钙网蛋白基因沉默对人肝癌细胞的增殖及侵袭能力的影响  

Effect of calreticulin gene silencing on proliferation and invaison in human hepatocellular carcinoma cells

作　　者：叶健文[1] 周闯[1] 阎冰[1] 杨佳[2] 傅哲[1] 唐文超[1] 翟文龙[1] 

机构地区：[1]郑州大学第一附属医院肝胆胰外科河南省消化器官移植重点实验室,450052 [2]郑州大学第一附属医院肿瘤科,450052

出　　处：《中华肝胆外科杂志》2015年第6期405-409,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金（U1204815）;河南省高校科技创新人才支持计划（14HASTIT030）

摘　　要：目的 探讨钙网蛋白（CRT）对人肝癌细胞株SMMC-7721、HepG2增殖及侵袭能力的影响.方法 利用siRNA沉默肝癌细胞株SMMC-7721、HepG2 CRT的表达.通过免疫荧光技术及蛋白印迹法检测CRT siRNA转染效果;Cell counting kit-8（CCK-8）检测细胞的增殖;采用流式细胞术检测细胞凋亡率变化;运用Transwell侵袭小室检测细胞侵袭能力;蛋白印迹法检测p-Akt、Akt蛋白表达.结果 siRNA实验组SMMC-7721和HepG2细胞24、36、48 h相对生长抑制率分别为（41.0±2.2）％、（46.5±1.6）％、（59.7±2.2）％和（36.8±2.7）％、（47.3±1.8）％、（61.5±3.2）％.与空白组及阴性对照组比较,siRNA实验组增殖能力明显降低（P＜0.05）.沉默CRT 36 h后SMMC-7721、HepG2细胞的凋亡率分别（45.2±9.1）％、（48.9±8.0）％.与空白组及阴性对照组比较,siRNA实验组凋亡率上调（P＜0.05）.Transwell实验显示：沉默CRT 36 h后SMMC-7721和HepG2细胞穿膜个数空白组、阴性对照组、siRNA实验组分别为：（96.8±7.3）、（95.6±5.4）、（34.0±4.2）和（124.0±9.9）、（121.6 ±7.0）、（70.4 ±9.5）.与空白组及阴性对照组比较,实验组侵袭能力降低（P＜0.05）.沉默CRT 36 h后,与空白组及阴性对照组比较,p-Akt蛋白表达水平均下调（P＜0.05）.结论 沉默肝癌细胞CRT对肝癌细胞的增殖、侵袭能力有明显的抑制作用,同时可诱导细胞的凋亡,且与PI3K/Akt信号通路相关.Objective To explore the effect of calreticulin （CRT） on cell proliferation and invasion of human hepatocellular carcinoma cell line SMMC-7721 and HepG2.Methods SMMC-7721 and HepG2 cells were transfected with small interfering RNA （siRNA）.The transfection rate was detected by immunoflurescence and western blot.The cell proliferation,invasion and apoptosis of SMMC-7721 and HepG2 cells were determined by using cell counting kit-8 （CCK-8） assays,transwell assays and flow cytometry,respectively.The p-Akt and Akt levels were detected by western blot.Results The growth inhibition rate in the siRNA experimental group of SMMC-7721 and HepG2 cells for 24,36 and 48 h were （41.0 ±2.2） ％,（46.5 ±1.6）％,（59.7 ±2.2）％ and （36.8 ±2.7）％,（47.3 ± 1.8）％,（61.5 ±3.2）％,respectively.The apoptosis rate after down-regulating the expression of CRT in SMMC-7721 and HepG2 cells for 36h were （45.2 ± 9.1） ％ and （48.9 ± 8.0） ％,respectively.Compared with the blank group and the negative control group,the growth inhibition rate in the siRNA experimental group was lower （P ＜0.05）,but the apoptosis rate was significantly higher （P ＜ 0.05）.Transwell experiments confirmed that the numbers of invaded SMMC-7721 and HepG2 cells in the blank group and the negative control group and siRNA experimental group were （96.8±7.3）,（95.6±5.4）,（34.0±4.2） and （124.0 ±9.9）,（121.6 ±7.0）,（70.4±9.5）,respectively,indicating that cell invasion in the siRNA experimental group was significantly suppressed （P ＜ 0.05）.The expression of p-Akt was decreased （P ＜ 0.05） after down-regulating the expression of CRT for 36h.Conclusion CRT gene silencing by siRNA can inhibit the SMMC-7721 and HepG2 cell proliferation and invasion,but increase the cell apoptosis by regulating PI3K/Akt signal pathway.

关 键 词：钙网蛋白 肝癌细胞 小干扰RNA p-Akt蛋白 

分 类 号：R735.7[医药卫生—肿瘤]