调节miR-27b-3p和Nrf2对人RPE细胞代谢记忆形成的抑制作用

Inhibitory effect of miR-27b-3p and Nrf2 regulation on metabolic memory formation in human RPE cells

目的探讨微小RNA-27b-3p(miR-27b-3p)/核因子E2相关因子2(Nrf2)对人视网膜色素上皮(RPE)细胞代谢记忆损伤的影响及其调控机制。方法取ARPE-19细胞,将其置于5.5 mmol/L葡萄糖培养基培养6 d作为正常对照组;于30 mmol/L高糖培养基中培养3 d后,换成5.5 mmol/L葡萄糖培养基继续培养3 d作为代谢记忆组;慢病毒转染细胞并加入嘌呤霉素,筛选转染成功的细胞,并将其置于30 mmol/L高糖培养3 d后换成5.5 mmol/L葡萄糖培养基继续培养3 d作为miR-27b-3p抑制剂组;细胞于30 mmol/L高糖+利拉鲁肽培养基培养3 d后换成5.5 mmol/L葡萄糖培养基继续培养3 d作为利拉鲁肽组。采用实时荧光定量PCR检测miR-27b-3p、Nrf2、醌氧化还原酶-1(NQO1)、血红素加氧酶-1(HO-1)mRNA表达水平;采用Western blot法检测Nrf2总蛋白、核蛋白水平;采用细胞免疫荧光检测Nrf2、NQO1、HO-1蛋白表达水平;采用细胞计数试剂盒8(CCK-8)检测细胞增生率以评估细胞活力;采用DHE试剂盒检测活性氧簇(ROS)水平。结果正常对照组、代谢记忆组、miR-27b-3p对照组和miR-27b-3p抑制剂组miR-27b-3p mRNA相对表达量分别为1.000±0.000、1.881±0.034、1.683±0.088和0.111±0.008,总体比较差异有统计学意义(F=850.815,P<0.001),其中,正常对照组miR-27b-3p mRNA相对表达量低于代谢记忆组,miR-27b-3p抑制剂组miR-27b-3p mRNA相对表达量低于正常对照组,差异均有统计学意义(均P<0.01)。代谢记忆组Nrf2 mRNA及总蛋白、核蛋白相对表达量较正常对照组降低,miR-27b-3p抑制剂组较miR-27b-3p对照组明显升高,差异均有统计学意义(均P<0.01);代谢记忆组NQO1、HO-1 mRNA相对表达量较正常对照组降低,miR-27b-3p抑制剂组较miR-27b-3p对照组明显升高,差异均有统计学意义(均P<0.01)。代谢记忆组Nrf2、NQO1、HO-1荧光强度均低于正常对照组,miR-27b-3p抑制剂组Nrf2、NQO1、HO-1荧光强度均高于miR-27b-3p对照组,差异均有统计学意义(均P<0.01)。与代谢记忆组相比,利拉鲁肽组miR-27b-3p mRNA相对表达量下降,差异有统计学意义(P<0.05)。利拉鲁肽组Nrf2、NQO1、HO-1 mRNA、Nrf2总蛋白及核蛋白相对表达水平均较代谢记忆组升高,差异均有统计学意义(均P<0.05),利拉鲁肽组荧光强度较代谢记忆组增强,差异均有统计学意义(均P<0.05)。与正常对照组及利拉鲁肽组相比,代谢记忆组细胞增生活力均下降,差异均有统计学意义(均P<0.01)。代谢记忆组ROS相对含量较正常对照组及利拉鲁肽组升高,差异均有统计学意义(均P<0.01)。结论利拉鲁肽通过下调miR-27b-3p逆转代谢记忆对Nrf2、NQO1和HO-1的抑制作用。

Objective To investigate the effect of microRNA-27b-3p(miR-27b-3p)/nuclear factor-E2-related factor 2(Nrf2)on metabolic memory impairment of human retinal pigment epithelial(RPE)cells and to explore its regulatory mechanism.Methods ARPE-19 cells were divided into normal control group,metabolic memory group,miR-27b-3p control group,miR-27b-3p inhibitor group,and liraglutide group.Cells in normal control group were cultured in 5.5 mmol/L normal glucose medium for 6 days.Cells in metabolic memory group were cultured in 30 mmol/L glucose for 3 days and changed to 5.5 mmol/L for 3 days.Cells in miR-27b-3p inhibitor group were added with puromycin after lentiviral transfection to select the successfully transfected cells,and were cultured in 30 mmol/L glucose for 3 days then 5.5 mmol/L glucose for 3 days.Cells in liraglutide group were cultured in 30 mmol/L glucose with liraglutide for 3 days then 5.5 mmol/L glucose for 3 days.The regulatory relationship between miR-27b-3p and Nrf2 was verified by lentiviral transfection.Expressions of miR-27b-3p,Nrf2,NAD(P)H dehydrogenase[quinone]1(NQO1),heme oxygenase-1(HO-1)mRNA and protein levels were analyzed by real-time quantitative PCR.Total and nuclear Nrf2 protein expressions were detected by Western blot.The cell proliferation rates of various groups were determined by cell counting kit-8(CCK-8).The reactive oxygen species(ROS)level was detected by the DHE kit.Results The miR-27b-3p mRNA relative expression of normal control group,metabolic memory group,miR-27b-3p control group,miR-27b-3p inhibitor group was 1.000±0.000,1.881±0.034,1.683±0.088 and 0.111±0.008,respectively,with a statistically significant difference(F=850.815,P<0.001).The miR-27b-3p mRNA relative expression level was lower in normal control group than in metabolic memory group,lower in miR-27b-3p inhibitor group than in normal control group,and the differences were statistically significant(both at P<0.01).The expression levels of Nrf2 mRNA,total protein,and nuclear protein were decreased in metabolic memory group in comparison with normal control group and were significantly increased in miR-27b-3p inhibitor group in comparison with miR-27b-3p control group,showing statistically significant differences(all at P<0.01).The NQO1 and HO-1 mRNA expressions were decreased in metabolic memory group in comparison with normal control group,and were significantly higher in miR-27b-3p inhibitor group compared with miR-27b-3p control group,showing statistically significant differences(all at P<0.01).The fluorescence intensity of Nrf2,NQO1,and HO-1 was lower in metabolic memory group than in normal control group,and was higher in miR-27b-3p inhibitor group than in miR-27b-3p control group,showing statistically significant differences(all at P<0.01).Compared with metabolic memory group,the relative expression of miR-27b-3p mRNA declined in liraglutide group,with a statistically significant difference(P<0.05).The relative expression levels of Nrf2 mRNA,NQO1 mRNA,HO-1 mRNA,total and nuclear Nrf2 protein of liraglutide group were enhanced in comparison with metabolic memory group,with statistically significant differences(all at P<0.05).The fluorescence intensity of Nrf2,NQO1,and HO-1 was enhanced in liraglutide group in comparison with metabolic memory group,and the differences were statistically significant(all at P<0.05).Compared with normal control group and liraglutide group,the cell proliferation viability was decreased in metabolic memory group,and the differences were statistically significant(both at P<0.01).The relative content of ROS was higher in metabolic memory group than in normal control group and liraglutide group,and the difference was significant(all at P<0.01).Conclusions Liraglutide reverses the inhibition of metabolic memory on Nrf2,NQO1,and HO-1 by downregulating miR-27b-3p.