RNA干扰介导的Adrm1基因沉默对大肠癌细胞增殖的抑制作用  

Silencing of Adrml by RNA interference suppresses proliferation of colorectal cancer cells

作　　者：陈薇[1] 胡晓彤[1] 石青岚[1] 张敷彪[1] 何超[1] 

机构地区：[1]浙江大学医学院附属邵逸夫医院生物医学研究(治疗)中心浙江省生物治疗重点实验室,杭州310016

出　　处：《中华肿瘤杂志》2009年第11期815-819,共5页Chinese Journal of Oncology

基　　金：浙江省自然科学基金（Y206090）

摘　　要：目的探讨RNA干扰使Adrm1犟因沉默后对大肠癌细胞增殖的影响。方法构建靶向Adrm1基因1的shRNA真核表达载体，转染大肠癌细胞RKO，筛选Adrm1基因沉默的稳定克隆。实验细胞分为3组，即稳定转染Adrm1-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Westenl blot法检测Adrm1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，Adrm1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G0／G1期和S／G2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G1期。Adrm1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K抑制作用，并引起大肠癌RKO绑胞大量凋亡。结论RNA干扰介导的Adrm1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Adrm1基因高表达的大肠癌患者，RNA干扰Adrm1基因的表达并结合传统化疗有望成为新的治疗手段。Objective To investigate the effects of the novel proteasome subunit Adrm1 knockdown by RNA interference on proliferation of colorectal cancer cells. Methods The shRNA eukaryotie expression vector against Adrm1 was constructed and transfected into colon cancer RKO cells. The Adrm1-shRNA stable transfected clones were selected. Experimental cells were divided into 3 groups： the experimental group cozltalning stabte Adrm1-shRNA transfected cells, the control group containing only RKO colon cancer cells and stable empty vector transfected control group. The Adrm1 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 （TUNEI.） assay. Cell cycle changes were assayed bv flow eytometry. Results Adrm1-shRNA effectively suppressed Adrm1 expression in the experimental group. Silencing of Adrm1 in RKO cells signifieantly inhibited their anehorage-lndependent 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 G0/G1 and S/G2 phase cells in the experimental group was （41.2 ±1. 1）% and （58.8± 1. 1 ）% , respectively. The cells were arrested at G1 phase. In addition, Adrm1 RNA interference combined with 5-Fu treatment significantly suppressed coloreetal cancer cell growth in vitro. Conclusion Silencing of Adnnl by RNA interference can significantly suppress proliferation of RKO cells through inducing apoptosis and arresting the cell cycle. The combined application of Adrm1 RNA interference and chemotherapy may become as a novel therapeutic strategy for Adrm1 overexpressed colorectal cancer.

关 键 词：Adrm1 蛋白酶体 结卣肠肿瘤 增殖 

分 类 号：R686[医药卫生—骨科学]