摘要
目的探讨Yes相关蛋白(Yes associated protein,YAP)对肝癌细胞凋亡的影响。方法 Western blotting方法检测人肝癌细胞Hu H-7、Hep G2、SNU-449和人正常肝细胞7702中YAP的表达水平。在肝癌细胞中转染YAP小干扰RNA1(YAP siRNA1组)和YAP小干扰RNA2(YAP siRNA2组),同时转染对照siRNA(siRNA-NC组),未转染组只加入转染试剂,培养48 h后,Western blotting检测细胞中YAP水平,筛选出干扰效率较高的YAP siRNA2继续研究。MTT检测细胞增殖,流式细胞术检测细胞凋亡,激光扫描共聚焦显微镜法检测活性氧(ROS)水平,Western blotting检测细胞中β-连环蛋白(β-catenin)、Wnt信号通路下游靶基因(C-myc)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)蛋白水平。结果人肝癌细胞Hu H-7、Hep G2、SNU-449中YAP表达水平均高于人正常肝细胞7702,且在Hep G2细胞中YAP的表达水平最高。YAP siRNA1、YAP siRNA2能够成功干扰肝癌细胞中YAP的表达水平,且YAP siRNA2的干扰效率最高。YAP siRNA2组细胞存活率和细胞中β-catenin、C-myc水平均低于未转染组,凋亡率和细胞中Cleaved Caspase-3、ROS水平均高于未转染组(P<0.01)。结论干扰YAP表达后能够促进肝癌细胞凋亡,抑制肝癌细胞增殖,Wnt信号通路是其可能的作用机制之一。
Objective To investigate the effects of Yes associated protein （YAP） on apoptosis of hepatocellular carcinoma cells. Methods Western blotting method was used to detect the expression of YAP in human hepatocellular carcinoma cell line HUH-7, HepG2, SNU-449 and human normal liver cell 7702. YAP small interfering siRNA1 （YAP siRNA1 group） and YAP small interfering RNA2 （YAP siRNA2 group） were transfected into hepatocellular carcinoma cells, at the same time, the control siRNA （siRNA-NC group） was transfeeted into hepatocellular carcinoma cells, the transfection group was only transfected with transfection reagent. After culturing 48 hours, YAP protein level in cells was detected by Western blotting, YAP siRNA2 with higher interference efficiency was screened out. Cell proliferation was detected by MTT. Cell apoptosis was detected by flow cytometry. ROS was detected by laser scanning confocal microsco- py. 13-catenin, C-myc and Cleaved Caspase-3 protein levels in cells were detected by Western blotting. Results The expression of YAP in human hepatoeellular carcinoma cells HUH-7, HepG2 and SNU-449 was higher than that in normal human hepatocytes 7702, the highest expression of YAP was observed in HepG2 cells. YAP siRNA1 and YAP siRNA2 could successfully interfere with the expression of YAP in hepatocellular carcinoma cells, the interference efficiency of YAP siRNA2 was the highest. The cell survival rate and β-catenin, C-myc levels in the YAP group were lower than those in the siRNA2 group, the apoptosis rate and the level of Cleaved Caspase-3, ROS in the cells were higher than those in the non-transfected group （P 〈 0.01 ）. Conclusion Interfering with the expression of YAP can promote the apoptosis of hepatoma ceils, inhibite proliferation of hepatoma cells, Wnt signaling pathway is one of the possible mecha- nisms.
作者
崔宏
杨楠木
贺涛
万百顺
韩风
CUI Hong YANG Nanmu HE Tao WAN Baishun HAN Feng(Department of Hepatobiliary and Pancreatic Surgery, the Cancer Hospital of He＇ nan, the Tumor Hospital Affiliated to Zhengzhou University, Zhengzhou 450052, Chin)
出处
《胃肠病学和肝病学杂志》
CAS
2017年第9期997-1001,共5页
Chinese Journal of Gastroenterology and Hepatology
关键词
肝癌
凋亡
增殖
Yes相关蛋白
Hepatocellular carcinoma
Apoptosis
Proliferation
Yes associated protein
