ShRNA靶向干扰EGFR抑制结直肠癌细胞增殖的机制

ShRNA-mediated silencing of the epidermal growth factor receptor gene inhibits proliferation of human HCT-15 colorectal cancer cells

目的:从细胞周期的角度探讨RNA干扰抑制表皮生长因子受体(epidermal growth factor receptor,EGFR)表达对结直肠癌细胞增殖影响的机制.方法:以人结直肠癌细胞HCT-15为研究对象,构建EGFR-短发夹RNA(short hairpin RNA,shRNA)载体,采用脂质体转染的方法转染细胞,G418筛选稳定细胞株以获得稳定的转染细胞模型,分为4组:转染shRNA-NC,转染shRNA-1组,转染shRNA-2组,转染shRNA-3组;应用半定量RT-PCR和流式细胞术检测转染细胞模型的mRNA和蛋白表达水平;应用平板克隆实验检测转染后细胞增殖能力;应用流式细胞术检测转染后细胞周期的变化.结果:正确构建了shRNA质粒表达载体,成功转染;转染shRNA-1、2、3组较转染shRNA-NC组mRNA、蛋白表达水平均有下降,以转染shRNA-1和2组的抑制效应好(40.2%±3.2%,52.8%±11.3%);转染shRNA-1、2两组较对照组增殖能力下降,G0/G1期细胞增多(63.69±2.75%,60.10±2.00%vs51.08±3.42%)、S期细胞减少(28.20%±2.42%,27.19%±1.95%vs36.11±1.84%),差异有统计学意义(P<0.05).结论:RNA干扰技术可以有效地抑制HCT-15细胞EGFR表达,并抑制了细胞增殖能力,这可能与诱导细胞出现G0/G1期阻滞有关.

AIM： To examine the effect of short hairpin RNA（shRNA）-mediated silencing of the epidermal growth factor receptor （EGFR） gene on the proliferation of human colorectal cancer cells and to explore the potential mechanisms involved. METHODS： Vectors containing shRNA targeting EGFR were constructed. HCT-15 cells were transfected with EGFR-shRNA-1, EGFR-shRNA-2, EGFR-shRNA-3 or shRNA-NC expression vectors, and stably transfected cell lines were screened. The mRNA level of EGFR was assessed by semi-quantitative RT-PCR. Protein expression and cell cycle changes were determined by flow cytometry. Cell proliferative capacity was assessed by colony formation assay. RESULTS： Compared to shRNA-NC, transfection of the three shRNA vectors significantly inhibited EGFR expression, especially shRNA-1 and shRNA-2 vectors （40.2% ± 3.2% and 52.8% ± 11.3%, respectively）. The mRNA expression of EGFR in shRNA-1-and shRNA-2-transfected cells also decreased significantly. The colony size and number decreased signifi cantly in cells tranfected with shRNA-1 or shRNA （P 0.05）. The percentage of cells in G0/G1 phase increased （63.69% ± 2.75%, 60.10% ± 2.00%; both P 0.05） and that of in S phase decreased （28.20% ± 2.42%, 27.19% ± 1.95%; both P 0.05） in shRNA-1- and shRNA-2-transfected cells. CONCLUSION： Transfection with shRNAs targeting the EGFR gene was capable of suppressing EGFR expression, decreasing cell proliferative capacity and inducing cycle arrest at G0/G1 phase.