短发夹RNA沉默YAP表达对肝癌细胞SMMC-7721增殖和凋亡的影响

目的本实验通过短发夹RNA(shRNA)抑制Yes相关蛋白(YAP)基因的表达,并观察其对肝癌细胞SMMC-7721的增殖、凋亡和周期影响。方法以人YAP mRNA编码区中的4条序列作为RNA干扰靶点,分别构建4个真核表达载体质粒YAP-shRNA-1、YAP-shRNA-2、YAP-shRNA-3、YAP-shRNA-4,用阳离子聚合物转染法瞬时转染,筛选出2个干扰效果较好的质粒,将它们瞬时转染肝癌细胞SMMC-7721,应用实时荧光定量聚合酶链反应(PCR)和蛋白印迹法检测SMMC-7721细胞中YAP在mRNA和蛋白水平表达的变化。噻唑蓝(MTT)法检测转染后细胞增殖能力,流式细胞术检测细胞凋亡和周期的变化。结果转染后293T细胞中shRNA-YAP-2组与shRNA-YAP-4组YAP mRNA表达明显降低,在SMMC-7721中shRNA-YAP-2组与shRNAYAP-4组的YAP mRNA及蛋白表达水平分别为(0.871±0.081,0.106±0.013)和(1.010±0.097,0.192±0.013),与未转染组(2.399±0.148,0.372±0.007)比较差异有统计学意义(P<0.01)。MTT显示,shRNA-YAP-2、shRNA-YAP-4组细胞生长明显抑制,但未出现阻滞;流式细胞仪分析显示,shRNA-YAP-2组和shRNA-YAP-4组细胞凋亡率分别为(12.0±0.81)%和(5.03±1.01)%,明显高于空质粒组的(2.89±0.08)%(P<0.01)。细胞被阻滞在G2期,G0/G1期出现DNA亚二倍体峰。结论运用shRNA干扰技术,可以有效地干扰肝癌SMMC-7721细胞YAP的表达并能降低YAP的生成,抑制SMMC-7721细胞的增殖和促进细胞的凋亡。

Effects of shRNA on human hepatocelinlar carcinoma cell line SMMC-7721 apoptosis and proliferation via Yes-associated protein expression silencing

Objective To investigate the effects of shRNA suppression-induced Yes-associated protein （YAP） expression on proliferation, apoptosis and cell cycle of human SMMC-7721 cell line. Methods The eukaryotic ex- pression vectors, YAP-shRNA-1, YAP-shRNA-2, YAP-shRNA-3 and YAP-shRNA-4 were constructed based on four sequences in human YAP mRNA coding region as the targets for shRNA interference. Cationic polymer transfection technique that entailed screening of two ideal plasmids with competitive interference was employed to transiently transfect into the hepatocellular carcinoma cell line SMMC-7721. Real-time fluorescent polymerase chain reaction and Western blotting were employed to assay the changes in YAP mRNA and protein expression in SMMC-7721 ceils. The cell proliferation following transfection was detected by methyl thiazoly tetrazolium （MTr） technique, and the cell apoptosis and cycle were measured by using flow cytometry （FCM）. Results Transfection of 293T cells resulted in at- tenuated shRNA-YAP-2 and shRNA-YAP-4 expression. In SMMC-7721 cells, the expression of YAP mRNA and pro- tein were, compared with non-transfection group （2.399±0.148） and （0.372±0.007）], significantly lower （all P〈0.01） in shRNA-YAP-2 group （0.871 ±0.081） and （0.106±0.013）] and shRNA-YAP-4 group （1.010±0.097） and （0.192±0.013）]. MTT assay showed marked cell suppression, but not retardation of growth, in groups shRNA YAP-2 and shRNA YAP-4. FCM evidenced substantially higher （P〈0.01） apoptotic ratio in groups shRNA-YAP-2 and shRNA- YAP-4 （12.01±0.81）% and （5.03±1.01）%] compared with blank plasmid group （2.89±0.08）%]. The ceils were re- tarded at phase G2, with a culmination of DNA hypodiploid at phase G0/G1. Conclusion The shRNA techniques ef- fectively attenuate YAP expression leading to reduced level of YAP synthesis, suppress proliferation and promote apoptosis in hepatocellular carcinoma SMMC-7721 cell line.