The Role And Mechanism Of Long Non-coding RNA H19 In The Process Of MSCs Chondrocytic Terminal Differentiation Induced By BMP2 And Fracture Repair

Objective: Elucidating the mechanism of terminal differentiation of mesenchymal stem cells(MSCs)induced by bone morphogenetic protein 2(BMP2)is very important for constructing BMP2-mediated tissue engineering cartilage and solving poor fracture healing.Previous studies have shown that LncRNA H19 can regulate a variety of cellular biological processes,including cell proliferation,apoptosis and differentiation.The purpose of this study is to explore:(1)whether LncRNA H19 can regulate the terminal differentiation of MSCs mediated by BMP2 and its related mechanism;(2)The role and mechanism of LncRNA H19 in endochondral ossificationt-mediated fracture repair by referring the regulatory effect of LncRNA H19 on chondrogenic terminal differentiation of MSCs.Materials and methods:(1)In this study,we established a chondrogenic differentiation model of MSCs induced by BMP2 in vitro and in vivo to explore the effect of LncRNA H19 on chondrogenic differentiation and chondrocyte terminal differentiation induced by BMP2.FISH analysis was used to detect the expression trend of LncRNA H19 at chondrocytes of growth plate in fetal mouse forelimb.The exogenous gene of BMP2 and LncRNA H19 was silenced or overexpressed by adenovirus vector.The factors expression of cartilage differentiation was analyzed by RT-qPCR,tissue-specific staining,western blotting and immunohistochemical assay.The correlations between the expression of LncRNA H19 and the specific markers of chondrogenic differentiation were analyzed by Spearman correlation.Mechanically,the binding relationship between LncRNA H19 and osteogenic transcription factor Runx2 was identified by RIP analysis.Then the differential miRNAs expression profiles after silencing LncRNA H19 were screened by miRNA microarray,and the downstream target genes of miRNA and miRNAs targeted by LncRNA H19 were identified by qPCR,western blotting,bioinformatics analysis and dual luciferase reporter gene assay.(2)After exploring the regulatory effect of LncRNA H19 on terminal differentiation of MSCs,we established a mouse tibial fracture model to explore the function of LncRNA H19 on fracture repair mediated by endochondral ossification.Intramedullary fixation of proximal tibial fracture in C57 mice was combined with adenovirus vector silencing or overexpression of exogenous gene GFP,LncRNA H19 and chemical transfection of AntagomiRNA.X-ray was used to detect the general healing of the fracture.?CT,ABH/OG and TRAP staining were performed to analyze theformation of callus and the quality of bone reconstruction during fracture healing.Western blotting,immunohistochemistry and immunofluorescence assay were used to detect the expression of specific markers in the process of fracture healing.Results: First of all,we found that the expression of LncRNA H19 was the highest in the area of proliferative chondrocytes of the growth plate in 14.5-day-old fetal mouse forelimb,while the expression decreased gradually from the area of pro-hypertrophic chondrocytes to hypertrophic chondrocytes.In addition,we found that under the induction of BMP2,the highest expression level of LncRNA H19 followed the peak expression of Sox9,and the expression of LncRNA H19 was positively correlated with the markers of chondrogenic differentiation of MSCs,such as Collagen2?1,Sox9 and Aggrecan,especially in the late stage of BMP2 differentiation.However,LncRNA H19 was negatively correlated with the expression of specific markers of terminal differentiation of MSCs,such as MMP13,Collagen10?1 and Adamst5.Then we further found that silencing LncRNA H19 can promote BMP2-triggered chondrocyte terminal differentiation through in vitro and in vivo experiments,indicating that LncRNA H19 plays an important role in maintaining BMP2-induced chondrocyte phenotype.In mechanism,we first confirmed that LncRNA H19 could regulate BMP2-mediated MSCs terminal differentiation by promoting Runx2 phosphorylation.In addition,we found that LncRNA H19 alsocontrol the process of BMP2-mediated MSCs terminal differentiation by regulating miRNA-21a-5p/Smad7 signal axis.In the fracture repair model,we found that silencing LncRNA H19 could delay the fracture healing process,although larger callus tissue formed rapidly around the fracture after silencing LncRNA H19,and the transformation process from cartilaginous callus to bony callus was enhanced,and the bony callus reconstruction process was inhibited.Finally,we verified that AntagomiRNA-21a-5p,an antagonist of miRNA-21a-5p,could partially reverse the delayed union caused by LncRNA H19.Conclusion: The results of this study show that LncRNA H19 can regulate the process of terminal differentiation of MSCs induced by BMP2,and its mechanism is mainly through:(1)Promoting the phosphorylation of Runx2;(2)Adjusting the miRNA-21a-5p/Smad7 signal axis.In addition,we also found that LncRNA H19 is a necessary regulator in the process of fracture healing,which can regulate the formation of normal callus and the process of bone remodeling.These findings provides a theoretical basis for reducing the terminal differentiation of cartilage in the process of constructing BMP2-mediated tissue engineering cartilage,and provides a theoretical reference to solve the bone defect repair caused by delayed fracture healing in clinic.