降低ROCK2通过调控MKP1增强肝癌细胞化疗敏感性的机制研究

Hepatocellular carcinoma (HCC) seriously threatens to people health with low sensitivity to chemotherapy.Our previous studies have found that down regulation of Rho associated coiled coil forming protein kinase 2 (ROCK2) can increase the sensitivity of HCC cells to adriamycin(ARD) and cisplatin(CDDP), but the specific mechanism is not clear. In addition, the study reported that the mitogen activated protein kinase (MKP1) was closely related to the chemotherapy sensitivity of liver cancer. We found that the expression of ROCK2 and MKP1 have positive correlation in hepatocellular carcinoma. Downregulation of ROCK2 in hepatocellular carcinoma cells can induce MKP1 expression , inhibit cell growth and promote cell apoptosis. Therefore, we speculate that ROCK2 influence the chemosensitivity of liver cancer cells by regulating MKP1 .First,we analyse the association of ROCK2 or MKP1 expression and clinicopathologic features of patients,and both as metastasis and prognostic markers for Hepatocellular carcinoma after operation ; Second,it,s confirmed that ROCK2 influence the chemosensitivity of hepatocellular carcinoma through the regulation of MKP1 by experiments in vivo and vitro ; Finally the mechanism can be determined that ROCK2 regulated MKP1 through the ubiquitination assay, immune coprecipitation and Luciferase Report Gene in vitro. The results of this study will provide a new theoretical basis for regulation of chemosensitivity in hepatocellular carcinoma.

肝癌严重危害人类健康且对化疗敏感性低。我们前期研究发现下调Rho相关卷曲螺旋形成蛋白激酶2（ROCK2）可提高肝癌细胞对阿霉素及顺铂的敏感性，但具体机制不清。另外，研究报道丝 裂 原 活 化 蛋 白 激 酶 磷 酸 酶（MKP1）与肝癌的化疗敏感性密切相关。我们发现肝癌组织中ROCK2及MKP1表达呈正相关；在肝癌细胞中下调ROCK2可引起MKP1表达下降，并抑制细胞生长和促进细胞凋亡。据此，我们推测ROCK2通过调控MKP1的表达而影响肝癌细胞的化疗敏感性。本项目拟先分析肝癌组织中ROCK2及MKP1的表达与患者临床病理特征的关联，以及两者作为肝癌术后转移和预后标志物的意义；再利用体内外实验明确ROCK2通过调控MKP1表达对肝癌化疗敏感性的影响；最后通过体内泛素化实验、免疫共沉淀及荧光素报告基因等方法确定ROCK2调控MKP1的机制。本研究结果将为调节肝癌化疗敏感性提供新的理论基础。

肝癌严重危害人类健康且对化疗敏感性低。我们前期研究发现下调Rho相关卷曲螺旋形成蛋白激酶2（ROCK2）可提高肝癌细胞对阿霉素及顺铂的敏感性，但具体机制不清。另外，研究报道丝 裂 原 活 化 蛋 白 激 酶 磷 酸 酶（MKP1）与肝癌的化疗敏感性密切相关。在国家自然基金资助下，我们证实肝癌组织中ROCK2及MKP1 mRNA表达增加，且两者呈正相关；在肝癌细胞中下调ROCK2可引起MKP1 mRNA表达下降，并抑制细胞生长和促进细胞凋亡。进一步展开了相关的实验，我们结果发现：在肝癌细胞中下调 ROCK2 的表达，P65 的表达也随之下降，而在利用 MG132 阻断蛋白酶体情况下，下调ROCK2的表达对 P65 蛋白的表达无明显影响，证实ROCK2通过调控 NF-κB/P65 的泛素化降解过程。在本研究中，我们证实 ROCK2 通过影响 NF-κB/P65 泛素化降解过程调控 MKP1 的表达从而影响肝癌化疗敏感性。另外，在完成原计划的基础上，拓展了研究内容，ROCK2激活ERK1/2-ATF2信号，导致MKP1的mRNA表达增加，从而促进肝癌的转移。综上所述，我们的研究结果证实ROCK2、MKP1作为是肝癌的一种潜在的生物标志物。