The Effect And Mechanism Of MiR-145 In Endochondral Ossification

The bone is mainly formed by intramembranous osteogenesis and endochondral ossification.The growth of limbs and trunk bones is mainly through endochondral ossification.The process begins with the accumulation,proliferation of mesenchymal cells,and differentiate into chondrocytes,which then proliferate,mature and hypertrophy into hypertrophic chondrocytes.And then undergo the stage of hypertrophic chondrocytes apoptosis,calcification,vascular invasion with osteoclasts and osteogenesis,followed by the degradation of the cartilage matrix and formation of trabecular bone.Finally,new bone tissue replaces hypertrophic cartilage tissue to form bone,forming new bone.Researchers have construct tissue engineering bone via re-enacting endochondral ossification,which is beneficial to vascularization,so it is expected to solve the problem of early blood supply deficiency in bone repair based on endochondral ossification.Mature chondrocyte hypertrophy changes and terminal differentiation is a key link in endochondral ossification.Hypertrophic chondrocytes secrete cartilage matrix rich in type X collagen,and then terminally differentiated hypertrophic chondrocytes secrete a variety of important factors with highly coordinated,comprehensive regulation.Therefore,it is of great value for endochondral ossification to study the regulation mechanism of chondrocyte hypertrophy and terminal differentiation.There is increasing evidence that miRNAs play an important regulatory role in endochondral ossification.Previously,our team had confirmed that miR-145 could target Sox9 to regulate mesenchymal stem cells to differentiate into cartilage,but whether miR-145 regulates related genes in the process of chondrocyte hypertrophy and affects endochondral ossification are still not clear.This study aims to investigate the role of miR-145 in endochondral ossification and its effect on bone repair,and also the regulation mechanism of TNFRSF11 B,a target gene of miR-145,in the process of chondrocyte hypertrophy.Materials and Methods:1.To observe the development of femur in newly born mice,morphological staining and immunohistochemistry were used to certain the chondrocyte quiescent area,chondrocyte proliferative area and chondrocyte hypertrophic area.The distribution of miR-145 was detected by fluorescence in situ hybridization.To observe the effect of miR-145 on chondrocyte hypertrophy,through overexpressing miR-145 in Bone marrow mesenchymal stem cells(BMSCs)-derived chondrocytes,then continuing hypertrophy induction,the markers gene related to hypertrophy were detected by RT-qPCR and western blot.And Balb/c Mice were contributed to construct a femoral fracture model to observe the role of miR-145 in endochondral ossification.2.Bioinformatics web-based software and dual luciferase reporter gene were combined to detect the targeting relationship between TNFRSF11 B and miR-145.Then the changes of TNFRSF11 B expression in hypertrophic chondrocytes were detected by RT-qPCR and Western blot,and the inhibition of TNFRSF11 B expression on BMSCs-derived chondrocytes was performed to detect the expression of hypertrophic related marker genes from mRNA and protein levels,respectively.RT-qPCR was used to detect the effect of mangiferin on miR-145 in BMSCs-derived chondrocytes.Results:1.Morphological results showed that the three layers of chondrocytes in the femur growth plate of the mice were arranged in a columnar shape and increased in size,which were divided into resting zone,proliferative zone and hypertrophic zone.In the hypertrophic zone of chondrocytes,the expression of Col10a1 and CD31 increased.Fluorescence in situ hybridization showed that miR-145 was most strongly expressed in the hypertrophic region of chondrocytes than in the resting and proliferative regions;2.RT-qPCR and Western blot showed that after overexpression of miR-145,the hypertrophic genes expression markers of Col10a1,MMP13,Runx2 were highly expressed in the control and inhibition groups(p<0.05),and the expression levels of Osterix,Col1a1 and Vegfa were also higher than those in the control and the inhibition group.In the mouse fracture model,HE staining,Masson staining and immunohistochemistry showed that the overexpressed miR-145 group can promote cartilage matrix degradation and trabecular bone formation,immunity Fluorescence showed that the expression of Vegfa was stronger than that of the control group.The results indicate that miR-145 enhances the process of endochondral ossification by promoting chondrocyte hypertrophy,thereby promoting bone repair;3.The dual luciferase reporter gene showed that miR-145 significantly down-regulated the expression of luciferase in TNFRSF11 B.In hypertrophic chondrocytes,TNFRSF11 B were decreased significantly in mRNA and protein levels;after inhibition of TNFRSF11 B expression,Col10a1,MMP13,Runx2 and Vegfa expression was higher than the control group(p<0.05).Therefore,these results showed that low expression of TNFRSF11 B could promote terminal differentiation of hypertrophic chondrocytes.The qPCR results showed that the expression of miR-145 was significantly higher than that of the control group in hypertrophic chondrocytes after MAG administration,and the expression of TNFRSF11 B was significantly lower than that in the control group(p<0.01).Conclusion:In this study,a bone repair system based on endochondral ossification was established by fracture model.It was proved that miR-145 can promote hypertrophy differentiation of chondrocytes,accelerate cartilage matrix degradation and trabecular bone formation during endochondral ossification by vitro and vivo experiments,thereby promoting new bone formation.In mechanism studies,TNFRSF11 B is a target gene of miR-145.miR-145 can inhibit the expression of TNFRSF11 B,increase the expression of Col10a1,MMP13,Runx2 and Vegfa at mRNA and protein levels,and promote the terminal differentiation of hypertrophic chondrocytes.In addition,it was preliminarily proved that mangiferin can promote the expression of miR-145 in hypertrophic chondrocytes derived from BMSCs and reduce the mRNA level of TNFRSF11 B.Further experiments are still needed to clarify and certain how mangiferin regulates the expression of miR-145 and the molecular mechanisms and signaling pathways of TNFRSF11 B during chondrocyte hypertrophy.