婆罗双树样基因4慢病毒干扰载体构建及对MGC80-3转移潜能的影响

Construction of an inducible SALL4 gene interfering lentiviral vector and its effect on the metastatic potential of MGC80-3 gastric cancer cells

目的人婆罗双树样基因4(spalt-like transcription factor 4,SALL4)是近年来新发现的癌基因,具有促进肿瘤生长和转移的作用。本研究旨在构建可诱导shRNA基因干扰SALL4慢病毒载体并转染胃癌细胞建立稳定细胞株,探讨SALL4基因沉默对胃癌细胞转移潜能的影响。方法合成特异性靶向人SALL4基因的shRNA序列,连接入TetpLKO-puro慢病毒载体;脂质体法介导重组慢病毒质粒系统转染293T细胞,包装产生慢病毒;收集病毒上清,转染人胃癌细胞株MGC80-3,嘌呤霉素筛选获得阳性克隆,采用四环素(tetracycline,Tet)诱导shRNA表达。同时设立shRNA对照组。荧光定量RT-PCR检测SALL4mRNA水平;蛋白质印迹检测SALL4蛋白水平;Transwell迁移实验检测细胞迁移能力;荧光定量RT-PCR和蛋白质印迹分别检测干性指标Oct4和上皮-间质转化(epithelial mesenchymal transition,EMT)指标E-cadherin、Slug和Twist基因及蛋白表达水平。结果 PCR结果表明,SALL4shRNA序列已成功连入Tet-pLKO-puro慢病毒载体。荧光定量RT-PCR结果表明,对照组Tet-PLKO-shCtrl细胞在Tet诱导前后,SALL4mRNA水平分别为1.015±0.150和0.932±0.127,P=0.580;实验组Tet-PLKO-shSALL4细胞在Tet诱导前后,SALL4mRNA水平分别为1.000±0.028和0.443±0.017,P<0.001。蛋白质印迹结果显示,SALL4蛋白与mRNA变化一致。Transwell迁移实验显示,对照组Tet-PLKO-shCtrl细胞Tet诱导前后,迁移细胞数分别为(327±66)和(330±50)个,P=0.856;Tet-PLKO-shSALL4细胞Tet诱导前后,迁移细胞数分别为(335±44)和(100±23)个,P<0.001。荧光定量RT-PCR检测结果表明,Tet-PLKO-shSALL4细胞Tet诱导后,干性指标Oct4及EMT指标Slug和Twist mRNA水平分别为0.140±0.003、0.532±0.047和0.652±0.033,P值分别为<0.001、0.008和0.035;E-cadherin mRNA水平增加至8.994±0.689,P<0.001。对照组经Tet诱导,Oct4、Slug、Twist和E-cadherin基因表达水平分别为1.180±0.010、0.931±0.016、0.978±0.036和1.330±0.371,P值分别为0.063、0.086、0.646和0.300。蛋白质印迹与荧光定量RT-PCR结果一致。结论成功构建可诱导shRNA基因干扰SALL4慢病毒载体,并建立稳定转染胃癌细胞株。Tet诱导SALL4shRNA表达可显著下调SALL4基因表达水平和抑制胃癌细胞MGC80-3的迁移能力。

OBJECTIVE Spalt-like transcription factor 4 （SALL4） is a newly identified oncogene which promotes cancer growth and metastasis. In the present study,we aimed to construct an inducible lentiviral vector for SALL4 genetargeting shRNA and establish a gastric cancer cell line that was stably transduced with the inducible SALL4 shRNA. The effects of SALL4 interference on the metastatic potential of gastric cancer cells were analyzed. METHODS SALL4 shRNA oligos were synthesized and inserted into the Tet-pLKO-puro vector. The packaging plasmid, envelope plasmid and the recombinant Tet-pLKO-shSALL4 plasmid were co-transfected into 293T cells using Lipofectamine 2000. Lentiviral particles were collected and used to infect human gastric adenocarcinoma cell line MGC80-3. The infected cells were selected with puromyein and SALL4 shRNA were induced by the addition of tetracycline （Tet）. Cells transduced with non-targeting shRNA served as the control. SALL4 mRNA and protein levels were analyzed by real-time PCR and western blot. The metastatic potential of cells was evaluated by transwell migration assay. The expressions of stemness gene Oct4 and EMT （epithelial mesenchymal transition）-associated genes were determined by real-time PCR and western blot. RESULTS The results of PCR showed that SALL4 shRNA oligos were inserted into Tet-pLKO-puro vector. After the selection with puromycin,the stably transduced MGC80-3 cells were established. Tet-PLKO-shCtrl cells showed a stable expression of SALL4 mRNA and protein when cultured with or without Tet. Compared to the uninduced cells,Tet-PL- KO-shSALL4 cells incubated with Tet showed a significantly lower level of SALL4 mRNA （1. 000 ± 0. 028 vs 0. 443 ± 0. 017,P〈0. 001）. The SALL4 protein level showed a similar change. When incubated with Tet,the number of Tet-PL- KO-shSALL4 migrated cells decreased from 335 ±44 to 100 ± 23 per high power field （HPF,P〈0. 001） while the number of Tet-PLKO-shCtrl migrated cells remains unchanged （327 ± 66 vs 330± 50, P = 0. 856）. In Tet-PLKO-shSALL4 cells treated with Tet,the mRNA levels of Oct4 ,Slug and Twist decreased to 0. 140 ±0.003,0. 532 ±0. 047 ,and 0. 652±0. 033 （P values were 〈0. 001,0. 008 and 0. 035,respectively）,while the mRNA level of E-cadherin raised to 8. 994±0. 689 （P〈0. 001）. The mRNA levels of Oct4, Slug, Twist, and E-cadherin in Tet-PLKO-shCtrl group were 1. 180±0. 100, 0. 931±0. 016,0. 978±0. 036 and 1. 330±0. 371 （P values were 0.06a,0. 086,0. 646 and 0. 300,respectively）. The results of western blot also showed the similar changes. CONCLUSIONS The inducible lentiviral system for SALL4 gene- targeting shRNA has been successfully constructed. The stably transduced gastric cancer cell line carrying the inducible SALL4 gene-targeting shRNA has been established. The inducible expression SALL4 shRNA resulted in a significant decrease in SALL4 mRNA and protein levels and the migration ability of gastric cancer cells.