【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。更多还原

【Abstract】 Objective:To investigate the mechanism of miR-495 and P4 HA2 in regulating hepatocellular carcinoma Methods:The 3’UTR reporter plasmid of P4 HA2 was constructed in vitro, and the regulatory relationship between miR-495 and P4 HA2 was detected using a luciferase reporter assay. HepG2 hepatoma cells were treated with miR-495 and its antisense strand to detect the expression of P4 HA2. The expression of miR-495 and P4 HA2 in HepG2 hepatoma cells and the cell proliferation response were detected under hypoxic conditions. The expression correlation of miR-495 and P4 HA2 were examined in 28 patients with hepatocellular carcinoma.Results:Luciferase reporter assay showed that miR-495 significantly inhibited the luciferase activity of pGL3-P4 HA2-wt in HepG2 cells, but did not inhibit the luciferase activity of pGL3-P4 HA2-mut. Overexpression of miR-495 was able to inhibit the expression of P4 HA2 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 P4 HA2 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 P4 HA2 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 P4 HA2 mRNA, while hypoxia can promote the expression of P4 HA2 by down-regulating miR-495, and then participate in the development of cirrhosis and liver cancer.更多还原