Ras/MAPK/ERK通过协同NF-κB促进肝癌细胞中cyclinD1的表达

Ras/MAPK/ERK Promote Expression of CyclinD1 Collaborating with NF-κB in Hepatoma Cells

目的探讨Ras癌基因在体内诱导肝肿瘤发生过程中促进cyclin D1表达的分子机制。方法利用H-ras12V转基因雄鼠肝肿瘤组织、肿瘤周围组织以及非转基因雄鼠肝组织进行病理分析和总RNA及蛋白质的提取。应用qRT-PCR和Western blot方法检测cyclin D1的表达以及调控cyclin D1表达的相关信号通路激活状态。采用miRNA测序、生物信息学分析和qRT-PCR验证方法检测miR-5102基因的表达水平。结果与非转基因雄鼠肝组织和肿瘤周围组织相比,肿瘤组织中cyclin D1基因的mRNA和蛋白表达水平显著升高。与非转基因雄鼠肝组织和肿瘤周围组织相比,在肿瘤组织中MAPK信号转导通路中的ERK信号分子的蛋白表达水平和磷酸化水平都显著升高,在肿瘤组织中成高激活状态,NF-κB信号通路中的抑制因子IκB蛋白水平显著降低。miR-5102的表达水平没有显著变化。结论在Ras癌基因诱导的肝肿瘤发生过程中,主要通过激活MAPK/ERK和NF-κB信号通路促进cyclin D1的表达。

Objective To investigate the regulatory mechanisms contributing to over-expressed cyclinD1 in H- rasl2V induced hepatocellular carcinoma. Method The liver tumor tissues and peri-tumor tissues of 9-month-old male H-rasl2V transgenic mice and normal liver tissues of male C57BL/6J mice were sampled. Part of each sample was fixed in 10% formalin and undergone paraffin-embedded tissue section, HE staining, and pathological analysis. Part of each sample was frozen in liquid nitrogen for extraction of total RNA and protein, miRNA expression was analyzed by High-Throughput Sequencing. The cyclin D1 and miR-5102 gene expression were examined by qRT-PCR. The protein levels of cyclin D1, p-ERK, ERK, and IKB were detected by Western blot. Result Compared with the normal liver tissues and peri-tumor tissues, the mRNA and protein expression levels of cyclin D1 in tumor tissues was significantly increased （P 〈 0.05） ; The protein and phosphorylation levels of signaling molecule ERK were significantly increased; And the protein level of inhibitor IKB were significantly reduced. The expression level of miR-5102 did not change significantly among the three sample groups. Conclusion In the process of H-rasl2V-induced liver tumorigenesis, activation of MAPK/ERK and NF-KB signaling pathways contribute to the over-expression of cyclin D1.