摘要
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.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第33期3402-3408,共7页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.81102078
苏州市科教兴卫青年科技基金资助项目
No.SWKQ1026
苏州市科技发展指导基金资助项目
No.SZD09132