摘要
目的:研究用小片段干扰RNA(small interfering RNA,siRNA)抑制肝癌细胞株中YAP(yes-associated protein)表达后肝癌细胞株对阿霉素(adriamycin,ADM)的敏感性变化。方法:设计YAP基因的siRNA序列,用脂质体LipofectamineTM2000将siRNA转染至4株肝癌细胞株PLC/PRF/5、Huh-7、MHCC97H、MHCC97L中,在荧光显微镜下观察并采用蛋白免疫印迹(Western blotting)法检测肝癌细胞株中YAP的表达。将细胞以不同浓度的ADM处理后,采用四甲基偶氮唑蓝(methyl thialzolyl tetrazolium,MTT)比色法检测细胞的存活率。结果:成功构建针对YAP的siRNA表达载体。Western blotting结果显示,肝癌细胞株经特异性siRNA转染后,YAP蛋白的表达受到抑制,表明RNA干扰(RNA interference,RNAi)能特异、有效地抑制肝癌细胞株中YAP的表达。MTT结果显示,转染YAP siRNA后肝癌细胞株的存活率受到明显抑制。结论:siRNA抑制肝癌细胞株中YAP的表达可增强肝癌细胞株对ADM的敏感性。
Objective：To investigate the inhibitory effect of small interfering RNA（siRNA）on yes-associated protein（YAP）expression and adriamycin（ADM）chemosensitivity of hepatoma cell lines.Methods：The siRNA sequences were designed based on YAP gene and were transfected into four different kinds of hepatoma cells（PLC/PRF/5,Huh-7,MHCC97 H,MHCC97L）through lipofectamineTM2000.The inhibition result of YAP expression in hepatoma cells was measured by Western blotting and fluorescence microscope.The viability rate of hepatoma cells was measured by methyl thiazolyl tetrazolium（MTT）colorimetry after treating with different concentrations of ADM.Results：The siRNA expression vector to aim directly at YAP gene was constructed successfully.Western blotting revealed that transfection with siRNA could inhibit the expression of YAP protein in hepatoma cell lines.The expression of YAP protein in hepatoma cell lines was specially and effectually inhibited by RNA interference（RNAi）.MTT colorimetry detection result revealed that the viability rate of hepatoma cell lines significantly decreased after transfection with YAP siRNA.Conclusions：Inhibiting the expression of YAP by siRNA can enhance the sensitivity of hepatoma cell lines to ADM.
出处
《中国临床医学》
2014年第6期643-646,共4页
Chinese Journal of Clinical Medicine
基金
江苏省南通市社会发展科技计划项目(编号:K2010011)
关键词
小干扰RNA
YAP
肝癌细胞
阿霉素
Small interfering RNA
Yes-associated protein
Hepatoma cells
Adriamycin
