VEGF_(165) RNA干涉诱导人宫颈癌HeLa细胞凋亡的研究

Study of apoptosis of human cervical carcinoma HeLa cellsby RNA interference targeting at VEGF_(165)

目的:利用RNA干涉技术抑制肿瘤细胞VEGF165基因表达,探讨其对肿瘤细胞凋亡的诱导作用。方法:设计针对VEGF165基因的小干扰RNA,合成DNA模板,体外转录合成siRNA(small interfering RNA),采用lipofectamine2000转染人宫颈癌HeLa细胞。通过Hoechst33258染色、流式细胞术、RT-PCR检测人宫颈癌HeLa细胞体外凋亡情况。在体内实验中建立子宫颈癌荷瘤裸鼠模型,将VEGF siRNA直接注入瘤体,观察siRNA对肿瘤生长的影响,蛋白印迹试验检测VEGF siRNA在体内条件下对VEGF、bcl-2蛋白表达的影响。结果:所设计的两个siRNA均能有效诱导细胞凋亡产生凋亡小体,并使细胞周期阻滞于G0/G1期;VEGF165和bcl-2在mRNA及蛋白水平的表达也受到有效抑制,明显减少。结论:针对人VEGF165基因体外转录合成的siRNA在体内外均可诱导HeLa细胞发生凋亡。

Objective：To study the induction of apoptosis by RNA interference -mediated inhibition of VEGF165expression in cancer cells. Methods： Small interfering RNA （siRNA） targeted VEGF165was designed and synthesized which were transfected into human cervical carcinoma HeLa cells with lipofectamine2000. By Hoechst33258 staining, RT-PCR, flow cytometry the apoptosis of HeLa cells was tested in vitro. Cervical cancer in nude mice was established, and siRNA was injected directly into tumor subcutaneously. Westernblot was used to evaluate the expressions of VEGF and bcl-2 proteins. Results： The siRNA targeting human VEGF165 effectively induced human cervical carcinoma HeLa cells apoptosis and affected the distribution of cell cycle, increased cell population in phase G0/G1 and decreased in S phase; the expressions of VEGF165and bcl-2 on mRNA and protein level were significantly inhibited, while no similar effects have been found downregulated in control scramble siRNA. Conclusion： Transfection of siRNA targeting VEGF165can induce apoptosis of HeLa cells in vitro and in vivo.