siRNA沉默STAT3基因对肝癌细胞株HepG2中STAT3及其下游相关基因表达的影响

Effect of silenced STAT3 gene with si RNA on STAT3 gene and its downstream genes in HepG2 cell line

【目的】探讨小分子干扰RNA片段(small interfering RNA,si RNA)沉默信号转导与转录活化因3(signal transducer and activator of transcription 3,STAT3)基因对肝癌细胞株Hep G2生长、凋亡及STAT3与其下游基因表达的影响。【方法】构建STAT3-si RNA,并将其稳定转染到肝癌细胞株Hep G2中,建立Hep G2/STAT3-si RNA细胞株。MTT比色法检测转染对细胞增殖的影响;RT-PCR和Western blotting分别检测STAT3基因及其下游细胞周期蛋白D1(cyclin D1)、存活蛋白(survivin)、血管内皮生长因子(vascular endothelial growth factor,VEGF)基因m RNA和蛋白表达水平;Hochest33342荧光染色法检测细胞凋亡。【结果】MTT结果显示,转染后Hep G2细胞存活率显著降低(t=4.262,P=0.001;t=25.330,P=0.000;t=50.288,P=0.000)。RT-PCR和Western blotting结果显示,转染后Hep G2细胞STAT3、cyclin D1、survivin、VEGF基因m RNA和蛋白表达水平显著降低(F=770.918,P=0.000;F=940.630,P=0.000;F=723.783,P=0.000;F=582.362,P=0.000)(F=496.363,P=0.000;F=484.556,P=0.000;F=468.469,P=0.000;F=193.845,P=0.000)。Hochest33342荧光染色结果显示,转染后细胞凋亡显著增加(F=3311.491,P=0.000)。【结论】通过RNA干扰技术沉默STAT3基因可有效下调肝癌细胞Hep G2中STAT3基因及cyclin D1、survivin、VEGF基因表达并抑制肝癌细胞存活增加细胞凋亡,STAT3-si RNA有望成为治疗原发性肝癌的新技术。

[Objective]To explore the proliferation and apoptosis in HepG2 cell line and the expression of STAT3 and its downstream genes by silencing the expression of signal transducer and activator d transcription 3（STAT3） gene with small interfering RNA（siRNA）. [ Methods ] HepG2/STAT3-siRNA cell line was constructed by transfecting HepG2 cells with STAT3-siRNA. The cell proliferation rate was detected by MTT colorimetric assay. RT-PCR and western blot were respectively used to detect the mRNA and protein expression levels of STAT3 gene and its downstream genes, such as cyclinD1, survivin and vascular endothelial growth factor（VEGF）. The apoptosis of cells was assessed by Hochest 33342 immunoflurescence staining. [ Results] The result of MTT suggested that the cell proliferation rate was significantly decreased ＂after the transfection（t=-4.262, P=0.001; t=25.330, P=-0.000; t=50.288, P=-0.000）. The resuhs of RT-PCR and western blotting demonstrated the mRNA and protein expression levels of STATS, cyclinD 1, survivin and VEGF gene in the transfected HepG2 ceils were significantly lowered（F=770.918, P=0.000; F=940.630, P=0.000; F=723.783, P=0.000; F=582.362, P=0.000）（F=496.363, P=-0.000; F=484.556, P=-0.000; F=468.469, P=-0.000; F=193.845, P=-0.000）. The results of Hochest33342 indicated that the apoptosis of transfected cells significantly increased（F=3311.491, P=0.000）. [ Conclusions ] Silencing the expression of STAT3 gene by RNA interference could effectively downregulate the expression eft STAT3, cyclinD1, survivin, and VEGF gene in HepG2 cells. The STAT3-siRNA may be a new technique in treating primary hepatic carcinoma.