调控miR-495、P4HA2的表达对肝纤维化和肝细胞癌发生机制研究  

Hypoxia promotes the expression of P4HA2 by down-regulating miR-495 and participates in the development of liver fibrosis and liver cancer

作　　者：张红宇[1] 李娟[1] 余祖江[1] 江河清[1] 梁红霞[1] 武淑环[1] ZHANG Hong-yu;LI Juan;YU Zu-jiang;WU Shu-huan(Department of infectious diseases,the First Affiliated Hospital of Zhengzhou University,Zhengzhou Henan,45005,China)

机构地区：[1]郑州大学第一附属医院感染性疾病科

出　　处：《中西医结合肝病杂志》2019年第4期313-316,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：河南省高等学校重点科研项目(No.18A320038)

摘　　要：目的:探究调控miR-495、脯氨酰4-羟化酶亚基α-1(P4HA2)的表达对肝纤维化、肝细胞癌的发生机制。方法:体外构建P4HA2的3’UTR报告基因质粒,使用荧光素酶报告基因检测miR-495与P4HA2的调控关系。使用miR-495及其反义链处理HepG2肝细胞癌细胞检测P4HA2的表达。低氧条件下检测HepG2肝细胞癌细胞中miR-495和P4HA2的表达,以及细胞增殖反应。在28例肝细胞癌患者中检测miR-495和P4HA2的表达,分析其相关性。结果:萤光素酶报告基因检测显示miR-495显著抑制了HepG2细胞中pGL3-P4HA2-wt的荧光素酶活性,但不能抑制pGL3-P4HA2-mut的荧光素酶活性。miR-495的过表达能够在HepG2细胞中以剂量依赖性方式抑制P4HA2的mRNA和蛋白表达。低氧组HepG2细胞中miR-495的表达显著低于正常组,而P4HA2的表达显著高于正常组。增殖检测表明低氧组HepG2细胞中EdU阳性细胞数显著增加,而在低氧组细胞中过表达miR-495可以明显逆转HepG2细胞的增殖。肝细胞癌中miR-495与P4HA2的mRNA的水平呈负相关(r=-0.683,P<0.05)。结论:miR-495能够通过靶向P4HA2 mRNA的3’UTR来抑制肝细胞癌细胞的增殖,而低氧可以通过下调miR-495来促进P4HA2的表达,进而参与肝纤维化和肝细胞癌的发生发展。Objective:To investigate the mechanism of miR-495 and P4HA2 in regulating hepatocellular carcinoma Methods:The 3 UTR reporter plasmid of P4HA2 was constructed in vitro,and the regulatory relationship between miR-495 and P4HA2 was detected using a luciferase reporter assay.HepG2 hepatoma cells were treated with miR-495 and its antisense strand to detect the expression of P4HA2.The expression of miR-495 and P4HA2 in HepG2 hepatoma cells and the cell proliferation response were detected under hypoxic conditions.The expression correlation of miR-495 and P4HA2 were examined in 28 patients with hepatocellular carcinoma.Results:Luciferase reporter assay showed that miR-495 significantly inhibited the luciferase activity of pGL3-P4HA2-wt in HepG2 cells,but did not inhibit the luciferase activity of pGL3-P4HA2-mut.Overexpression of miR-495 was able to inhibit the expression of P4HA2 mRNA and protein levels in HepG2 cells in a dose-dependent manner.The expression of miR-495 in HepG2 cells in hypoxia group was significantly lower than that in normal group,and the expression of P4HA2 was significantly higher than that in normal control group.Proliferation assay showed a significant increase in the number of EdU-positive cells in the hypoxic group,whereas overexpression of miR-495 in the hypoxic group significantly reversed the proliferation of HepG2 cells.There was a negative correlation between miR-495 and P4HA2 mRNA levels in hepatocellular carcinoma(r=-0.683,P<0.05).Conclusion:miR-495 can inhibit the proliferation of hepatoma cells by targeting the 3 UTR of P4HA2 mRNA,while hypoxia can promote the expression of P4HA2 by down-regulating miR-495,and then participate in the development of cirrhosis and liver cancer.

关 键 词：低氧 miR-495 P4HA2 肝细胞癌 肝纤维化 

分 类 号：R73[医药卫生—肿瘤]