迁移诱导基因-7-ShRNA重组逆转录病毒联合血管内皮抑素抑制肝癌移植瘤的生长及转移

Inhibitory effect of migration-inducing gene-7-shRNA recombinant retrovirus combined with endostalin ongrowth and metastasis of hepatoma xenograft

目的探讨逆转录病毒介导的RNA（shRNA）干扰迁移诱导基因-7（Mig-7）联合重组人血管内皮抑素（ES）对人肝癌细胞裸鼠皮下移植瘤生长、转移的抑制作用。方法设计2条Mig-7-mRNA寡核苷酸序列（Mig-7-shRNA-1、Mig-7-shRNA-2）和1条作为负对照的无关序列（Mig-7-shRNA-N）。构建特异性M迫-7-shRNA逆转录病毒表达载体质粒，转染Mig-7高表达人肝癌细胞MHCC-97H。建立人肝细胞癌（HCC）裸鼠皮下移植瘤模型，根据转染情况和给药不同分为4组：pSIREN-M1组；pSIREN-MN组；ES组；pSIREN-Ml＋ES组。比较各组移植瘤体积、质量、转移情况；免疫组织化学观察各组移植瘤中血管生成拟态（VM）形成及肿瘤微血管密度（MVD）的差异；Westernblot检测各组Mig-7及血管内皮生长因子（VEGF）的表达。组间比较采用单因素方差分析，计量资料的组间比较采用Fisher精确概率法检验。结果PSIREN-M1组移植瘤体积、质量、转移率、Mig-7表达及VM形成明显低于PSIREN-MN组（尸〈0．05），VEGF表达及MVD明显高于PSIREN-MN组（P〈0．05）；ES组移植瘤体积、质量及转移率、VEGF表达及MVD明显低于PSIREN-MN组（P〈0．05），Mig-7表达及VM形成明显高于PSIREN-MN组（P〈0．05）；DSIREN-Ml＋ES组的移植瘤体积、质量及转移率、Mig-7表达与VM形成、VEGF表达与MVD明显低于PSIREN-M1组及ES组（P〈0．05）。结论Mig-7-shRNA重组逆转录病毒联合ES抑制HCC移植瘤生长及转移的效果明显优于两者单用。体内单用靶向肿瘤血管内皮细胞的“抗肿瘤血管生成治疗”效果有限的原因可能是其促进了VM的形成。

Objective To investigate the inhibitory effect of migration-inducing gene-7 （Mig- 7） interfered with retrovirus-mediated RNA （shRNA） combined with recombinant human endostatin （ES） on the growth and metastasis of subcutaneous xenograft of human hepatoma cells in nude mice. Methods Two Mig-7-mRNA oligonucleotide sequences （Mig-7-shRNA-1 and Mig-？-shRNA-2） and one sequence as a negative control （Mig-7-shRNA-N） were designed. The specific Mig-7-shRNA recombinant retrovirus expression vector plasmid was constructed and used for the transfection of human hepatoma MHCC-97H cells with high expression of Mig-7. The subcutaneous xenogratt tumor model of human hepatocellular carcinoma （HCC） in nude mice was established, and according to the condition of transfection and administration, the nude mice were divided into pSTREN-M1 group, pSIREN-MN group, ES group, and pSIREN-MI＋ES group. The xenografl tumor volume, mass, and metastasis were compared between groups. Immunohistochemistry was used to observe the formation of vasculogenic mimicry （VM） in xenograft tumor and the difference in tumor microvascular density （MVD）, and Western blot was used to measure the expression of Mig-7 and vascular endothelial growth factor （VEGF） in each group. A one-way analysis of variance was used for comparison between groups, and the Fisher＇s exact test was used for comparison of continuous data between groups. Results Compared with the pSIREN-MN group, the pSIREN-M1 group had significantly lower xenografi tumor volume, mass, and metastasis rate, Mig-7 expression, and formation of VM （P 〈 0.05）, as well as significantly higher VEGF expression and MVD （P 〈 0.05）. Compared with the pSIREN-MN group, the ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, VEGF expression, and MVD （P 〈 0.05）, as well as significantly higher Mig-7 expression and formation of VM （P 〈 0.05）. Compared with the pSIREN-M1 group and the ES group, the pSIREN-MI＋ES group had significantly lower xenografl tumor volume, mass, and metastasis rate, Mig-7 expression, formation of VM, VEGF expression, and MVD （P 〈 0.05）. Conclusion Mig-7-shRNA recombinant retrovirus combined with ES has a better inhibitory effect on the growth and metastasis of HCC xenograft tumor than Mig-7-shRNA recombinant retrovirus or ES alone. The anti-tumor angiogenesis therapy alone, which targets vascular endothelial cells in vivo, has a limited effect, since it may promote the formation of VM.