干扰人ATP1B1基因对胃腺癌SGC-7901细胞增殖和迁移能力的影响  

Interference of human Na/K-ATPaseB1 subunit on proliferation and migration of gastric adenocarcinoma cell line SGC-7901

作　　者：刘凯[1] 张洁[1] 任婧婧[1] 王修杰[1] 杨洪亮[1] 林苹[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室老年医学研究室,四川成都610041

出　　处：《癌症》2009年第3期225-231,共7页Chinese Journal of Cancer

基　　金：华西医院回国人员启动基金资助(No.136050132)~~

摘　　要：背景与目的:Na+-K+ATP酶B1亚基基因ATP1B1在高分化的肿瘤细胞中表达高,低分化的表达低,并且ATP1B1的表达与细胞紧密连接和上皮细胞的极性有关。因此,我们构建了特异性干扰ATP1B1 mRNA的短发夹RNA,以探讨干扰ATP1B1对人胃腺癌细胞株SGC-7901增殖和迁移能力的影响。方法:构建特异性干扰ATP1B1 mRNA的短发夹RNA质粒表达载体,转染SGC-7901细胞,G418筛选阳性克隆细胞,RT-PCR和real-timePCR法检测ATP1B1 mRNA的表达。通过MTT法检测细胞增殖,流式细胞术检测细胞周期,以及克隆形成实验、Transwell小室迁移实验等方法观察ATP1B1对细胞增殖和迁移的影响。结果:瞬间转染后24h,sh150,sh295,sh562,sh765干扰位点及shNC对SGC-7901细胞ATP1B1mRNA的抑制率分别为(60.87±4.38)%,(44.93±2.24)%,(49.28±2.02)%,(52.17±2.60)%,(3±0.15)%,4个位点对ATB1B1 mRNA均有抑制效应。150位点对ATB1B1基因的抑制效应强于其它3个位点,用作后续实验。G418筛选后,获得了具有稳定干扰ATP1B1 mRNA效应的细胞株shATP1B1-7901,RT-PCR初步分析,sh150位点对ATP1B1-7901细胞的ATP1B1 mRNA的抑制率为(85.72±5.22)%,与阴性对照组的(3.3±0.22)%相比,P<0.05;Real-timePCR检测结果显示,shATP1B1-7901细胞中ATP1β1 mRNA的抑制率为(87.53±3.23)%,高于阴性对照组shNC-7901的(4.17±0.33)%,P<0.05。MTT法细胞增殖实验中,第三天后,shATP1B1-7901细胞的增殖率大于阴性对照组和空白对照组,经统计学分析,P<0.05,差异有统计学意义。流式细胞术结果显示,shATP1B1-7901的S期和G2/M期细胞比例增加,大于阴性对照组和空白对照组,P<0.05,差异有统计学意义,阴性对照组和空白对照组的周期分布差异无统计学意义。shATP1B1-7901稳定株细胞的克隆形成率为(68.50±2.65)%,大于阴性对照组的(50.00±2.53)%及空白对照组的(52.50±2.11)%,P<0.05。shATP1B1-7901稳定株细胞的迁移率(2.80±0.02)%,大于阴性对照组的(1.15±0.05)%和空白对照组的(1.25±0.02)%,P<0.05�Background and Objective. The Na^＋/K^＋ ATPaseB1 （ATP1B1） subunit gene is highly expressed in well-differentiated tumor cells, while it is hypoexpressed in poorly differentiated tumor cells. The expression of ATP1B1 is closely related to cell tight junction and polarity of epithelial cells. This study was to investigate the effect of specific interference of human Na^＋/K^＋ ATP1B1 using shRNA on cell proliferation and migration of gastric adenocarcinoma cell line SGC-7901. Methods. Four shRNA plasmids specifically targeting different fragments of ATP1B1, sh150, sh295, sh562, sh765, were constructed and transiently transfected into SGC-7901 cells. Stable positive clones, shATP1B1-7901 cells, were sorted out by G418. The expression of ATP1B1 mRNA was detected by semi-quantitative RT-PCR and real-time PCR. Cell proliferation was measured by MTT, cell cycle distribution was assessed by flow cytometry, and clone forming was analyzed by the colony formation assay. Cellular migration was observed using the Transwell experiment. Results： At 24 h after transfection, the inhibition ratios of sh150, sh295, sh562, sh765 on ATP1B1 mRNA were （60.87±4.38）%, （44.93±2.24）%, （52.17±2.60）% and （52.17±2.60）% respectively in SGC-7901 cells, which were significantly higher than that of shNC control （3.00±0.15）% （P〈0.05）. Among the four ATP1B1 shRNAs, sh150 exerted the strongest effect （P〈0.05） and was used in the following study. Assessed by RT-PCR and real-time PCR, the expression of ATP1B1 mRNA was inhibited by （85.72±5.22）% and （87.53±3.23）% in the shATP1B1-7901 group, in comparison with （3.3±0.22）% and （4.17±0.33）% in the shNC-7901 group （P〈0.05）. The proliferation ratio was higher in the shATP1B1-7901 group than in the shNC-7901 and SGC-7901 groups 3 days after transfection （P〈0.05）. Cells at S and G2/M phases in the shATP1B1-7901 group were significantly increased compared with those in the shNC-7901 and SGC-7901 groups （P〈 0.05）. The clone fo

关 键 词：ATP1B1 胃肿瘤 腺癌细胞 RNA干扰 细胞增殖 细胞迁移 

分 类 号：R735.2[医药卫生—肿瘤]