下调miRNA-217对UVB诱导人皮肤成纤维细胞氧化损伤及Sirt1/FOXO1通路的影响

Effects of down-regulating miRNA-217 on UVB-induced oxidative damage and Sirt1/FOXO1 pathway in human skin fibroblasts

目的探讨下调微RNA-217(miRNA-217)对中波紫外线(UVB)诱导的人皮肤成纤维细胞(HFF-1)氧化损伤及沉默信息转录调节因子1(Sirt1)/叉形头转录因子1(FOXO1)信号通路的影响。方法将未损伤和UVB损伤的HFF-1细胞分为正常组、对照组、miRNA-217阴性对照(NC)组和miNAR-217抑制剂组。实时荧光定量PCR(RT-qPCR)法检测miRNA-217表达情况;细胞计数试剂盒8(CCK-8)法检测细胞生长情况;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)法检测细胞凋亡情况;2′,7′-二氯荧光黄双乙酸盐(DCHF-DA)荧光探针法检测活性氧(ROS)表达;过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和丙二醛(MDA)试剂盒检测该指标水平;Western blot检测Sirt1、FOXO1和乙酰化FOXO1(Ac-FOXO1)蛋白表达。结果与对照组和miRNA-217 NC组比较,miRNA-217抑制剂组HFF-1细胞miRNA-217、凋亡率及MDA、ROS、Ac-FOXO1蛋白表达和Ac-FOXO1/FOXO1比值明显降低(P<0.05),细胞存活率、CAT、SOD表达水平、Sirt1和FOXO1蛋白表达均明显升高(P<0.05);对照组与miRNA-217 NC组上述各指标水平比较,差异均无统计学意义(P>0.05);与正常组比较,对照组上述各指标变化呈相反趋势。结论下调miRNA-217可能通过促进Sirt1/FOXO1信号通路,保护UVB诱导下HFF-1细胞免受氧化损伤。

Objective To explore the effect of down-regulating microrna-217(miRNA-217)on medium wave ultraviolet B ray(UVB)-induced oxidative damage and silencing information transcription regulator 1(Sirt1)/forkhead transcription factor 1(FOXO1)signal pathway of human skin fibroblasts(HFF-1).Methods The undamaged and UVB damaged HFF-1 cells were divided into the normal group,control group,miRNA-217 negative control(NC)group and miRNA-217 inhibitor group.The expression of miRNA-217 was detected by real-time quantitative PCR(RT-qPCR);the cell counting kit 8(CCK-8)method was used to detect the growth of cells;the Annexin V-fluorescin isothiocyanate/propidium iodide(Annexin V-FITC/PI)assay was used to detect the apoptosis;the expression of reactive oxygen species(ROS)in HFF-1 cells was detected by 2′,7′-dichlorofluorescein diacetate(DCHF-DA);the catalase(CAT),superoxide dismutase(SOD)and malondialdehyde(MDA)kits were used to detect the levels of above indexes;and Western blot analysis was used to detect the expression of Sirt1,FOXO1 and acetylated FOXO1(Ac-FOXO1).Results Compared with the control group and miRNA-217 NC group,the miRNA-217,apoptosis rate,MDA expression,ROS level,Ac-FOXO1 protein expression and Ac-FOXO1/FOXO1 ratio in HFF-1 cells of the miRNA-217 inhibitor group were significantly decreased(P<0.05),the survival rate,CAT and SOD expression levels,Sirt1 and FOXO1 protein expressions were significantly increased(P<0.05);there was no significant difference in the above indexes between the control group and miRNA-217 NC group(P>0.05);compared with the normal group,the changes of above indexes in the control group showed an opposite trend.Conclusion The down-regulation of miRNA-217 protects HFF-1 cells from oxidative damage induced by UVB possibly by promoting Sirt1/FOXO1 signaling pathway.