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目的观察RNA干扰(RNAi)技术能否有效抑制大肠癌LoVo细胞株表皮生长因子受体(EGFR)的表达以及对细胞增殖的影响。方法构建质粒表达载体pU6-EGFR-shRNA-1和pU6-EGFR-shRNA-2；脂质体瞬时转染后24、48、72、96、144 h实时荧光定量PCR(Real Time PCR)和Western blot检测EGFR表达,流式细胞仪检测细胞周期和凋亡,并绘制细胞生长曲线；分5组：A组：LoVo细胞组；B组：Lipofectamine2000组；C组：对照载体HK组；D组：pU6-EGFR-shRNA-1组；E组：pU6-EGFR-shRNA-2组。结果D组E组细胞转染shRNA后mRNA和蛋白表达降低,G_0～G_1期细胞增加,S期细胞减少,细胞凋亡率增加,对数生长期延迟,以48 h效果最显著,和A组B组C组比较差异有统计学意义(P＜0．05)；但随着时间的推移其干扰作用降低。结论两条EGFR-shRNA均可有效抑制大肠癌LoVo细胞EGFR表达,阻滞更多的细胞位于G_0～G_1期,促进细胞凋亡而抑制细胞增殖。
Objective To observe whether RNA interference (RNAi) can effectively inhibit the expression of epidermal growth factor receptor (EGFR) in colorectal cancer LoVo cell line and its effect on cell proliferation. Methods Plasmid expression vectors pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were constructed. Real-time PCR and Western blot were performed 24, 48, 72, 96 and 144 h after transfection. Cell cycle and apoptosis were detected by flow cytometry and cell growth curve was drawn. The cells were divided into 5 groups: group A: LoVo cell group; group B: Lipofectamine2000 group; group C: control vector HK group; group D: pU6 -EGFR-shRNA-1 group; Group E: pU6-EGFR-shRNA-2 group. Results The expression of mRNA and protein in group E of E group decreased after transfected with shRNA, the number of G 0 ~ G 1 phase increased, the number of S phase decreased, the rate of apoptosis increased, and the logarithmic growth phase delayed. The difference between group B and group C was statistically significant (P <0.05), but the interference decreased with the passage of time. Conclusion Both EGFR-shRNA can effectively inhibit EGFR expression in colorectal cancer LoVo cells, block more cells in G_0 ~ G_1 phase, promote apoptosis and inhibit cell proliferation.