RhoC高表达诱导人正常肝细胞迁移和侵袭的研究

RhoC overexpression-induced migration and invasion of human normal hepatocytes

目的观察RhoC高表达诱导人正常肝细胞迁移和侵袭恶性转化。方法pcDNA3-RhoC转染HL7702细胞，划痕实验和Transwell小室检测细胞迁移和侵袭能力；明胶酶谱分析检测基质金属蛋白酶（MMPs）活性；逆转录一聚合酶链反应（RT—PCR）检测细胞迁移和侵袭相关基因表达；Westernblot检测相关蛋白水平；接种裸鼠检测活体成瘤率。结果转染RhoC细胞组与HL7702细胞组和空质粒转染细胞组比较获得了迁移和侵袭能力，相对迁移距离显著增大，分别为（63．33±10．07）％、（28．67±7．02）％和（28．33±6．66）％（P〈0．01）；穿过Transwell微孔滤膜细胞显著增多，分别为（65．33±6．80）％、（24．33±5．79）％和（23．33±5．73）％（P〈0．01），侵袭能力显著增强，分别为43．67±7．59、13．33±3．40和15．33±4．11（P〈0．01）；MMPs活力增强；迁移和侵袭相关基因MMP-2、MMP-9、血管内皮生长因子（VEGF）、P27RF．Rho和Survivin表达上调，基质金属蛋白酶组织抑制剂1（TIMP1）、TIMP2、p53和人第10号染色体缺失的磷酸酶及张力蛋白同源的基因（PTEN）表达水平下调；接种裸鼠成瘤率为40％。结论RhoC高表达诱导人正常肝细胞迁移和侵袭的恶性转化。

Objective To investigate the role of Ras-homologous GTPase C （RhoC） overexpression in inducing migration and invasion for malignant transformation in normal human hepatocytes. Methods pcDNA3-RhoC vector was transfected into human normal liver cell HL7702. Scratch-healing test combined with Transwell was used to examine cell migration and invasion, and matrix metalloproteinases （ MMPs ） gelatinase activities were assayed through zymography. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting were used to detect the migration and invasion associated genes. RhoC transfected HL7702 cells were inoculated into the nude mice to test tumor formation. Results Cells transfected with pcDNA3-RhoC acquired migration capacity, and the migration distance was notably increased as compared with parental cells and negative control groups [ （63.33 ± 10. 07 ）% vs. （28.67 ±7.02）% and （28. 33 ±6. 66 ） % , P 〈 0. 01 ]. The number of pcDNA3-RhoC cells through the Transwell was increased significantly （65.33 ± 6. 80 vs. 24. 33 ± 5.79 and 23.33 ± 5.73 ,P 〈 0. 01 ）, and the invasion capacity was significantly enhanced in pcDNA3-RhoC-transfected group as compared with control groups （43.67 ± 7.59 vs. 13.33 ±3.40 and 15.33 ±4. 11 ,P 〈0.01 ）. MMPs gelatinase activities were increased, the expression of migration and invasion associated genes and proteins such as MMP-2, MMP-9, vascular endothelial growth factor （VEGF） , P27RF-Rho and Survivin was significantly up-regulated, and the expression of tissue inhibitor of tissue inhibitorof metalloproteinase 1 （ TIMP1 ） , TIMP2, p53 and phosphatase and tensin homolog deleted on chromosome ten （PTEN） was down-regulated in pcDNA3-RhoC-transfected group as compared with control group. The tumor formation ratio in nude mice was 40%. Conclusion RhoC overexpression can induce migration and invasion for malignant transformation in human normal hepatocytes.