特异性ILK siRNA对膀胱癌EJ细胞EMT及移植瘤生长的影响

基础医学与临床 ›› 2016, Vol. 36 ›› Issue (2): 191-198.

特异性ILK siRNA对膀胱癌EJ细胞EMT及移植瘤生长的影响

庄翔,朱军,吕梦欣,陈俊霞

重庆医科大学

收稿日期:2015-06-25 修回日期:2015-09-30 出版日期:2016-02-05 发布日期:2016-01-21
通讯作者: 陈俊霞 E-mail:chjunxia@126.com
基金资助:
国家自然科学基金

Effect of specific integrin-linked kinase siRNA on EMT of human EJ bladder cancer cells and growth of transplanted bladder cancer

Received:2015-06-25 Revised:2015-09-30 Online:2016-02-05 Published:2016-01-21
Supported by:
National Natural Science Foundation of China

摘要/Abstract

摘要： 目的研究整合素连接激酶（ILK）特异siRNA沉默ILK基因对膀胱癌EJ细胞上皮间质转化（EMT）及移植瘤生长的影响。方法用特异性ILK siRNA表达载体pGensil-1siRNA1质粒和对照质粒pGensil-1siRNA3稳定转染EJ细胞，实验分为EJ siRNA组、EJ vector组和EJ细胞组；激光共聚焦显微镜下观察细胞骨架；Western检测蛋白p-AKT、p-GSK-3β蛋白的表达；细胞免疫荧光检测p-AKT、p-GSK-3β的表达；用免疫荧光检测整合素连接激酶和核糖核酸酶抑制因子在EJ细胞中的共定位；MTT法检测细胞增殖；流式细胞术检测细胞周期；建立裸鼠移植瘤模型，肿瘤和肺组织切片HE染色，免疫组化检测瘤组织中ILK、RI、NM23-H1和 E-Cadherin蛋白的表达；免疫荧光检测p-AKT、p-GSK-3β和CD31蛋白的表达。结果 EJ siRNA组细胞运动能力减弱；EJ siRNA组p-AKT、p-GSK-3β蛋白表达明显降低（P＜0.05）；MTT法结果提示实验组细胞增殖明显受到抑制；下调ILK通过阻滞G0/G1期抑制细胞增殖；下调ILK抑制膀胱癌EJ细胞移植瘤生长转移。结论特异性ILK siRNA可以改变EJ细胞特性，抑制EMT的发生，引起增殖侵袭能力下降。

关键词: 整合素连接激酶, siRNA, EMT, EJ细胞, 移植瘤

Abstract: Objective To investigate the effect of specific integrin-linked kinase siRNA on proliferation and metastasis of human EJ bladder cancer cells and growth of transplanted bladder cancer. Methods Specific ILK siRNA plasmid and negative control plasmid were stably transfected into bladder cancer EJ cells.Then the effect of ILK siRNA on cytoskeleton was observed under laser confocal microscope.The expression of p-AKT，p-GSK-3β was evaluated by Western Blot.And the expression of p-AKT，p-GSK-3β of transfected cells was detected by immunofluorescence staining. RI co-localized with ILK in EJ cells by immunofluorescence experiments.The EJ cells was determined for proliferative activity by MTT and the distribution of cell cycle by flow cytometry.EJ cells stably expressing ILK siRNA were injected into nude mice.Morphology of tumor and lung tissue was observed with H&E staining.The expression of ILK，RI，NM23-H1 and E-Cadherin in tumor tissue was detected by immunohistochemistry. The change of p-AKT，p-GSK-3β and CD31 of transplanted bladder cancer was determined by immunofluorescence. Results ILK siRNA can inhibit the movement of EJ cells. Compared with control,EJ siRNA cells obviously decreased the expression of p-AKT，p-GSK-3β（P＜0.05）. The specific ILK siRNA can weaken the proliferating ability of EJ cells.ILK siRNA can inhibit the growth and metastasis of transplanted bladder cancer in nude mice. Conclusion The siRNA plasmid for ILK gene can inhibit EMT and decrease proliferation and metastasis ability significantly in EJ cells.

Key words: integrin-linked kinase, siRNA , EMT, EJ cells, transplanted tumor

中图分类号:

庄翔朱军吕梦欣陈俊霞. 特异性ILK siRNA对膀胱癌EJ细胞EMT及移植瘤生长的影响[J]. 基础医学与临床, 2016, 36(2): 191-198.

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