shRNA沉默基因DNMT1的表达对膀胱癌T24细胞增殖与凋亡的影响  

Influence of Silencing DNMT1 with shRNA on Proliferation and Apoptosis of Bladder Cancer Cell Line T24

作　　者：张士龙[1] 曾甫清[1] 彭世波[1] 董继华[2] 廖贵益[1] 汪良[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,武汉430022 [2]华中科技大学同济医学院附属协和医院中心实验室,武汉430022

出　　处：《华中科技大学学报（医学版）》2009年第2期165-168,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省科技攻关计划资助项目(No.2006AA301B56-1)

摘　　要：目的体外研究shRNA(short hairpin RNA)沉默基因DNMT1的表达对膀胱癌T24细胞增殖和凋亡的影响,初步探讨DNMT1在膀胱肿瘤形成过程中的作用机制。方法实验分为空白对照组(给予脂质体)、HK组(含无效干扰序列的质粒)及pshRNA-DNMT1组;常规培养膀胱癌T24细胞株,用脂质体Lipofectamine 2000将构建成功的重组质粒pshRNA-DNMT1转染进入T24细胞,然后进行RT-PCR、Western blot检测各组DNMT1 mRNA及蛋白质水平变化;进行MTT、AnnexinⅤ-FITC/PI双染法流式细胞术检测各组T24细胞增殖和凋亡情况。结果重组质粒pshR-NA-DNMT1成功转染进入膀胱癌T24细胞;经RT-PCR检测pshRNA-DNMT1组DNMT1 mRNA表达减少,24、48、72h的抑制率分别是28.44%、52.48%、70.91%;转染pshRNA-DNMT1后,T24细胞中DNMT1蛋白24、48和72 h的抑制率分别为24.27%、57.79%、69.74%;在MTT检测中,pshRNA-DNMT1组的细胞增殖抑制率在24、48、72 h分别为(4.34±0.76)%,(9.87±1.54)%和(13.78±1.93)%,与空白对照组比较各时间点差异均有极显著性意义(均P<0.01);转染24、48和72 h后,pshRNA-DNMT1组细胞凋亡率分别为(3.87±0.81)%、(8.69±1.23)%和(11.46±1.24)%,与空白对照组比较差异均有极显著性意义(均P<0.01)。结论重组质粒pshRNA-DNMT1能有效降低膀胱癌细胞中DNMT1基因mRNA及蛋白的表达,并在一定程度上抑制T24细胞的增殖和促进凋亡。Objective To investigate the influences of silencing DNMT1 gene by RNA interference（RNAi） on the proliferation and apoptosis of bladder cancer cell line T24,and study the mechanism of DNMT1 in promoting bladder tumor formation. Methods Eukaryotic vectors HK and pshRNA-DNMT1 were transfected into T24 cells. Untransfected cells were used as blank controls. The expression of DNMT1 mRNA and protein was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot,respectively. Cell proliferation was tested by MTT assay. Cell apoptosis was measured by flow cytometry with Annexin V-FITC/PI staining. Results Eukaryotic vectors HK and pshRNA-DNMT1 were transfected into T24 cells successfully. Relative to controls,24,48 and 72 h after transfection of pshRNA-DNMTI,the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44 %, 52.48 %, 70.91%, respectively. And those of DNMT1 proteins were 24.27 %, 57.79% ,and 69.74%, respectively. The growth inhibition rate of pshRNA-DNMT1 at 24,48 and 72 h after transfection of pshRNA-DNMT1 was（4.34±0.76）% ,（9.87±1.54）% and（13.78±1.93）% ,respectively. There was statistically significant difference between pshRNA-DNMT1 and control blank groups at each time point（P〈0.01）. After T24 cells were transfeeted for 24,48 and 72 h,the apoptosis rate of pshRNA-DNMT1 was（3.87±0.81）%, （8.69± 1.23）% and（11. 46±1. 24）% respectively（P〈0.01 vs blank control）. Conclusion The recombinant plasmid pshRNA-DNMT1 can silence the expression of DNMT1 mRNA and protein effectively,and to some extent,it also can inhibit the proliferation of bladder cancer cells and promote the apoptosis.

关 键 词：DNA甲基转移酶1基因 RNA干扰 细胞凋亡 细胞增殖 膀胱肿瘤 

分 类 号：R737.14[医药卫生—肿瘤]