ABSTRACT
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.