MiR-142 Regulates FOXO1 Promotes Osteoclast Differentiation

Objective:Osteoporosis OP is a metabolic bone disease characterized by high risk,high incidence,and prone to fracture.The pathogenesis is mainly caused by the imbalance between bone resorption of osteoclasts(Osteoclast OC)and bone formation of osteoblasts(Osteoblast OB).Studies have shown that microRNA plays an important role in the differentiation of osteoclasts.miRNA is a non-coding single-stranded RNA consisting of about 20-24 nucleotides.It exerts a gene silencing effect on mRNA mainly at the post-transcriptional level through the sponge adsorption effect.MiR-183,miR-214,and miR-142 can promote osteoclastogenesis.Cell differentiation then affects diseases such as osteoporosis.FOXO1 is a member of the forkhead transcription factor(FOXO)that regulates biological processes such as cell proliferation,gluconeogenesis,energy metabolism,and oxidative stress.FOXO1 can mediate osteoclast maturation and differentiation induced by RANKL pathway.This study used cell transfection techniques to observe the effect of miR-142 overexpression or silencing on osteoclast differentiation and FOXO1.Methods:1.Osteoclast-inducing culture:6-8 week-old KM mice were sacrificed by cervical dislocation,femoral bone and tibial bone marrow cells were extracted,and 50ng/ml RANKL was induced to incubate BMMS in vitro for 5 days,respectively,at 0,1,3,and 5 days.The osteoclastogenesis was observed under a microscope.2.Differential expression of miR-142:During the differentiation of BMMs to osteoclasts,the expression of miR-142-3p and miR-142-5p gradually increased over time;by the fifth day,the expression level of miR-142 was increased.About 20 times the first day.3.Verify that miR-142 promotes osteoclast differentiation:Transfection of BMMs into cells using Lipofectamine 2000 plasmid is divided into(1)blank control group;(2)NC group;(3)miR-142mimics group;(4)miR-142 inhibitor group;50 ng/ml RANKL induction after 5 days real-time quantification The expression levels of miR-142,MMP-9,Acp-5,Integrin av and Cathepsin K mRNA in each group were detected by PCR;osteoclastogenesis was observed by TRAP staining;actin in osteoclasts was observed by circular actin staining.The optical density of rings was observed to observe the bone resorption function of osteoclasts.4.Verify that miR-142 regulates FOXO1:NC group,miR-142mimics group,and miR-142inhibitor group cells were induced by 50ng/ml RANKL for 2 days.Real-time quantitative PCR and Western-blot were used to detect the mRNA and protein expression of FOXO1 in each group.5.verify FOXOl promote osteoclast differentiation:BMMs are divided into three groups:blank control group,RANKL group,AS 1842856 group;Western-blot detection of FOXO1,Nfatc1 protein expression levels.6.Verify that miR-142 regulates FoxO1 pathway to promote osteoclast differentiation:5uM AS1842856+20nM miR-142mimics co-transfect BMMs cells by Lipofectamine 2000 plasmid,and stimulated by 50ng/ml RANKL for 2 days,qRT-PCR,Western-blot detection The expression levels of FOXO1,Nfatc1,CathepsinK,Accp-5,MMP-9 mRNA and protein in each group.Results:1.Differentiation of BMMs into osteoclasts induced by RANKL:BMMs cells were almost unchanged at 1d,a few polynuclear giant cells formed at 3d,and some small cells aggregated at the same time.There was a large number of multinucleated mature osteoclasts at 5d.The cells multiply and form.2.Differential expression of miR-142:During the differentiation of BMMs to osteoclasts,the expression of miR-142-3p and miR-142-5p gradually increased over time;by the fifth day,the expression level of miR-142 was increased.About 20 times the first day.3.miR-142 promoted the differentiation of osteoclasts:When miR-142 was overexpressed,the levels of miR-142,Acp-5,MMP9,Integrin av,and Cathepsin K in the cells were significantly higher than those in the control group(P<0.001).However,miR-142 decreased when it was inhibited(P<0.01),and the miR-142 inhibitory group miR-142 was consistent with the down-regulation of the osteoclast differentiation marker;tartaric acid acid phosphatase showed that the miR-142 overexpression group had a large number of TRAPs.Staining-positive multinucleated giant cells formed;while the inhibition group had only a few and almost no TRAP-positive cells;circular actin staining results miR-142mimics group can produce a complete and clear fusion Actin ring structure,while the inhibition group Actin ring number was significant Decrease,and there is a loop in,a contraction,or even a rupture.4.miR-142 regulates FOXO1 expression:At the gene level,FOXO1 mRNA expression was up-regulated in miR-142 overexpression group and downregulated in miR-142 inhibition group(P<0.05);FOXO1 protein expression was upregulated when miR-142 protein was overexpressed(P<0.05),the inhibition group decreased.5,FOXO1 promotes osteoclast differentiation:compared with the blank group,RANKL group,AS 1842856 group FOXO1 protein expression levels were significantly reduced(P<0.05),Nfatcl protein expression was also significantly reduced(P<0.05).6.miR-142 promotes osteoclast differentiation through the FOXO1 pathway:?Gene expression levels of FOXO1(P<0.001),Nfatc1(P<0.01),CathepsinK(P<0.001),Accp-5(P<0.001),MMP-9 mRNA(P<0.001)in miR-142mimics group Significantly increased;and AS 1842856 + miR-142mimics group,no significant difference in mRNA expression.?Protein level,FOXO1(P<0.001),Nfatc1(P<0.001),CathepsinK(P<0.05),Accp-5(P<0.001),MMP-9(P<0.001)protein expression levels in miR-142mimics group The expression of FOXO1 and Accp-5 proteins in the AS 1842856+miR-142mimics group was not significantly different,with Nfatc1(P<0.01),CathepsinK(P<0.05),and MMP-9(P<0.001).Protein expression was significantly reduced.Conclusion:(1)High expression of miR-142 promotes osteoclast differentiation.(2)miR-142 regulates the FOXO1 axis to promote osteoclast differentiation.(3)miR-142 as a research target for the treatment of osteoporosis,reduce bone loss.