Nup88-shRNA对人结肠癌细胞株HT-29生长及侵袭力影响的实验研究  

Experimental effect of Nup88-shRNA on growth and invasiveness of human colon cancer cell line HT-29

作　　者：李男[1] 胡占升[1] 王宝权[1] 孙宏治[2] 朱志图[3] 李玉强[4] 李谌[4] 王钰[4] 

机构地区：[1]辽宁医学院附属第一医院重症医学科,辽宁锦州121001 [2]辽宁医学院附属第一医院普外科,辽宁锦州121001 [3]辽宁医学院附属第一医院肿瘤科,辽宁锦州121001 [4]辽宁医学院附属第一医院临床生物样本中心,辽宁锦州121001

出　　处：《生物医学工程与临床》2016年第4期414-421,共8页Biomedical Engineering and Clinical Medicine

基　　金：辽宁省高等学校优秀人才支持计划资助项目(LJQ2011091)

摘　　要：目的构建重组慢病毒介导的Nup88-sh RNA载体,由RNA干扰(RNAi)技术沉默Nup88,研究其对结肠癌HT-29细胞的增殖、黏附、侵袭和转移的影响,为结肠癌的临床基因治疗寻找新的靶点。方法 Nup88重组慢病毒载体的构建后包装检验效价,以最佳感染复数转染结肠癌HT-29细胞,实验分为沉默组、阴性对照组和空白组3组,其中沉默组分为Nup88-sh RNA1、Nup88-sh RNA2、Nup88-sh RNA3、Nup88-sh RNA4 4个亚组。通过逆转录-聚合酶链反应(RT-PCR)和Western blot印迹检测各组表达效率,主要是HT-29细胞的m RNA和蛋白;四甲基偶氮唑盐(MTT)法和流式细胞术检测Nup88基因被干扰后对HT-29细胞增殖和凋亡的影响;细胞侵袭实验检测Nup88基因被干扰后对HT-29细胞侵袭力的影响。结果沉默4组病毒及1组阴性对照均构建成功,滴度均为4E+8 TU/m L。沉默组Nup88 m RNA与阴性对照组和空白组相比,蛋白表达差异均有显著统计学意义(P<0.01)。Nup88-sh RNA1转染后,沉默率可达到86%。沉默组细胞增殖程度显著减少,与空白组和阴性对照组相比,差异有统计学意义(P<0.05)。沉默组细胞凋亡率(30.28%±0.19%)显著增加,与阴性对照组(4.15%±0.24%)和空白组(2.98%±0.27%)相比,差异有统计学意义(P<0.05)。培养肿瘤细胞常规24 h后,沉默组穿膜细胞数(120.5±8.7)和抑制率(49.22%)与阴性对照组(232.2±13.4,-2.14%)和空白组(276.4±10.2,0)相比,穿膜细胞数明显减少,抑制率明显增加,差异具有统计学意义(P<0.05)。结论 Nup88重组慢病毒可以通过RNAi成功抑制HT-29细胞中Nup88基因的表达,并能显著抑制其增殖及远处的侵袭能力。Objective To construct recombinant lentivirus-mediated vector for Nup88-shRNA and study its impact on proliferation, adhesion, invasion and metastasis of HT-29, and find new gene therapy targets for colon cancer. Methods The potency test was performed after construction and package of the recombinant lentivirus-mediated vector for Nup88, and HT-29 cells were transfected with the best multiple infection index. The test was divided into 3 groups, silence group, negative control group and blank group, and the silence group were further divided into 4 sub-group, Nup88-shRNA1, Nup88-shRNA2,Nup88-shRNA3 and Nup88-shRNA4. The efficiency of each group was evaluated by detecting mRNA and protein expression in HT-29 cells by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot. MTT method and flow cytome-try were performed to test the impact on proliferation and apoptosis of HT-29 cells after Nup88 silencing. Cell invasion assay was performed to evaluate the invasiveness of HT-29 after Nup88 silencing. Results Four silence sub-group and negative control group were successfully constructed, and the titer was 4E ＋ 8 TU/mL for all of them. Compared with negative control group and blank group, the expression of Nup88 mRNA of silence group were significantly different（P〈0.01）. After transfection with Nup88 shRNA1, the silencing rate reached to 86 %. The degree of cell proliferation was significantly decreased in silence group,and as compared with negative control group and blank group, there were significant differences（P〈0.05）. The apoptotic rate in silence group（30.28 % ± 0.19 %） was increased, and when compared with negative control group（4.15 % ± 0.24 %） and blank group（2.98 % ± 0.27 %）, there were significant differences（P〈0.05）. After 24-hour of tumor cell culture, trans-membrane cell（120.5 ± 8.7） was decreased and inhibition rate（49.22 %） was increased in silence group, and when compared with negative control group（232.2 ± 13.4,-2.14 %） and blank gr

关 键 词：NUP88 人结肠癌细胞HT-29 RNA干扰(RNAI) 慢病毒载体 

分 类 号：R735.35[医药卫生—肿瘤]