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目的:构建以STAT3为靶基因的短发夹状小干扰RNA表达载体,并探讨STAT3小干扰RNA表达载体对人结肠癌HCT116细胞增殖和凋亡的影响。方法:以pGPU6/GFP/STAT3质粒为载体,构建STAT3的小干扰RNA表达载体,使用脂质体法转染人结肠癌HCT116细胞系,用MTT法检测空白对照组、小干扰RNA组、阴性对照组与脂质体对照组的增殖活性,48 h后流式细胞仪分析4组细胞周期分布与凋亡的变化,并且通过RTPCR和Western blot检测结肠癌HCT116细胞STAT3基因mRNA和蛋白表达水平。结果:酶切鉴定和测序分析表明靶向STAT3的RNA干扰重组体构建成功;重组体转染HCT116后,STAT3的mRNA和蛋白水平较空载体转染组显著下降;小干扰RNA组与3对照组相比细胞增殖活性明显受到抑制(P<0.01);细胞生长明显减缓,G0/G1期细胞比例增加(P<0.01)。结论:沉默STAT3基因可有效控制结肠癌细胞的增殖,使癌细胞阻滞于G0/G1期并诱导其凋亡,可望成为结肠癌治疗的新方法。
OBJECTIVE: To construct a short hairpin RNA interference expression vector targeting STAT3 and to investigate the effect of STAT3 siRNA on the proliferation and apoptosis of human colon cancer HCT116 cells. METHODS: Small interfering RNA expression vector of STAT3 was constructed with plasmid pGPU6 / GFP / STAT3 as a vector and transfected into human colon cancer HCT116 cell line by lipofectamine. MTT assay was used to detect the expression of STAT3 in the blank control group, small interfering RNA group and negative control group The proliferation and proliferation of HCT116 cells were detected by flow cytometry. The cell cycle distribution and apoptosis were analyzed by flow cytometry 48 h later. The STAT3 mRNA and protein expression in HCT116 cells were detected by RTPCR and Western blot. Results: The restriction endonucleases and sequencing analysis showed that the RNA interference recombinant plasmids targeting STAT3 were constructed successfully. The mRNA and protein levels of STAT3 mRNA and protein were significantly decreased after transfected with HCT116. Compared with the control group (P <0.01). The cell growth slowed down significantly and the proportion of cells in G0 / G1 phase increased (P <0.01). Conclusion: Silencing STAT3 gene can effectively control the proliferation of colon cancer cells, arrest the cancer cells in G0 / G1 phase and induce their apoptosis, which is expected to become a new method for the treatment of colon cancer.