针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体的构建及其对肝癌细胞的抑制作用

Construction of adenovirus carrying dual-target shRNA for Oct-4 and Survivin and its inhibitory effect on human hepatocellular carcinoma cells

摘要:

转录因子Oct-4和Survivin是细胞增殖的关键调控因子，构建针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体Ad5-Dual-shRNA，并研究其对肝癌细胞及移植瘤的生长抑制作用。合成Oct-4和Survivin基因的shRNA序列，插入腺病毒穿梭载体pDC312，含有shRNA的穿梭载体与腺病毒骨架载体pBHGloxdeltaE13Cre共转染HEK293细胞，经Cre/LoxP位点特异性重组获得重组腺病毒Ad5-Dual-shRNA；腺病毒Ad5-Dual-shRNA感染肝癌细胞

Abstract:

The transcriptional factor Oct-4 and Survivin are the key regulatory factors in cancer cell proliferation and mitosis. A dual cancer-specific shRNA adenovirus vector, Ad5-Dual-shRNA, targeting Oct-4 and Survivin genes was constructed by molecular cloning and recombination. After cells were infected with virus, hepatocellular carcinoma cell line EHBH-H1 was used for detecting the expression of Oct-4 and Survivin proteins by Western blotting. The viral cytotoxic effect on cancer cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay in vitro, and the inhibition effect on tumor xenografts was observed in nude mice. The results showed that the expression of Oct-4 and Survivin in cancer cell line EHBH-H1 could be silenced markedly by Ad5-Dual-shRNA. In MTT and animal experiments, Ad5-Dual-shRNA also represented much stronger anti-tumor effect on tumor growth than Ad5-Surv-shRNA and Ad5-Oct4-shRNA. From this research we can draw a conclusion that the cancer-specific adenovirus vector expressing dual-shRNA targeting Oct-4 and Survivin genes may provide us a more effective, specific and convenient gene therapy method.