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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 investigate the effect of RNA interference on the transcription and protein expression of hypoxia inducible factor-1α (HIF-1α) in gastric cancer cells and on the proliferation and apoptosis of gastric cancer cells. Methods The human hairpin RNA (shRNA) eukaryotic expression vector of human HIF-1α gene was constructed and transfected into BGC-823 cells. The expression of HIF-1α mRNA and protein was detected by RT-PCR and Western blot. After the inhibition of gastric cancer cell growth; TUNEL method observed cell apoptosis; flow cytometry analysis of gastric cancer cells after transfection cell cycle and apoptosis rate. Results The pshRNA-HIF-1α1 and pshRNA-HIF-1α2 recombinant plasmids were constructed successfully. After transfection with BGC-823, pshRNA-HIF-1α1 could effectively inhibit the transcription of HIF-1α gene in BGC-823 cells The protein expression and the inhibition rates were 93.4% and 55.7% respectively. The MTT assay showed that the cell growth was significantly inhibited after transfection. The results of flow cytometry showed that the transfection rate was significantly higher than that of empty plasmid group ), The proliferation activity of BGC-823 cells was significantly decreased, the proportion of cells in G0-G1 phase increased significantly, the proportion of cells in S phase and G2-M phase decreased; TUNEL method showed nuclear condensation and nuclear chromatin aggregation or fragmentation after transfection; The inhibition rate of pshRNA-HIF-1α1 was 41.75%. Conclusions Both pshRNA-HIF-1α1 and pshRNA-HIF-1α1 can effectively inhibit the gene expression of BGC-823 HIF-1α in gastric cancer cells and inhibit the proliferation and induce the apoptosis of gastric cancer cells in vitro and in vivo.