摘要
目的:近年来胆管癌的发病率和病死率呈上升的趋势,5年生存期低于5%,因此亟需利用现代生物技术探索新的治疗方法。探讨针对肝细胞生长因子(HGF)受体c-Met的RNA干扰对人胆管癌(CCA)QBC939细胞增殖的影响。方法:构建针对人c-Met基因的小干扰RNA(siRNA),转染体外培养的人CCA QBC939细胞,应用MTT法检测细胞增殖,Western blot检测c-Met、细胞周期素(Cyclin)D1和A表达以及磷酸肌醇-3激酶/蛋白激酶B(PI3K/Akt)和胞外信号调节激酶(ERK1/2)信号通路活化。结果:c-Met siRNA显著抑制c-Met蛋白表达,且随着其浓度的增加,c-Met蛋白表达逐渐下降,500 nmol/L c-Met siRNA对c-Met蛋白表达的抑制率达65%。HGF刺激24 h,细胞增殖是对照组的4.54倍,100 nmol/L c-Met siRNA预处理即显著抑制HGF诱导的细胞增殖,其抑制率达26%,且随浓度增加,其抑制作用逐渐增强,500 nmol/L c-Met siRNA对HGF诱导的细胞增殖的抑制率达85%。HGF刺激4h,PI3K和Akt的活性即显著增强,分别是对照组的4.09和4.54倍,c-Met siRNA可呈浓度依赖性的抑制HGF诱导的PI3K/Akt活化,500 nmol/L c-Met siRNA几乎完全抑制HGF诱导的PI3K/Akt活化。HGF显著诱导人CCAQBC939细胞ERK1/2活化,50 ng/ml HGF刺激4 h,磷酸化ERK1/2表达是对照组的3.63倍,c-Met siRNA可呈浓度依赖性的抑制HGF诱导的ERK1/2活化。HGF显著诱导人CCA QBC939细胞细胞周期素D1和A表达,分别是对照组的2.83和3.16倍,c-Met siRNA可呈浓度依赖性的抑制HGF诱导的细胞周期素D1和A表达。结论:针对人c-Met基因的RNA干扰可有效阻断人CCA QBC939细胞c-Met表达,并通过阻断PI3K/Akt和ERK1/2信号通路活化而抑制HGF诱导的细胞增殖。
Objective： The incidence and mortality of cholangiocarcinoma are increasing worldwide in recent years, with a 5 - year survival of less than 5%. Therefore, it is imperative to search for new options based on modern biotechnology. This study aimed to investigate the effect of the hepatocyte growth factor （HGF） receptor c-Met small interference RNA （siRNA） on the growth of human bile duct carcino- ma QBC939 cells. Methods： We constructed c-Met siRNA and transfected it to human bile duct carcinoma QBC939 cells, followed by detection of the growth of the cells by MTr, examination of the expressions of c-Met, cyclin D1 and cyclin A, and determination of phosphorylation of PI3K/Akt and ERK1/2 by immunoblotting. Results： The c-Met siRNA significantly reduced the expression of the c-Met protein, which was gradually decreased with the increasing of the c-Met siRNA concentration, and inhibited by 65% in the presence of 500 nmoL/L c-Met siRNA. Incubation with 5 ng/ml HGF for 24 h induced cell proliferation by 4.54 times, which was significantly inhibited by 100 nmol/L c-Met siRNA. The cell growth was inhibited by 85% in the presence of 500 nmol/L c-Met siRNA. HGF induced PI3K/Akt activation by 4.09 and 4.54 times. The c-Met siRNA inhibited HGF-stimulated PI3K/Akt activation in a dose-dependent manner, and almost completely blocked it at 500 nmo]/L. HGF increased ERK1/2 phosphorylation and the expressions of cyclin D1 and cyclin A by 3.63,2.83, and 3.16 times, respectively. The c-Met siRNA inhibited HGF-stimulated ERK1/2 phosphorylation and the expressions of cyclin D1 and cyclin A in a dose-dependent manner. Conclusion： The c-Met siRNA effectively reduced c-Met protein expression in human bile duct carcinoma QBC939 cells and blocked HGF-induced cell growth by inhibiting the activation of PI3K/Akt and ERK1/2 and the expression of cyclin D1 and cyclin A.
出处
《医学研究生学报》
CAS
2009年第11期1154-1158,共5页
Journal of Medical Postgraduates
基金
南京医科大学科技发展基金重点项目资助(批准号:2005NYDZD15)
