慢病毒载体介导的dbpA基因沉默对结直肠癌细胞生物学行为的影响  

Effect of lentivirus vector-mediated RNA interference dbpA gene silencing on the biological behavior of colorectal cancer cells

作　　者：刘瑞廷[1] 侯亚莉[2] 吴向天 王国荣[1] 刘昌[3] 白继荣 邱健[1] 闫立昆[1] 李小军[1] 王小强[1] Liu Ruiting;Hou Yali;Wu Xiangtian;Wang Guorong;Liu Chang;Bai Jirong;Qiu Jian;Yan Likun;Li Xiaojun;Wang Xiaoqiang(Department of General Surgery, Shanxi Provincial People's Hospital, Xi'an 710068, China;Department of Endoscopy Center, Shanxi Provincial People's Hospital, Xi'an 710068, China;Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi' an Jiaotong University, Xir an 710061, China;Department of Pathology, University of Nebraska Medical Center, Omaha 68101, USA)

机构地区：[1]陕西省人民医院普外一科,西安710068 [2]陕西省人民医院腔镜中心,西安710068 [3]西安交通大学第一附属医院肝胆外科,710061 [4]美国内布拉斯加大学医学中心病理科,奥马哈68101

出　　处：《中华普通外科杂志》2019年第7期613-617,共5页Chinese Journal of General Surgery

基　　金：国家自然科学基金项目(81172363);陕西省自然科学基金资助项目(2014JM4089);陕西省国际科技合作与交流计划面上项目(2017KW046).

摘　　要：目的探讨慢病毒介导的RNA干扰(RNA interference, RNAi)沉默DNA结合蛋白dbpA基因对大肠癌细胞生物学行为的影响。方法实验分为3组:siRNA-dbpA慢病毒干扰组、非特异性序列组和空白对照组。制备siRNA-dbpA慢病毒表达载体,转染SW620后,采用RT-PCR法对细胞中dbpA mRNA表达水平进行分析,Western blot检测SW620细胞的dbpA蛋白表达水平。采用流式细胞仪检测细胞凋亡和细胞周期变化,MTT法检测细胞增殖抑制率,并检测细胞克隆形成能力。通过裸鼠移植瘤实验观察沉默dbpA后SW620细胞在体内成瘤能力。结果siRNA-dbpA显著下调了 SW620细胞中dbpA的表达,所构建的重组慢病毒有较高的基因沉默效率。siRNA-dbpA组的dbpA表达水平下调。各组的细胞凋亡率分别为26. 6%±0. 38%,12. 54%± 0. 25%和4. 46%± 0. 19%,siRNA-dbpA组高于其他两组,差异有统计学意义(F = 28. 159,P <0.01);各组的细胞增殖第5天吸光度值分别为(0. 194 ±0. 037),(0. 814 ± 0. 043)、( 1.625 ±0.061), siRNA-dbpA 组低于其他两组,差异有统计学意义(F = 23.214,P<0.01);细胞克隆数分别为(37±3)、(64±5)和(175 ±10)个,siRNAdbpA组低于其他两组,差异有统计学意义(F = 40. 254,P <0. 01)。siRNA-dbpA组裸鼠移植瘤体积在14.21,35 d后与非特异性序列组和空白对照组相比差异均有统计学意义(F= 38. 256、40. 241、30.257,均P<0.05)。结论以dbpA为靶标的RNA干扰能下调大肠癌细胞株SW620中的dbpA表达,显著增加细胞的凋亡,降低了细胞的增殖克隆能力。dbpA沉默后对活体肿瘤细胞的生长有明显的抑制作用。Objective To investigate the effects of lentivirus-mediated RNA interference ( RNAi) targeting DNA binding protein A (dbpA ) on the proliferation and the biological behavior of colorectal cancer cell line SW620. Methods The experiment was divided into 3 groups: KD group ( siRNA-dbpA, lentivirus interference group), CON group ( non-specific sequence group) and NC group (blank control group). The lentiviral vector siRNA-dbpA was constructed and verified by PCR and DNA sequencing. SW620 cells were transfected with siRNA-dbpA plasmid, nontargeting siRNA plasmid, or empty plasmid. After 48 h the transfection, the cells were examined for dbpA expression using Western blot. After 72 hrs transfection, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate was detected by MTT (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2?H-tetrazolium bromide ) assay, and then clone formation was detected, and the ability of SW620 cells to form tumors in vivo after dbpA was silenced was studied in nude mice. Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector. siRNA-dbpA transfection resulted in reduced expression of dbpA in SW620 cells. After transfection, the apoptosis rate of siRNA-dbpAtransfected cells increased to 26. 60%± 0. 38%, significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid 12. 54%± 0. 25% and 4. 46%± 0. 19%, respectively ( F = 28. 159 ,P <0. 01 ). The growth inhibition test indicate that the OD value of the fifth day in siRNA-dbpA group was 0. 194 土 0. 037 , significantly lower than that in the other two groups 0. 814 ±0. 043 and 1. 625 ± 0. 061 , respectively ( F =23. 214 ,P < 0. 01 ). The colony formation number is 37 ± 3 , 64 ± 5and 175 ± 10 respectively, siRNA-dbpA is significantly higher than that in the other two groups (F = 40. 254, P < 0. 01 ). After the completion of nude mouse transplantation tumor model, throu

关 键 词：结直肠肿瘤 DNA结合蛋白质类 细胞凋亡 细胞增殖 

分 类 号：R735.34[医药卫生—肿瘤]