建立人肝细胞癌索拉非尼耐药细胞株Huh7-SR;用蛋白质印迹法(Western blotting)检测WIPI2蛋白在Huh7细胞与Huh7-SR细胞中的表达情况;用siRNA转染法敲低Huh7-SR细胞中的WIPI2基因表达,用平板克隆实验检测Huh7-SR细胞对索拉非尼的敏感性改变。结果 WIPI2蛋白在耐药株Huh7-SR细胞中为高表达[蛋白相对表达值为(2.18±0.23)],明显高于其在Huh7细胞中的表达水平(1.02±0.07),二者差异具有统计学意义(P<0.05);在si-WIPI2转染后的Huh7-SR细胞中WIPI2蛋白表达水平降低(0.44±0.03),而si-NC转染后的Huh7-SR细胞中WIPI2蛋白仍为高表达(1.21±0.08);在索拉非尼的作用下,Huh7-SR的si-WIPI2转染组的细胞克隆数为(60±4)个,较si-NC转染组的细胞克隆数[(102±3)个]明显减少(P<0.05)。结论自噬基因WIPI2在人肝细胞癌耐药株Huh7-SR中的高表达对其耐药形成具有重要作用,敲低自噬基因WIPI2可恢复Huh7-SR细胞对索拉非尼的敏感性,这表明WIPI2有望成为肝细胞癌治疗的新的分子靶点。Objective To explore the expression and significance of autophagy gene WIPI2 in sorafenibresistant human hepatocellular carcinoma cell strain Huh7-SR.Methods With human hepatocellular carcinoma Huh7 cells as parent cells,drug concentration-gradient method was used to establish a sorafenib-resistant human hepatocellular carcinoma cell strain Huh7-SR.Western blotting was employed to detect the expression of WIPI2 protein in Huh7 and Huh7-SR cells.The expression of WIPI2 gene in Huh7-SR cells was knockeddown by siRNA transfection,and the sensitivity of the Huh7-SR cells to sorafenib was detected by plate cloning experiments.Results WIPI2 protein was highly expressed in drug-resistant cell strain Huh7-SR cells,with the relative protein expression value of(2.18±0.23),significantly higher than that in Huh7 cells(1.02±0.07),and the difference was statistically significant(P<0.05).The expression level of WIPI2 protein was decreased in Huh7-SR cells transfected with si-WIPI2(0.44±0.03),while WIPI2 protein was still highly expressed in Huh7-SR cells transfected with si-NC(1.21±0.08).Under the effect of sorafenib on Huh7-SR cells,the number of cell clones in the si-WIPI2 transfected group was(60±4),which was significantly less than that in the si-NC transfected group(102±3,P<0.05).Conclusion High expression of autophagy gene WIPI2 in drug-resistant human hepatocellular carcinoma cell strain Huh7-SR plays an important role in drug resistance formation.Knock-down of autophagy gene WIPI2 can restore the sensitivity of Huh7-SR cells to sorafenib,which indicates that WIPI2 gene is hopefully to become a new molecular target for the treatment of hepatocellular carcinoma.

关 键 词：肝肿瘤 索拉非尼耐药性 WIPI2基因 自噬 

分 类 号：R735.7[医药卫生—肿瘤]