miR-21对肝细胞缺氧复氧损伤的影响

Effect of miR-21 on hypoxia-reoxygenation injury in hepatocellular carcinoma cells

目的探讨miR-21对肝细胞缺氧复氧损伤的影响。方法将Hep G2细胞随机分为正常组、缺氧复氧组、miR-21 mimic处理组(实验组)和NC组,正常组不予任何处理,缺氧复氧组及实验组建立肝细胞缺氧复氧损伤模型,实验组在进行缺氧复氧前2 h,Hep G2细胞内加入miR-21 mimic 100 nmol/L。NC组在缺氧复氧2 h前,加入等体积的无效miRNA。采用CCK-8法测算细胞存活率、双染法测算细胞凋亡率,采用Western blotting法检测细胞蛋白表达,同时测定乳酸脱氢酶(LDH)活性,且使用实时荧光定量PCR法检测miR-21表达。结果实验组、NC组、缺氧复氧组、正常组miR-21相对表达量分别为145.33±22.18、2.33±1.02、2.71±1.22、16.65±0.98,缺氧复氧组的miR-21表达量低于正常组(P<0.05),实验组高于缺氧复氧组(P<0.05)。缺氧复氧组的细胞存活率低于正常组(P<0.05),LDH活性高于正常组(P<0.05);实验组的细胞存活率高于缺氧复氧组(P<0.05),LDH活性低于缺氧复氧组(P<0.05)。实验组、NC组、缺氧复氧组、正常组细胞凋亡率分别为20.21%±1.73%、45.46%±1.63%、47.83%±1.45%、2.14%±0.33%,与正常组相比,缺氧复氧组、实验组、NC组的细胞凋亡率升高(P均<0.05),其中缺氧复氧组的细胞凋亡率最高(P<0.05)。缺氧复氧组的PTEN、AKT蛋白表达高于正常组(P均<0.05),实验组的PTEN、AKT蛋白低于缺氧复氧组(P均<0.05)。结论 miR-21可减轻缺氧复氧导致的肝细胞凋亡,提高肝细胞存活率,抑制LDH的释放,从而减轻肝细胞损伤。

Objective To investigate the effect of microRNA-21（ miR-21） on the hypoxia-reoxygenation injury in hepatocellular carcinoma cells. Methods The hepatocellular carcinoma Hep G2 cells were divided into the normal group（ without treatment）,hypoxia-reoxygenation group,miR-21 mimic treatment group（ experimental group）,and NC group.We established the models of hypoxia-reoxygenation injury in the hypoxia reoxygenation group and experimental group,and the cells in the experimental group were given miR-21 mimic 100 nmol/L at 2 h before hypoxia-reoxygenation. The equal volume ineffective miRNA was added to the cells in the NC group at 2 h before hypoxia-reoxygenation. The cell survival rate and apoptosis were detected by the CCK-8 assay and double-staining,the protein expression and the lactate dehydrogenase（ LDH） activity were detected by Western blotting,and the miR-21 expression was determined by the real-time fluorescent quantitative RT-PCR. Results The miR-21 expression in the experimental group,NC group,hypoxia-reoxygenation group and normal group were 145. 33 ± 22. 18,2. 33 ± 1. 02,2. 71 ± 1. 22,and 16. 65 ± 0. 98,respectively; the miR-21 expression of the hypoxia-reoxygenation group was lower than that of the normal group,and the experimental group was higher than the hypoxia-reoxygenation group（ both P〈0. 05）. The cell survival rate in the hypoxia-reoxygenation group was lower than that of the normal group,and the activity of LDH was higher than that of the normal group（ both P〈0. 05）. The cell survival rate in the experimental group was higher than that of the hypoxia-reoxygenation group,LDH activity was lower than that of the hypoxia-reoxygenation group（ both P〈0. 05）. The apoptosis rates in the experimental group,NC group,hypoxia-reoxygenation group,and normal group were 20. 21% ± 1. 73%,45. 46% ± 1. 63%,47. 83% ± 1. 45%,and 2. 14% ±0. 33%. The apoptosis rates of the hypoxia-reoxygenation group,experimental group,and NC group significantly increased as compared with the normal group,and the hypoxia-reoxygenation group was the highest. The expression levels of PTEN and AKT protein in the hypoxia-reoxygenation group were higher than those of the normal group,and the expression levels of PTEN and AKT of the experimental group were lower than those of the hypoxia-reoxygenation group（ both P〈0. 05）.Conclusion miR-21 can alleviate the apoptosis of hepatocytes induced by anoxia reoxygenation,increase the survival rate of hepatocytes,inhibit the release of LDH,and thereby lessen the injury of hepatocytes.