RNA干扰抑制表皮生长因子受体表达对Colo-16细胞凋亡及化疗敏感性的影响

Effects of RNA interference targeting epidermal growth factor receptor on the apoptosis in and chemosensitivity of a human cutaneous carcinoma cell line Colo-16

目的 探讨靶向表皮生长因子受体（EGFR）基因的短发夹RNA（EGFR-shRNA）对皮肤鳞状细胞癌Colo-16细胞生长和雷帕霉素敏感性的影响.方法 构建针对EGFR序列特异的shRNA表达载体,用脂质体转染Colo-16细胞.实验分正常细胞组、脂质体组、阴性对照组、阳性干扰组.分别采用免疫细胞化学和Western印迹测定EGFR蛋白的表达;噻唑蓝（MTT）法检测Colo-16细胞对雷帕霉素的敏感性;流式细胞仪检测细胞凋亡率.结果 EGFR-shRNA可明显抑制Colo-16细胞EGFR的表达,抑制率达43.3%（F=44.6,P〈0.05）,并将Colo-16细胞对雷帕霉素的敏感性提高2.44倍.阳性干扰组Colo-16细胞的凋亡率为（12.65±0.091）%,与正常细胞组差异有统计学意义（F=2042.9,P〈0.05）.结论 针对EGFR的质粒表达载体可以有效抑制Colo-16细胞EGFR的表达,增强Colo-16细胞对雷帕霉素的敏感性,并诱导其凋亡.

Objective To investigate the effects of a short hairpin RNA targeting epidermal growth factor recereceptor （EGFR-shRNA） on Colo-16 cell apoptosis and sensitivity to rapamycin. Methods The expression vector of EGFR-specific shRNA was constructed. Colo-16 cells were classified into 4 groups, normal control group remaining untreated, liposome group transfected with lipofectamine 2000, negative control group transfected with shRNA-NC/Iipofectamine 2000 and positive interference group transfected with the expression vector of shRNA-EGFR/Lipofectamine 2000. After additional culture, immunocytochemistry and Western blot were conducted to detect the protein expression of EGFR, and flow cytometry to measure the apoptosis in Colo-16cells. MTT assay was performed to measure the sensitivity of Colo-16 cells to rapamycin. Results Compared with the normal control group, the expression of EGFR was down-regulated by 43.3% in positive interference group （F= 44.6, P〈 0.05）, and the sensitivity to rapamycin was increased by 2.44 folds. The apoptosis rate in positive interference group was （12.65±0.091）%, significantly different from that in the normal control group （F = 2042.9, P 〈 0.05）. Conclusion The plasmid expression vector containing shRNA targeting EGFR can effectively suppress the expression of EGFR by Colo-16 cells, enhance the sensitivity of Colo-16 cells to rapamycin and induce the apoptosis in Colo-16 cells.