MiRNA-1224-5p Promote Bone Repair By Regulate Osteoblast/osteoclast Differentiation Via The RAP1 Signaling Target ADCY2

Objective: mi RNA,as a class of small non-coding RNAs with regulatory effects,have been widely confirmed to be involved in the regulation of a variety of cell biological functions and have attracted more scientist’s attention as new therapeutic methods.Nonunion is one of the difficult problems in clinical orthopedic treatment.Peripheral blood of fracture patients contains a large number of mi RNA,and the functional study of abnormally expressed mi RNA may provide new therapeutic methods for the treatment of nonunion and osteoporosis.Materials and Methods: After ethical approval,3 fracture patients and 3 volunteers of the same age and classification were enrolled(removed with internal fixation after fracture healing).10 ml peripheral venous serum was collected,and differentially expressed mi RNA in peripheral blood were identified by RNA high-throughput sequencing.After significantly up-regulated mi RNA were analyzed and selected,10 patients with fractures patients and 10 volunteers were enrolled again.Peripheral venous serum was collected at different times after fracture,and real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)was used to verify the expression mi RNA-1224-5p in peripheral blood of patients with fractures and the correlation between mi RNA-1224-5p and fracture healing process.Exogenous overexpressed and silenced mi RNA were constructed,and BMSCs and BMMs were isolated from mouse.First,mi RNA-1224-5p was transfected into BMSCs via Lipo3000,and then BMSCs were induced to differentiate into osteoblasts(OB)and adipocytes(AP).ALP staining,ARS,ORS,quantitative detection ALP concentration,calcium concentration,ORS staining and adipocyte quantitative analysis was used to observe the effect of mi RNA-1224-5p on the osteogenic and adipogenic differentiation of BMSCs.WB and q RT-PCR were used to verify the effects of mi RNA on the expression of key target genes and proteins(ALP,OCN,RUNX2,COL1A1),and to observe the effects of mi RNA on the differentiation of BMSCs into OB and AP.Secondly,mi RNA were transfected into BMMs with transfection reagent Lipo3000 and induced to differentiate into osteoclasts(OC).The expression levels of key target genes and proteins(CTSK,C-FOS,ACP5 and NFATC1)in OC differentiation were determined by WB and q RT-PCR.Finally,the selected mi RNA were administered to mouse fracture site,normal and aging mouse models.Finally,mi RNA-1224-5p were injected into mouse fracture models,young and aging mouse models,and mi RNA-1224-5p was assessed using small animal CT(μCT),hematoxylin-eosin staining(H&E),and TRAP immunohistochemical staining preliminary effects of agomi RNA-1224-5p on toxic and side effects to vital organs,fracture healing process and bone mass deposition in young and aged mouse.Results: The expression of miRNA-1224-5p in peripheral blood of fracture patients was found to be abnormally elevated by high-throughput sequencing.q RT-PCR results confirmed that the expression of mi RNA-1224-5p in peripheral patients after fracture was increased and the expression trend was consistent with the process of fracture healing.WB and a RT-PCR results confirmed that overexpression of mi RNA-1224-5p accelerated the differentiation of BMSCs to OB and inhibited the differentiation of BMSCs to AP.BMMs differentiation test to OC and WB and q RT-PCR results confirmed that overexpression of mi RNA-1224-5p inhibited osteoclast differentiation.μCT and H&E results of mouse bone repair model showed that the experimental dose of mi RNA-1224-5p has no obvious toxic and side effects on important organs,and overexpression of mi RNA-1224-5p could promote fracture healing and increase bone mass in young and aging mouse.Conclusion: The expression of mi RNA-1224-5p in peripheral blood of patients with fracture was abnormally elevated.In vitro overexpression of mi RNA-1224-5p could promote the differentiation of BMSCs into osteoblasts,inhibit the differentiation of BMMs into OC cells,and safely promote fracture healing and bone deposition in young and aging mouse.Objective: In the previous part,overexpression of mi RNA-1224-5p has been confirmed to promote bone mass deposition and bone healing and inhibit the differentiation of BMMs to OC.However,the specific mechanism of overexpression of mi RNA-1224-5p on fracture healing and bone mass deposition has not been reported in the literature.Therefore,the effect and mechanism of mi RNA-1224-5p on OC differentiation were studied in order to unravel the specific mechanism of mi RNA-1224-5p affecting bone metabolism.Materials and Methods: Lipo3000 was used to transfect overexpressed and silenced mi RNA-1224-5p into BMMs cells.Firstly,the bone slice "absorption pit" test,TRAP test and flow cytometry test were used to detect the effect of mi RNA-1224-5p on the differentiation ability of BMMs into OC,the bone resorption function of OC and the apoptosis.Then,RNA-Seq sequencing technology was used to identify the effect of mi RNA-1224-5p on key target genes and signaling pathways in OC differentiation,and then select the key target genes abnormally expressed in OC differentiation(NFATC1,C-FOS,SRC,ACP5)by q RT-PCR verification.The effect of overexpression of mi RNA-1224-5p on RAP1 and ADCY2 protein expression was verified by WB,and the expression of OC cytoskeleton and cell behavior proteins(RAC1,FAK,RHO-A,P-RHO-A,CDC42,P-RAC1)was explored by WB.Next,immunohistochemical staining(IHC)was used to determine the in vivo