逆转录病毒介导shRNA抑制Mig-7基因对人肝癌细胞血管生成拟态及体外侵袭转移的抑制作用

Inhibitory effect of Mig-7 silencing by retrovirus-mediated shRNA on vasculogenic mimicry,invasion and metastasis of human hepatocellular carcinoma cells in vitro

目的探讨逆转录病毒介导的短发夹RNA(shRNA)干扰迁移诱导基因7(Mig-7)对肝细胞癌(HCC)细胞血管生成拟态(VM)形成及体外侵袭转移能力的影响。方法设计2条Mig-7 m RNA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Mig-7shRNA逆转录病毒表达载体质粒,并将要接受转染的人肝癌细胞MHCC-97H分为6组:转染Mig-7 shRNA-1组;转染Mig-7 shRNA-2组;转染Mig-7 shRNA-N组;转染空载质粒组(Vector);重组人血管内皮抑素(ES,商品名:恩度)组;MHCC-97H细胞对照组(Control)。半定量PCR、Western blot检测其对Mig-7表达的影响;三维细胞培养观察其对VM形成的影响;细胞间粘附实验、Transwell侵袭实验及迁移实验观察其对细胞粘附、侵袭及迁移能力的影响。结果转染后,Mig-7 shRNA-1组与Mig-7 shRNA-2组中Mig-7 m RNA与蛋白的表达、MHCC-97H细胞形成VM能力、细胞侵袭、迁移能力明显减低(P<0.05),细胞间粘附能力明显增加(P<0.05);Mig-7 shRNA-N组、Vector组、ES组中MHCC-97H细胞Mig-7的表达、VM形成、细胞间粘附、迁移、侵袭能力较MHCC-97H细胞组均无明显变化。结论逆转录病毒介导的shRNA能够有效下调Mig-7的表达并明显抑制HCC细胞VM形成能力及侵袭转移能力,增加细胞间的粘附作用;ES对HCC细胞的Mig-7表达、VM形成、侵袭转移及粘附能力无明显影响。

Objective To explore the inhibitory effect of migration-inducing gene 7（Mig-7） gene silencing induced by retroviralmediated small hairpin RNA（shRNA） on vasculogenic mimicry（VM）, invasion and metastasis of human hepatocellular carcinoma（HCC） cells in vitro. Methods Two target sequences（Mig-7 shRNA-1 and Mig-7 shRNA-2） and one negative control sequence（Mig-7 shRNA-N） were synthesized. The recombinant retroviral vectors carrying Mig-7 shRNA were constructed,and HCC cell line MHCC-97 H were transfected with Mig-7 shRNA-1, Mig-7 shRNA-2, Mig-7 shRNA-N, or the empty vector,or treated with 125 μg/m L recombinant human endostatin（ES）. Mig-7 expression in the treated cells was detected using semiquantitative PCR and Western blotting. The inhibitory effect of Mig-7 silencing on VM formation was investigated in a 3-dimensional cell culture system; the changes in cell adhesion, invasion and migration were assessed with intercellular adhesion assay, Transwell invasion assay and Transwell migration assay, respectively. Results The expression of Mig-7 at both m RNA and protein levels decreased significantly, VM formation, invasion and metastasis were suppressed, while intercellular adhesion increased significantly in MHCC-97 H cells in Mig-7 shRNA-1 and Mig-7 shRNA-2 groups（P〈0.05）; such changes were not observed in cells transfected with Mig-7 shRNA-N or the empty vector, nor in cells treated with ES. Conclusions Mig-7 silencing by retroviral-mediated shRNA significantly inhibits VM formation, invasion and metastasis and increases the intercellular adhesion of the HCC cells, while ES does not have such inhibitory effects.