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原发性肝癌细胞中Rock2调控MMP2对其侵袭迁移的作用

Effects of Rock2 Regulating MMP2 on Invasion and Metastasis in Hepatocellular Carcinoma

  • 摘要: 目的 探讨Rho相关卷曲螺旋形成蛋白激酶2 (Rho associated coiled coil containing protein kinase 2,Rock2)调控基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)对肝癌细胞侵袭迁移能力的影响。方法 用Western blot法检测30例肝癌组织及对应癌旁组织中Rock2和MMP2的蛋白表达,同时分析它们之间的关联;用荧光定量PCR和Western blot法观察稳定降低Rock2细胞中MMP2 mRNA及蛋白表达变化;利用PCR定点突变技术构建Rock2同义突变质粒“恢复”稳定干扰Rock2的肝癌细胞中Rock2 表达后检测MMP2蛋白表达变化;采用Transwell侵袭实验和划痕实验观察空白对照组、稳定低表达Rock2组、“恢复”Rock2组HepG2细胞侵袭和迁移能力变化。结果 Rock2和MMP2蛋白在肝癌组织中均比对应癌旁组织明显增高,且两者的表达呈正相关性。稳定降低肝癌细胞中Rock2的表达后发现MMP2的mRNA和蛋白表达也随之降低;另外,“恢复”稳定干扰Rock2的肝癌细胞中Rock2的表达,MMP2表达也“恢复”;同时证实Rock2通过调节MMP2表达从而影响肝癌细胞的侵袭和迁移。结论 在原发性肝癌细胞中降低Rock2可引起MMP2表达下降,进而抑制肝癌细胞的侵袭和迁移。

     

    Abstract: Objective To investigate the effects of Rho associated coiled coil containing protein kinase2 (Rock2) on matrix metalloproteinase2 (MMP2) in hepatocellular carcinoma(HCC). Methods The expressions of Rock2 and MMP2 protein were detected by Western blot in 30 cases of HCC tissues, and the correlation between them was also analyzed. The mRNA and protein expression of MMP2 were detected by qRT-PCR and Western blot in ROCK2 stable knockdown cells. The silent mutation plasmid of Rock2 was constructed by PCR site-directed mutagenesis. The expression of MMP2 protein was determined after the Rock2 expression restored in stable knockdown Rock2 cells. The invasion and migration abilities of HepG2 in blank control group, shRock2 group and Rock2 restored group were measured by Transwell assay and wound healing assay. Results Compared with corresponding adjacent tissues, Rock2 and MMP2 were remarkably overexpressed in HCCs and demonstrated a significantly positive correlation. The expressions of MMP2 mRNA and protein were signifi cantly downregulated in Rock2 stable knockdown cells. We also confi rmed that Rock2 regulated cell invasion and migration abilities through regulating MMP2 expression. Conclusion Reduction of Rock2 could decrease the expression of MMP2 in HCC cells, thereby inhibiting HCC cell invasion and migration.

     

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