LncRNA-NEF通过吸附miR-21调节绝经后骨质疏松小鼠的免疫失衡和PD-1/PD-1L介导的Treg-Th17细胞平衡

LncRNA-NEF improves immune imbalance and PD-1/PD-1L-mediated Treg-Th17 cell balance in postmenopausal osteoporosis mice by miR-21 adsorption

目的研究长链非编码RNA(lncRNA)NEF对绝经后骨质疏松症(PMOP)小鼠T细胞免疫功能的调节机制。方法使用Balb/c小鼠建立去卵巢模型(OVX,模拟PMOP)(46只),设立假手术(sham)对照组(16只),从这两组小鼠中用随机数字表法各选6只安乐死后分离培养骨髓间充质干细胞(BMSCs);构建NEF的重组表达载体(pIRSE2-NEF)并转染BMSCs,使用RT-qPCR检测sham组、OVX组和pIRSE2-NEF组的BMSCs细胞中的NEF和miR-21水平;荧光素酶基因报告实验验证NEF和miR-21的结合作用。将剩余的40只OVX小鼠分为4组,包括OVX组(10只),pIRSE2-NEF注射组(pIRSE2-NEF组,10只),pIRSE2-NEF联合PD-1抑制剂组(pIRSE2-NEF+PD-L1-IN-1组,10只),pIRSE2-NEF联合miR-21拟似物(mimic)组(pIRSE2-NEF+mimic组,10只)。sham组剩余的10只小鼠作为对照组。ELISA检测外周血IFN-γ、IL-2、IL-4、IL-13、PD-1/PD-1L信号水平,流式细胞术检测血清Treg-Th17细胞亚群偏移情况变化。结果与sham组(1.01±0.04,1.00±0.03)比,OVX组的BMSCs中NEF的表达下调(0.23±0.01),miR-21上调(2.96±0.05)(P<0.05)。与OVX组(1.23±0.15,5.20±0.31)比,pIRSE2-NEF组BMSCs细胞中NEF明显上调(6.83±0.35)(P<0.05),而miR-21下调(0.29±0.11)(P<0.05)。NEF与miR-21具有直接结合碱基位点。OVX组小鼠外周血的IFN-γ(3.25±0.21)、IL-2(2.44±0.06)水平,Th17/Treg比率(3.18±0.65)与sham组(1.03±0.02、1.00±0.01、0.86±0.09)比上升(均P<0.05),而IL-4(0.45±0.02)、IL-13(0.43±0.07)、PD-1(0.24±0.03)及PD-1L(0.51±0.06)的水平与sham组(1.00±0.04、1.00±0.02、1.00±0.03、1.00±0.00)比降低(均P<0.05);而与OVX比,pIRSE2-NEF组的IFN-γ(2.02±0.06)、IL-2水平(0.88±0.01)、Th17/Treg比率(1.43±0.22)降低,IL-4(0.87±0.03)、IL-13(0.84±0.07)、PD-1(0.79±0.06)及PD-1L(0.77±0.06)的水平升高(均P<0.05);与pIRSE2-NEF组比,pIRSE2-NEF+PD-L1-IN-1组中的IFN-γ(2.89±0.06)、IL-2水平(2.07±0.07)、Th17/Treg比率(2.39±0.38)上升,IL-4(0.68±0.03)、IL-13(0.76±0.08)、PD-1(0.52±0.02)及PD-1L(0.83±0.04)的水平降低(均P<0.05);且pIRSE2-NEF+mimic组与pIRSE2-NEF+PD-L1-IN-1组的调节效果一致。结论lncRNA-NEF通过吸附miR-21改善绝经后骨质疏松小鼠免疫失衡和PD-1/PD-1L介导的Treg-Th17细胞平衡。

Objective To investigate the regulatory mechanism of long non-coding RNA(lncRNA)NEF on T cell immune function in postmenopausal osteoporosis(PMOP)mice.Methods Female Balb/c mice were used to construct OVX model(n=46)and sham control group(n=16).Bone marrow mesenchymal stem cells(BM-SCs)from these two groups of mice were cultured.NEF recombinant expression vector(pIRSE2-NEF)was constructed and transfected into BMSCs.RT-qPCR was used to detect NEF and miR-21 levels in BMSCs cells in sham group,OVX group,and pIRSE2-NEF group.Luciferase gene report experiment was used to verify the binding effect of NEF and miR-21.The remaining 40 OVX mice were divided into 4 groups,including OVX group(n=10),pIRSE2-NEF injection group(pIRSE2-NEF group,n=10),pIRSE2-NEF combined with PD-1 inhibitor group(pIRSE2-NEF+PD-L1-IN-1 group,n=10),and pIRSE2-NEF combined with miR-21 mimic(mimic)group(pIRSE2-NEF+mimic group,n=10).The remaining 10 mice in sham group were used as the control group.ELI-SA was used to detect the levels of IFN-γ,IL-2,IL-4,IL-13 and PD-1/PD-1L in peripheral blood.Flow cytometry was used to detect the shift of serum Treg-Th17 cell subsets.Results Compared with the Sham group(1.01±0.04,1.00±0.03),the expression of NEF in BMSCs of OVX group was down-regulated(0.23±0.01),and miR-21 was up-regulated(2.96±0.05)(P<0.05).Compared with OVX group(1.23±0.15,5.20±0.31),NEF in BMSCs cells of Pirse2-nef group was significantly up-regulated(6.83±0.35)(P<0.05),while miR-21 was down-regulated(0.29±0.11)(P<0.05).NEF has a direct binding base site with miR-21.The levels of IFN-γ(3.25±0.21),IL-2(2.44±0.06)and Th17/Treg ratio(3.18±0.65)in peripheral blood of mice in OVX group were significantly higher than those in Sham group(1.03±0.02,1.00±0.01,0.86±0.09)(all P<0.05).The levels of IL-4(0.45±0.02),IL-13(0.43±0.07),PD-1(0.24±0.03)and PD-1L(0.51±0.06)were significantly lower than those of Sham group(1.00±0.04,1.00±0.02,1.00±0.03,1.00±0.00)(P<0.05);Compared with OVX,IFN-γ(2.02±0.06),IL-2(0.88±0.01)and Th17/Treg ratio(1.43±0.22)in Pirse2-nef group were decreased.The levels of IL-4(0.87±0.03),IL-13(0.84±0.07),PD-1(0.79±0.06)and PD-1L(0.77±0.06)were increased(all P<0.05);Compared with Pirse2-nef group,IFN-γ(2.89±0.06),IL-2(2.07±0.07)and Th17/Treg ratio(2.39±0.38)were increased in Pirse2-nef+PD-L1-in-1 group.The levels of IL-4(0.68±0.03),IL-13(0.76±0.08),PD-1(0.52±0.02)and PD-1L(0.83±0.04)were decreased(all P<0.05).Moreover,the pIRSE2-NEF+mimic group had the same adjustment effect as the pIRSE2-NEF+PD-L1-IN-1 group.Conclusion lncRNA-NEF improves immune imbalance and PD-1/PD-1L-mediated Treg-Th17 cell balance in postmenopausal osteoporosis mice by sponging miR-21.