MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation By Targeting Tumor Necrosis Factor Receptor 1

Background and Objectives:Postmenopausal osteoporosis(PMOP)is a systemic metabolic bone disease associated with estrogen deficiency leading to bone loss and bone tissue changes,causing bone fragility and increased risk of fracture,seriously affecting the health and quality of life of the elderly,rendering it an important health issue today.The dynamic balance of bone tissue requires the maintenance of osteoblast bone formation and osteoclast bone resorption.The reduction of estrogen levels can lead to a break in the balance between bone absorption and bone formation.When bone resorption in osteoclasts exceeds the osteogenesis of osteoblasts,it will lead to the occurrence of osteoporosis,resulting in osteopenia and traumatic fracture.MicroRNA(miRNA)is a class of noncoding single strand RNA encoded by endogenous genes,with approximately ~22 nucleotides,which are paired with 3'UTR of the target gene by the complementary pairing of bases,thus negatively regulating the transcription and translation of target genes.Previous studies have shown that miRNAs can play an important role in the development of postmenopausal osteoporosis by regulating the differentiation and activity of osteoblasts and osteoclasts.Previous studies were mainly focused on the relationship between miR-218 and osteogenesis in osteoblasts.However,the mechanism of miR-218 on osteoclast differentiation is still unclear.The purpose of this study is to explore the role of miR-218 in osteoclast differentiation and the molecular mechanism of osteoclast differentiation.Material and Methods:Firstly,RAW 264.7 cells were induced to differentiate into osteoclasts stimulated with RANKL(receptor activator for nuclear factor-? B ligand),and the changes of miR-218 expression level in RAW 264.7 cells during osteoclast differentiation were explored.Positive Trap staining shows the success of osteoclast induction.Realtime PCR and western blot preliminarily validated the expression levels of osteoclast related markers NFATc1(nuclear factor of activated T cells 1)and TRAF6(tumor necrosis factor receptor-associated factor 6)during osteoclastic differentiation.Next,we designed miR-218 mimics,inhibitors and negative control(miR-NC).MiR-218 mimics,inhibitors or negative control were transfected in RAW 264.7 cells to overexpress or inhibit the expression of miR-218,and then RANKL induced osteoclast differentiation,and the effect of miR-218 on osteoclast differentiation was studied.The expression levels of osteoclast related markers NFATc1 and TRAF6 were altered,when miR-218 was overexpressed or inhibited.TargetScan,miRanda and PicTar were used to predict the potential target genes for miR-218.In the candidate target genes we found,the 3 'UTR of TNFR1(tumor necrosis factor receptor 1)has a miR-218 binding site,and TNFR1 was closely related to the signaling pathway of osteoclast differentiation.Wild type(WT)or mutant type(Mut)TNFR1 3'UTR and miR-218 were transfected in HEK293 T cells,and luciferase activity was detected to verify the targeting relationship between miR-218 and TNFR1.Results:In the process of RAW 264.7 cells differentiated into osteoclasts,the expression level of miR-218 decreased.When miR-218 was overexpressed,the differentiation of RAW 264.7 cells to osteoclasts was inhibited.The mRNA level and protein level of the osteoclast related marker genes,NFATc1 and TRAF6,were all decreased,and the count of Trap positive cells decreased significantly.However,inhibition of the expression of miR-218 promoted the differentiation of RAW 264.7 cells to osteoclasts.The mRNA level and protein level of the osteoclast related marker genes,NFATc1 and TRAF6,were increased,and the count of Trap positive cells increased significantly.Among the candidate genes predicted by TargetScan,miRanda and PicTar,TNFR1 has a miR-218 binding site and is closely related to the signaling pathway of osteoclast differentiation.When the wild-type or mutant TNFR1 3'UTR and miR-218 were co transfected in the luciferase reporter gene experiment,miR-218 could significantly inhibit the expression of wild type TNFR1,but it could not inhibit the expression of mutant TNFR1.Bioinformatics analysis and luciferase reporter gene experiments showed that TNFR1 is a direct target of miR-218.Conclusion:Our results show that miR-218 inhibits the downstream NF-?B signaling pathway by targeting TNFR1,negatively regulates osteoclast differentiation and inhibits the occurrence and development of osteoporosis.The results suggest that targeting miR-218 may be a potential therapy for postmenopausal osteoporosis.