shRNA沉默PLCε基因表达对肾癌细胞增殖的抑制及作用机制  

The inhibitory effect of silencing PLCε gene expression by shRNA on the proliferation of renal carcinoma cells and its action mechanism

作　　者：谢建红[1] 罗春丽[1] 郭永灿[1] 冀慧莹[1] 吕纯芳[1] 荀春华[1] 

机构地区：[1]重庆医科大学检验系,临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《肿瘤》2008年第11期916-920,共5页Tumor

摘　　要：目的:探讨经短发夹RNA(short hairpin RNA,shRNA)干扰技术沉默磷脂酶Cε(phospholipase C epsilon,PLCε)基因表达后对肾癌786-0细胞增殖的抑制及其作用机制。方法:脂质体介导重组质粒(pGenesil-PLCε)和空质粒(pGenesil-NP)转染786-0细胞48 h后,RT-PCR检测PLCεmRNA的表达;MTT法检测转染24、48和72 h后细胞增殖的抑制率;转染48 h后采用FCM方法分析细胞周期;RT-PCR和免疫细胞化学法分析转染48 h后p27和Ki67的表达情况。结果:转染重组质粒后可明显抑制PLCεmRNA的表达,其抑制率为69.7%;转染24、48和72 h后细胞增殖抑制率分别为21.2%,31.6%和32.7%;FCM结果显示转染重组质粒后细胞周期的分布发生了改变,G0/G1期细胞增多,S期和G2/M期细胞减少,细胞阻滞于G0/G1期,并出现了亚二倍体"凋亡峰"。RT-PCR和免疫细胞化学结果均表明p27表达上调,而Ki67表达下调。结论:RNA干扰技术阻断PLCε基因表达后可抑制肾癌786-0细胞增殖,其作用机制可能是诱导p27表达上调和Ki67表达下调。Objective： To investigate the inhibitory effect of short hairpin RNA （shRNA）-mediated silence of phospholipase C epsilon （PLCε） gene expression on the proliferation of renal carcinoma 786-0 cells and its action mechanism, nethods：Liposome was employed to mediate the transfection of both the recombinant plasmid （pGenesil-PLCε） and the control plasmid（pGenesil-NP）into the 786-0 cells. RT-PCR was used to detect the CPLCε mRNA expression after being transfected for 48 h. MTT assay was conducted to detect the proliferation inhibitory rate at 24, 48, and 72 h after transfection, respectively. Flow cytometry （FCM） was performed to analyze cell cycle after 48-h transfection. RT-PCR and immunocytochemistry were used to analyze the expression levels of both p27 and Ki67. Results： The expression of PLCε mRNA was significantly inhibited by recombinant plasmid transfection with the inhibitory rate of 69.7%. The proliferation inhibitory rates were 21.2% , 31.6% , and 32.7% after being transfected for 24, 48, and 72 h, respectively. FCM analysis demonstrated that the distribution of cell cycle changed. The number of cells in the G0/G1 phase increased, and that in both the S phase and G2/M phase decreased. Cells were arrested at the G0/G1 phase, and the subdiploid ＂apoptotic peak＂ appeared at the same time. RT-PCR and immunocytochemistry indicated that the expression of p27 was up-regulated and that of Ki67 was downregulated. Conclusion：The proliferation of the renal carcinoma 786-0 cell line is inhibited by interference of PLCε gene expression, and the underlying mechanism may partially be related with up-regulation of P27 and down-regulation of Ki67.

关 键 词：癌 肾细胞 基因沉默 磷脂酶C 细胞增殖 

分 类 号：R737.11[医药卫生—肿瘤]