RNA干扰介导的Adrml基因沉默对大肠癌细胞增殖的抑制作用

摘要

Objective To investigate the effects of the novel proteasome subunit Adrml knockdown by RNA interference on proliferation of colorectal cancer cells.Methods The shRNA eukaryotic expression vector against Adrml was constructed and transfected into colon cancer RKO cells.The Adrml-shRNA stable transfected clones were selected.Experimental cells were divided into 3 groups:the experimental group containing stable Adrml-shRNA transfected cells,the control group containing only RKO colon cancer cells and stable empty vector transfected control group.The Adrml protein expression level was analyzed by Western blot.The colony-forming ability of the three groups was assessed by soft agar assay.The cell proliferation and apoptosis were analyzed by methyl thiazolyl tetrazolium(MTT)method and in situ end labeling(TUNEL)assay.Cell cycle changes were assayed by flow cytometry.Results Adrml-shRNA effectively suppressed Adrml expression in the experimental group.Silencing of Adrml in RKO cells significantly inhibited their anchorage-independent growth,only occasional individual colonies were formed.The apoptosis rate of experimental group was(12.4±1.1)%,significantly higher than that of the stable empty vector transfected control group.The proportion of G_0/G_1 and S/G_2 phase cells in the experimental group was(41.2±1.1)%and(58.8±1.1)%,respectively.The cells were arrested at G_1 phase.In addition.Adrml RNA interferenCe combined with 5-Fu treatment significantly suppressed colorectal cancer cell growth in vitro.Conclusion Silencing of Adrml by RNA interference can significantly suppress proliferation of RKO cells throush inducing apoptosis and arresting the cell cycle.The combined application of Adrml RNA interference and chemothempy may become as a novel therapeutic strategy for Adrml overexpressed colorectal cancer.%目的 探讨RNA干扰使Adrml基因沉默后对大肠癌细胞增殖的影响.方法 构建靶向Adrml基因的shRNA真核表达载体,转染大肠癌细胞RKO,筛选Adrml基因沉默的稳定克隆.实验细胞分为3组,即稳定转染Adrml-shRNA的实验组、仅含大肠癌RKO细胞的空白对照组和转染空载体的阴性对照组.采用Western blot法检测Adrml蛋白的表达水平.通过软琼脂细胞集落形成实验观察3组细胞的集落形成能力.采用四甲基偶氮唑蓝(MTT)法和原位末端标记(TUNEL)法检测细胞的增殖和凋亡水平.应用流式细胞仪检测细胞周期的变化情况.结果 实验组大肠癌RKO细胞中,Adrml蛋白的表达受到明显抑制.实验组细胞的非贴壁依赖生长能力明显下降,偶见个别集落形成.实验组细胞的凋亡百分率为(12.4±1.1)%,明显高于阴性对照组[(1.3±0.2)%,P＜0.05].实验组G_0/G_1期和S/G_2期细胞所占的比例分别为(41.2±1.1)%和(58.8±1.1)%,实验组细胞被阻滞在G_1期.Adrml基因沉默能明显增强5-氟尿嘧啶(5-Fu)对大肠癌RKO细胞的生长抑制作用,并引起大肠癌RKO细胞大量凋亡.结论 RNA干扰介导的Adrml基因沉默能诱导大肠癌细胞发生凋亡并出现细胞周期阻滞,从而抑制肿瘤细胞的增殖.对于Adrml基因高表达的大肠癌患者,RNA干扰Adrml基因的表达并结合传统化疗有望成为新的治疗手段.