靶向MTDH的小干扰RNA对人原发性肝癌细胞HepG2骨架重排的影响

Inhibition of MTDH expression by shRNA induces skeletal rearrangement of human hepatocellular carcinoma cell strains HepG2

目的构建人MTDH基因的shRNA干扰载体,并观察其对原发性肝癌细胞HepG2骨架重排的影响。方法针对MTDH基因的不同部位设计3对shRNA的寡核苷酸片段,克隆到载体pGCsilencerTM H1/Neo中,RT-PCR、Western blotting及免疫荧光检测转染后MTDH mRNA和蛋白表达变化情况,并筛选最佳抑制效率的shRNA干扰载体,并将其转染人原发性肝癌细胞HepG2,用罗丹明标记的鬼笔环肽显示沉默MTDH基因后对人原发性肝癌细胞HepG2骨架蛋白F_actin的改变。结果靶向MTDH干扰质粒构建成功。与转空载体的阴性对照组及未转染组比较,转染pGCsilencerTM H1/Neo-MTDH-shRNA后,HepG2中MTDH mRNA和蛋白表达明显降低,其中以pGCsilencerTM H1/Neo-MTDH-S1最为明显,达到90%以上;细胞形态及细胞骨架显示转染pGCsilencerTMH1/Neo-MTDH-S1的细胞骨架蛋白F_actin发生重排。结论成功构建并筛选最佳抑制效率的靶向MTDH干扰pGCsilencerTM H1/Neo-MTDH-S1,该载体能有效重排肝癌细胞骨架。

Objective To construct a recombinant short hairpin RNA （shRNA） vector carrying MTDH gene, and to investigate its effect on skeletal rearrangement of hepatocellular carcinoma cell strain HepG2 by silencing MTDH. Methods Three oligonucleotides targeting MTDH gene were synthesized and cloned into vector pGCsilencerTM H1/ Neo. The shRNA vector with best transfection efficiency was detected and identified by RT-PCR, Western blotting and immunofl uorescence staining analysis, which was transfected into hepatocellular carcinoma cell strain HepG2, and its effect on skeletal rearrangement of hepatocellular carcinoma cell strain HepG2 was measured by confocal laser scanning microscopy. Results The recombinant vector pGCsilencerTM H1/Neo-MTDH-shRNA was constructed successfully. Af ter silencing MTDH, compared with the blank and control groups, MTDH mRNA and protein were decreased significant ly in HepG2, especially pGCsilencerTM H1/Neo-MTDH-S1, whose transfection effieiency was more than 90% , and the skeletal of HepG2 cells was rearranged. Conclusion The recombinant shRNA vector targeting MTDH, pGCsilencerTM H1/Neo-MTDH-S1, with best transfection efficiency, is constructed successfully, pGCsilencerTM H1/Neo-MTDH-SI aheres the skeletal rearrangement of HepG2.