UHRF2通过PI3K/Akt信号通路促进肝细胞癌细胞的增殖和转移

UHRF2 promotes migration and proliferation of human hepatocellular carcinoma cells by PI3K/Akt signaling pathway

目的探讨泛素样含PHD和环指域蛋白2(ubiquitin-like with PHD and RING finger domains 2,UHRF2)在肝细胞癌(hepatocellular carcinoma,HCC)增殖和转移中的作用及其机制。方法检索TCGA数据库中HCC的基因表达谱数据和患者的临床数据,分析UHRF2的表达差异与HCC患者的预后情况,采用Cox比例风险模型探索UHRF2对HCC患者预后的独立作用;收集2018年7月至2019年12月在重庆医科大学附属第一医院确诊的20例肝癌患者的肿瘤组织和癌旁组织,采用免疫组化检测UHRF2蛋白的表达,分析蛋白的表达与患者临床病理参数的关系;构建UHRF2表达质粒,通过RT-qPCR和Western blot检测转染效率,在HCC细胞系中过表达UHRF2进行划痕及平板克隆实验检测肝细胞癌细胞的迁移和增殖情况;基因富集分析探讨UHRF2与PI3K/Akt通路活化的关系,Western blot检测Akt、PI3K、p-Akt、p-PI3K的表达。结果 TCGA数据库分析显示UHRF2 mRNA在HCC组织中表达明显升高并提示预后不良;Cox多因素回归分析发现UHRF2的表达是影响患者预后的独立危险因素。免疫组化检测结果显示UHRF2蛋白在HCC中的表达明显高于癌旁组织。UHRF2的高表达与肿瘤大小(P=0.002)、肿瘤转移(P=0.002)密切相关。在迁移实验和平板克隆实验中,UHRF2过表达组的划痕愈合能力及增殖能力较对照组显著增强(P<0.01);PI3K/Akt通路在UHRF2高表达HCC患者中显著富集;Western blot检测结果显示,UHRF2过表达组的p-Akt、p-PI3K的表达水平明显上调(P<0.05)。结论 UHRF2的表达水平与HCC的预后密切相关,是影响患者预后的独立危险因素;过表达UHRF2通过PI3K/Akt信号通路促进HCC细胞的增殖和转移。

Objective To explore the role of Ubiquitin-like with PHD and RING finger domains 2 (UHRF2) on the migration and proliferation of human hepatocellular carcinoma (HCC) cells and investigate its underlying mechanism. Methods The HCC gene expression data and patient clinical data were searched in the TCGA database, the differences of UHRF2 expression were analyzed between normal liver tissue and HCC tissue, and the correlation of its level with prognosis was also analyzed. Liver tumor tissues and adjacent normal tissues were harvested from 20 HCC patients diagnosed in the First Affiliated Hospital of Chongqing Medical University between July, 2008 and December, 2013 to detect UHRF2 expression by immunohistochemistry. The relationship between UHRF2 expression and clinical pathological parameters of these patients was analyzed. After UHRF2 expression plasmid was constructed and transfected into HepG2 and HepG2.2.15 cells, the transfection efficiency was measured by RT-qPCR and Western blotting. Then cell scratch test and plate cloning was employed to detect the effect of UHRF2 overexpression on the migration and proliferation of the cells. Gene set enrichment analysis was applied to further explore the relationship between UHRF2 and activation of PI3K/Akt signaling pathway. The expression of Akt, PI3K, p-Akt and p-PI3K was detected with Western blotting. Results TCGA database analysis revealed that the expression of UHRF2 in HCC tissues was higher than that in normal liver tissues, and indicated a poor prognosis. Cox multivariate regression analysis found that UHRF2 expression was an independent risk factor for the prognosis. Immunohistochemistry showed that the expression level of UHRF2 was significantly higher in the HCC tissues than that in the adjacent normal tissues. The high expression of UHRF2 was closely related to tumor size (P=0.002) and tumor metastasis (P=0.002). In wound healing test and plate cloning test, the HCC cells with UHRF2 overexpression showed significantly stronger migration and proliferation abilities as compared with the control cells. PI3K/Akt pathway was significantly enriched in the HCC patients with higher expression of UHRF2. Western blotting results showed that UHRF2 overexpression significantly activated the phosphorylation of PI3K and Akt proteins in the HCC cells. Conclusion The expression of UHRF2 is closely related to the prognosis of HCC patients, and is an independent risk factor for HCC patients. Overexpression of UHRF2 promotes the proliferation and metastasis of HCC cells through the PI3K/Akt signaling pathway.