摘要
目的:探讨RNA干扰技术沉默STAT3基因表达对人肝癌细胞的抑制作用及对相关生长调控基因的调节.方法:构建pSilencer3.0-H1-siRNA-STAT3重组质粒,转染人肝癌细胞株SMMC 7721,采用MTT法观察重组质粒对肝癌细胞的生长抑制,RT-PCR和Western blot及免疫组化法分别观察STAT3基因和蛋白水平的变化,同时检测survivin,c-myc,VEGF,p53,caspase3生长调控基因的mRNA,并用流式细胞技术(FCM)及AO/EB染色方法观察细胞凋亡.结果:pSilencer 3.0-H1-siRNA-STAT3重组质粒对肝癌细胞的生长有抑制作用,实验组48h和72h抑制率分别为59.32%,76.49%,与空白组及阴性组细胞相比有显著性差异(P<0.01);在重组质粒组,STAT3基因mRNA及蛋白水平表达降低,surviving,VEGF的mRNA表达下调,p53,caspase3的mRNA表达上调(P<0.01),c-myc的mRNA表达却无明显改变;重组质粒可诱导SMMC 7721细胞凋亡,凋亡率达21.6%(P<0.01),细胞周期分析显示细胞阻滞于G2期.结论:pSilencer3.0-H1-STAT3-siRNA可能通过下调基因survivin和VEGF mRNA表达,上调p53和caspase3 mRNA表达来抑制STAT3基因在人肝癌细胞中的表达.
AIM： To explore the effect of silencing STAT3 expression by siRNA on the growth of human hepatocellular carcinoma cells and the regulation of genes related to growth control.
METHODS： We reconstructed the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP, which was then transfected into SMMC 7721 cells. MTT assay was applied to investigate the proliferation of transfected SMMC 7721 cells, and the protein expression level of STAT3 was determined by Western blotting and immunohistochemical staining. The transcription of the STAT3 gene was detected by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）, mRNA expression for growth-controlrelated genes such as survivin, c-myc, VEGF, p53 and caspase3 was detected in transfected cells at the same time. Flow cytometry （FCM） and AO/ EB double staining were used to observe apoptosis in transfected cells.
RESULTS： MTT assay demonstrated that cell growth was inhibited in transfected cells, and the 48-hour and 72-hour inhibition rates of the test group were 59.32 and 76.49%, respectively. There were significant differences compared to those of the mock-treated and negative groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in transfected cells. Genes related to growth control also changed at the mRNA level, and expression of survivin and VEGF in transfected cells was obviously reduced. The expression of p53 and caspase3 in transfected cells increased. The ratios of the above expression products in the test group were significantly different compared to those of the negative and mock-treated groups, while the expression of c-myc in transfected cells did not change. The results of FCM and AO/EB double staining indicated that there was apparent cell apoptosis in the test group; the apoptosis rate was 21.6% （P 〈 0.01）. There were significant dif- ferences compared to those of the mock-treated and negative groups. Analysis of the cell cycle showed that cells were inhibited in the G2 stage.
CONCLUSION： The recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP can significantly inhibit the expression of the STAT3 gene in SMMC 7721 cells. The mechanism may be related to the down-regulation of survivin and VEGF mRNA expression and the up-regulation of p53 and caspase3 mRNA expression.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第19期2101-2107,共7页
World Chinese Journal of Digestology
