miR-125a-5p通过靶向HIF1AN调控HIF-1α参与小胶质细胞活化

miR-125a-5p is involved in microglial activation by regulating HIF-1αthrough targeting HIF1AN

目的 观察miR-125a-5p通过靶向HIF1AN调控HIF-1α在BV-2小胶质细胞炎症模型中对小胶质细胞的活化作用。方法 首先用LPS 150μg/L+IFN-γ 20μg/L建立BV-2小胶质细胞炎症模型;将BV-2小胶质细胞分为对照组(Con组)、小胶质细胞炎症模型组(LPS+IFN-γ组),用实时荧光定量PCR检测miR-125a-5p、HIF1AN mRNA表达水平,用Western blotting检测iNOS和HIF-1α蛋白的表达。再通过调控miR-125a-5p、HIF-1α的表达,用荧光定量PCR法检测miR-125a-5p,HIF-1α、促炎因子(包括IL-1β、TNF-α)、M1极化标志物iNOS mRNA表达水平,用Western blotting法检测HIF1AN、HIF-1α的蛋白表达量。结果 与Con组相比,LPS+IFN-γ组miR-125a-5p,iNOS、IL-1β和TNF-α mRNA表达升高(P<0.05),HIF1AN mRNA表达下降(P<0.05),HIF-1α、iNOS蛋白表达升高(P<0.05)。上调miR-125a-5p后,与NC agomir+LPS+IFN-γ组相比,miR agomir+LPS+IFN-γ组miR-125a-5p,IL-1β、TNF-α和iNOS mRNA表达升高(P<0.05),HIF1AN蛋白表达下降(P<0.05),HIF-1α蛋白表达升高(P<0.05);下调miR-125a-5p表达后,与NC antagomir+LPS+IFN-γ组相比,miR antagomir+LPS+IFN-γ组miR-125a-5p,IL-1β、TNF-α和iNOS mRNA表达下降(P<0.05),HIF1AN蛋白表达上升(P<0.05),HIF-1α蛋白表达下降(P<0.05);敲低HIF-1α的表达后,结果显示,与NC siRNA+LPS+IFN-γ组相比,HIF-1α siRNA+LPS+IFN-γ组HIF-1α、IL-1β、TNF-α和iNOS mRNA表达下降(P<0.05)。结论 miR-125a-5p通过靶向HIF1AN调控HIF-1α参与了BV-2小胶质细胞活化。

Objective To observe the effect of miR-125a-5p on microglial activation in BV-2 microglial inflammation model by regulating HIF-1αthrough targeting HIF1AN.Methods First,BV-2 microglial inflammation model was established with LPS 150μg/L+IFN-γ20μg/L.BV-2 microglia were divided into control group(Con group)and microglial inflammation model group(LPS+IFN-γgroup).The expressions of miR-125a-5p and HIF1AN mRNA were detected by real-time quantitative PCR,and the protein expressions of iNOS and HIF-1αwere detected by Western blotting.Then,by regulating the expressions of miR-125a-5p and HIF-1α,the expression level of miR-125a-5p and the mRNA expression level of HIF-1α,pro-inflammatory factors(including IL-1βand TNF-α)and M1 polarization marker iNOS were detected by fluorescence quantitative PCR.The protein expression levels of HIF1AN and HIF-1αwere detected by Western blotting.Results Compared with Con group,the mRNA expressions of miR-125a-5p,iNOS,IL-1βand TNF-αincreased(P<0.05),the mRNA expression of HIF1AN decreased(P<0.05),and the protein expressions of HIF-1αand iNOS increased(P<0.05)in LPS+IFN-γgroup.After up-regulation of miR-125a-5p expression,compared with NC agomir+LPS+IFN-γgroup,the mRNA expressions of miR-125a-5p,IL-1β,TNF-αand iNOS increased(P<0.05),the protein expression of HIF1AN decreased(P<0.05),and the protein expression of HIF-1αincreased(P<0.05)in miR agomir+LPS+IFN-γgroup.After down-regulation of miR-125a-5p expression,compared with NC antagomir+LPS+IFN-γgroup,the mRNA expressions of miR-125a-5p,IL-1β,TNF-αand iNOS decreased(P<0.05),the protein expression of HIF1AN increased(P<0.05),and the protein expression of HIF-1αdecreased(P<0.05)in miR antagomir+LPS+IFN-γgroup.After knockdown of HIF-1αexpression,the results showed that compared with NC+LPS+IFN-γgroup,the mRNA expressions of HIF-1α,IL-1β,TNF-αand iNOS decreased(P<0.05)in HIF-1αsiRNA+LPS+IFN-γgroup.Conclusion miR-125a-5p is involved in BV-2 microglial activation by regulating HIF-1αthrough targeting HIF1AN.