短发夹RNA干扰胃癌低氧诱导因子-1α表达及其临床意义

RNA interference targeting hypoxia inducible factor-1α in BGC-823 cells inhibits cell proliferation and induces cell apoptosis

目的探讨RNA干扰对胃癌细胞低氧诱导因子-1α(HIF-1α)转录和蛋白表达的作用以及对胃癌细胞增殖和凋亡的影响。方法构建人HIF-1α基因的短发夹RNA(shRNA)真核表达载体并转染胃癌细胞BGC-823;采用RT-PCR和Western blot法检测HIF-1αmRNA、蛋白表达水平;MTT法检测转染后对胃癌细胞生长的抑制;TUNEL法观察细胞的凋亡情况;流式细胞术分析转染后胃癌细胞的细胞周期和凋亡率。结果成功构建了pshRNA-HIF-1α1和pshRNA-HIF-1α2重组质粒;转染BGC-823后,与空质粒组相比,pshRNA-HIF-1α1可有效抑制BGC-823细胞HIF-1α基因转录和蛋白质表达,抑制率分别可达93.4%和55.7%;MTT法显示转染后细胞生长明显受到抑制;流式细胞术结果表明转染后,其凋亡率明显高于空质粒组(P<0.05),BGC-823细胞增殖活性显著降低,G0～G1期细胞比例明显增加,S期与G2～M期细胞比例减少;TUNEL法显示转染后细胞核固缩,核染色质聚集或断裂;体内实验显示,pshRNA-HIF-1α1的抑瘤率为41.75%。结论体内、外初步实验证实pshRNA-HIF-1α1能有效抑制胃癌细胞BGC-823 HIF-1α基因表达,抑制胃癌细胞增殖,诱导其凋亡。

Objective To study the expression of hypoxia inducible factor-1α （HIF-1α） in BGC-823 cells after RNA interference and the proliferation and apoptosis of BGC-823 cells. Methods The recombinant plasmid pshRNA-HIF-1α was constructed and transfected into gastric cancer cells BGC-823 by LipofectamineTM 2000. Then the expressions of HIF-1α in BGC-823 cells were observed by RT-PCR and Western blotting, the cell proliferation was examined by MTF assay and the cell apoptosis by TUNEL and flow eytometry. The trans- fected cells were transplanted to nude mice to observe the inhibitory effect of transfection. Results The restriction enzyme digestion and sequencing result showed that the recombinant plasmid of pshRNA-HIF-1α had been constructed successfully. After it was transfeeted into BGC-823 cells, the genetic transcription and protein expression of HIF-1α were inhibited significantily （inhibitory ratio： 93.4% and 55.7% ）, compared with controis. The inhibitory ratio of cell proliferation in the pshRNA-HIF-1αtransfected ceils was higher than that of controls, and the cell ratio was increased in G0/G1 phase, and decreased in S and G2/M phases. The apoptotic ratio of the cells transfected with pshRNA-HIF-1α was also higher than that of controls. Karyopycnosis, nuclear chromosomal condensation and segmentation were observed by TUNEL. In vivo, pshRNA-HIF-1α was proved to be able to inhibit the proliferation and induce the apoptosis of BGC-823 cells by restraining the expressions of HIF-1α, whose tumor inhibition rate was 41.75%. Conclusion Our results show that the recombinant plas- mid pshRNA-HIF-1α can inhibit the genetic transcription and protein expression of HIF-1α, and suppress the proliferation and induce the apoptosis of gastric cancer cells BGC-823 in vivo and in vitro.