CAJ | 학술논문

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.
목적 탐토RNA간우사Adrml기인침묵후대대장암세포증식적영향.방법 구건파향Adrml기인적shRNA진핵표체재체,전염대장암세포RKO,사선Adrml기인침묵적은정극륭.실험세포분위3조,즉은정전염Adrml-shRNA적실험조、부함대장암RKO세포적공백대조조화전염공재체적음성대조조.채용Western blot법검측Adrml단백적표체수평.통과연경지세포집락형성실험관찰3조세포적집락형성능력.채용사갑기우담서람(MTT)법화원위말단표기(TUNEL)법검측세포적증식화조망수평.응용류식세포의검측세포주기적변화정황.결과 실험조대장암RKO세포중,Adrml단백적표체수도명현억제.실험조세포적비첩벽의뢰생장능력명현하강,우견개별집락형성.실험조세포적조망백분솔위(12.4±1.1)%,명현고우음성대조조[(1.3±0.2)%,P＜0.05].실험조G_0/G_1기화S/G_2기세포소점적비례분별위(41.2±1.1)%화(58.8±1.1)%,실험조세포피조체재G_1기.Adrml기인침묵능명현증강5-불뇨밀정(5-Fu)대대장암RKO세포적생장억제작용,병인기대장암RKO세포대량조망.결론 RNA간우개도적Adrml기인침묵능유도대장암세포발생조망병출현세포주기조체,종이억제종류세포적증식.대우Adrml기인고표체적대장암환자,RNA간우Adrml기인적표체병결합전통화료유망성위신적치료수단.
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.