磷脂酰肌醇蛋白多糖-3对Hippo信号通路的调控机制及其对肝癌Huh7细胞生物学行为的影响  

Regulation Mechanism of Glypican-3 on Hippo Signaling Pathway and Its Effects on Biological Behavior of Hepatocellular Carcinoma Huh7 Cells

作　　者：杨民[1] 李媛[2] 雷长江[2] 李磊[2] 冯加锐[2] 龚小军[2] 杨锐[2] 

机构地区：[1]江汉大学第二附属医院(武汉市第五医院)二分院综合科,湖北武汉430050 [2]江汉大学第二附属医院(武汉市第五医院)普外科,湖北武汉430050

出　　处：《中国普外基础与临床杂志》2016年第11期1315-1320,共6页Chinese Journal of Bases and Clinics In General Surgery

基　　金：武汉市科技局应用基础研究项目(编号:2015061701011630)~~

摘　　要：目的探讨磷脂酰肌醇蛋白多糖-3(GPC3)对Hippo信号通路的调控机制及其对人肝癌Huh7细胞生物学行为的影响。方法分别构建4种靶向GPC3和YAP1基因的sh RNA序列,然后转染入肝癌Huh7细胞,并筛选稳定表达的细胞株。采用荧光定量PCR法和蛋白印迹法检测转染后的Huh7细胞中GPC3和YAP1的表达,以筛选有效沉默GPC3和YAP1的sh RNA。观察沉默GPC3和YAP1以及人重组YAP1(rh YAP1)对肝癌细胞增殖、侵袭和凋亡的影响。GPC3 sh RNA转染实验分为6组:未转染组、空载体组、GPC3-714-sh RNA组、GPC3-647-sh RNA组、GPC3-1718-sh RNA组、GPC3-2134-sh RNA组。YAP1 sh RNA转染实验分为6组:未转染组、空载体组、YAP1-906-sh RNA组、YAP1-1363-sh RNA组、YAP1-1666-sh RNA组、YAP1-2895-sh RNA组。GPC3对YAP1的调控实验分为5组:未转染组、空载体组、GPC3-1718-sh RNA组、GPC3-1718-sh RNA+rh YAP1组、YAP1-1666-sh RNA组。结果 1成功构建了可有效沉默GPC3和YAP1表达的GPC3-1718-sh RNA和YAP1-1666-sh RNA质粒。2 GPC3sh RNA各转染组细胞中GPC3 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05),而GPC3-1718-sh RNA组又明显低于其他转染组(P<0.05)。YAP1 sh RNA各转染组细胞中YAP1 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05),而YAP1-1666-sh RNA组又明显低于其他转染组(P<0.05)。3 GPC3-1718-sh RNA组和YAP1-1666-sh RNA组细胞中YAP1 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05);而GPC3-1718-sh RNA+rh YAP1组细胞中YAP1 m RNA和蛋白表达明显高于GPC3-1718-sh RNA组(P<0.05)和YAP1-1666-sh RNA组(P<0.05)。4与未转染组和空载体组比较,GPC3-1718-sh RNA组和YAP1-1666-sh RNA组的细胞增殖和侵袭能力明显降低(P<0.05),细胞凋亡明显增加(P<0.05);而GPC3-1718-sh RNA+rh YAP1组细胞增殖、侵袭和凋亡均得到明显改善(P<0.05)。结论 GPC3很有可能通过对Hippo信号通路YAP1的调控,实现其对人肝癌细胞Huh7生物学行为的Objective To investigate regulation mechanism of glypican-3 （GPC3） on Hippo signaling pathway and its effects on biological behavior of hepatocellular carcinoma Huh7 cells. Methods Short hairpin RNAs （shRNA） targeting GPC3 and Yes-associated protein 1 （YAP1） genes were constructed. All of the shRNAs were transfected into Huh7 cells by liposome transfection in order to screen out the stable expression cell lines. The expressions of GPC3 and YAP1 in Huh7 cells were detected by PCR and Western blot in order to screen out the effective shRNAs. The proliferation, invasion, and apoptosis of Huh7 cells were detected by EdU cell proliferation assay, Transwell, and flow cytometry. GPC3 shRNA transfection experiments were divided into 6 groups： non-transfection group, empty vector group, GPC3-714-shRNA group, GPC3-647-shRNA group, GPC3-1718-shRNA group, and GPC3-2134-shRNA group. YAP1 shRNA transfection experiments were divided into 6 groups： non-transfection group, empty vector group, YAP1-906- shRNA group, YAP 1-1363 -shRNA group, YAP 1-1666-shRNA group, and YAP 1-2895 - shRNA group. GPC3 regulation experiments were divided into 5 groups： non-transfection group, empty vector group, GPC3-1718-shRNA group, GPC3- 1718-shRNA＋ rhYAP1 group, and YAPl-1666-shRNA group. Results （1） GPC3-1718-shRNA and YAPl-1666-shRNA plasmids were successfully constructed to silence the expressions of GPC3 and YAP 1. （2） The expressions of GPC3 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group （P〈0.05） and the empty vector group （P〈0.05）, while which in the GPC3-1718-shRNA group was significantly lower than those in all the other transfection groups （P〈0.05）. The expressions of YAP1 mRNA and protein in each transfection group were significantly lower than those in the non-transfection group and empty vector group （P〈0.05）, while which in the YAP1- 1666-shRNA group was significantly lower than those in all the other transfection groups

关 键 词：磷脂酰肌醇蛋白多糖-3 Hippo信号通路 肝癌细胞 

分 类 号：R735.7[医药卫生—肿瘤]