regulation of mi RNA-1224-5p on key genes(RAP1,ADCY2,NFATC1)in the RAP1 signaling pathway.Subsequently,WB was used to verify the inhibitory effect of overexpression of mi RNA-1224-5p on NFATC1,a key target gene of osteoclast differentiation,and CHIP was used to verify the binding effect of overexpression of mi RNA-1224-5p on ADCY2,RAP1 and NFATC1.Finally,si RNA-ADCY2 was used to silence ADCY2,and the effect of mi RNA-1224-5p targeting ADCY2 and RAP1 signaling pathway on the expression of NFATC1 was verified by WB and CHIP.Results: The bone slice "resorption pit" test,TRAP staining and flow cytometry results showed that overexpression of mi RNA-1224-5p inhibited the differentiation of OC and the bone resorption function of OC,but did not lead to the apoptosis of BMSCs.RNA-Seq sequencing and q RT-PCR showed overexpression of mi RNA-1224-5p led to the down-regulation of genes critical for osteoclast differentiation(NFATC1,C-FOS,SRC,ACP5,CTSK),and the RAP1 signaling pathway and corresponding genes significantly decreased.si RNA-ADCY2 silencing ADCY2 reverse validation and CHIP test confirmed that mi RNA-1224-5p targeted and inhibited ADCY2 and RAP1 protein expression,reduced RAC1 and RHO-A protein and RANKL-induced FAK protein expression.Conclusion: mi RNA-1224-5p targets and inhibits ADCY2 gene to affect the normal expression of osteoclast differentiation genes through RAP1 signaling pathway,thereby inhibiting osteoclast differentiation and bone resorption function.Objective: Overexpression of mi RNA-1224-5p in mouse has been confirmed to promote bone mass deposition and bone healing,and promote the differentiation of BMSCs into OB.However,the specific mechanism by which mi RNA-1224-5p regulates the differentiation of BMSCs into osteoblasts is not clear.Therefore,the research on the effect and mechanism of mi RNA-1224-5p on BMSCs differentiation may reveal the specific mechanism of mi RNA-1224-5p promoting bone repair.Materials and Methods: First,we overexpressed and silenced mi RNA-1224-5p in BMSCs and calvarial osteogenic precursor cells(Pre-Ob),and then used ALP and ARS staining to quantify ALP and calcium nodule concentrations to observe the effect of mi RNA-1224-5p on osteogenesis differentiation effects.Then,WB,q RT-PCR and CHIP techniques were used to observe whether mi RNA-1224-5p affects osteogenic differentiation through RAP1 signaling pathway.Finally,the specific target proteins of mi RNA-1224-5p affecting osteoblast differentiation were investigated by WB to analysis of p-RHO-A,osteocalcin(OCN)and RAC1 proteins.Results: In vitro osteoblast differentiation assay and WB and qRT-PCR results showed that overexpression of mi RNA-1224-5p could promote osteogenic differentiation.Subsequently,WB,q RT-PCR,and CHIP results confirmed that mi RNA-1224-5p regulated the expression of p-RHO-A protein through the RAP1 signaling pathway,and increased the expression of OCN protein.In addition,mi RNA-1224-5p attenuates the phosphorylation of RAC1 protein by BMP2 and FBS,and activates the BMP signaling pathway.Conclusion: mi RNA-1224-5p regulates osteoblast differentiation through RAP1 signaling pathway,and alleviates the inhibition of BMP signaling pathway by RAC1 to activate BMP signaling pathway,and synergistically promote osteoblast differentiation.Objective: In the second and third parts,mi RNA-1224-5p has been confirmed to have an important regulatory effect on osteogenic and osteoclast differentiation.However,the therapeutic effect in the in vivo bone repair model needs further verification.Materials and Methods: Firstly,the distal femoral defect model,skull defect model,OVX(ovarianectomy)and aging osteoporosis model were established,and mi RNA-1224-5p was overexpressed and silenced in vivo,and the blank control group was treated with PBS.Next,ELISA kits were used to detect the expression of the bone resorption marker type I collagen carboxy-terminal peptide beta specific sequence(CTX)and the bone formation marker type I procollagen N-terminal propeptide(PINP)in serum of mouse.Subsequently,bone repair in the cortical / cancellous bone and bone defect sites was observed by μCT,and ADCY2 protein expression in bone tissue was observed by IHC.Finally,the morphological structure of new bone tissue was observed by H&E,the osteoclast differentiation of bone tissue was observed by TRAP staining,and the changes of bone metabolism parameters(BV/TV,BMD,Oc.S/BS,Ob.S/BS)were quantitatively analyzed by SPSS V22.0.Results: The results of μCT 3D reconstruction and H&E showed that overexpression of mi RNA-1224-5p could accelerate bone regeneration in mouse femoral defect model and calvarial defect model,and slow down the progression of osteoporosis caused by aging and ovarianectomy.The quantitative analysis results of bone metabolism parameters,bone resorption and bone formation markers were consistent with the experimental results of animal models.IHC results confirmed that overexpression of mi RNA-1224-5p could significantly reduce ADCY2 protein expression in bone tissue.The results of TRAP and bone metabolism parameters showed that overexpression of mi RNA-1224-5p could significantly reduce the number of osteoclasts and increase the number of osteoblasts in vivo.Conclusion: The experimental results confirmed that mi RNA-1224-5p inhibited osteoclast differentiation,increased osteoblast differentiation,accelerate bone defect regeneration and delayed osteoporosis progression by targeting ADCY2 in vivo.The experimental result laid a theoretical foundation for the clinical trials of mi RNA-1224-5p.