| Background:In clinical work,bone fracture caused by violence is one of the most common diseases of the trauma repair department such as orthopedics and hand surgery.It is characterized by complex causes,diverse injuries,and a wide range of people,which often brings heavy physical and mental disorder to patients and families as well as economic burden.Despite the strong healing ability of the bone,about 10% of the fracture healing will be interfered with different degrees.Such as osteomyelitis,osteonecrosis,fracture nonunion or deformity,complications of fracture are also difficulties in treatment.Therefore,it’s important to explore the physiological process of osteogenesis and its regulatory mechanism,which can provide new ideas and targets for clinical treatment.Fracture refers to the complete or partial destruction of the continuity of the bone structure.The restoration of the original function and structure of the bone will trigger a complex regeneration and repair process with multiple factors.Giannoudis et al.have proposed four major factors affecting the fracture healing process: related growth factors,bone tissue scaffolds,mesenchymal stem cells and mechanical environment.And these aspects are also critical points for the clinical treatment of fractures.Among them,differentiation of stem cells plays an important role in promoting bone tissue formation and fracture healing.Bone marrow mesenchymal stem cells(BMSCs),also known as bone marrow-derived mesenchymal stem cells,play a crucial role in the formation of osteoblasts and the maintenance of bone tissue osteogenic activity.When bone destruction occurs in a certain part,the body can recruit bone marrow mesenchymal stem cells to the damaged area,and synergize with many growth factors of osteoblasts to differentiate osteoblasts,and then synthesize bone matrix collagen or other proteins.The bone matrix gradually mineralizes and matures to form new bones.On the other hand,the differentiation regulation function of osteoclasts and the initiation of bone remodeling coordinate the complete healing of bones.Osteogenic differentiation involves many genes,hormones,growth factors and other aspects.It mediates multiple signaling pathways to jointly regulate the differentiation process of bone marrow mesenchymal stem cells.In recent years,with the rapid development of high-throughput gene sequencing technology in molecular biology,researches on non-coding RNA have been deepened.And many long non-coding RNAs(Lnc RNAs)and micro RNAs(mi RNAs)have been discovered to be involved in the regulation of cell differentiation,growth and activity of osteogenic tissues.In bone marrow mesenchymal stem cells,mi RNAs regulate the activity of signaling pathways or key transcription factors to regulate BMSCs osteogenic differentiation by affecting the expression of certain important signaling molecules.However,the mode of action of Lnc RNAs on osteogenic differentiation is more complicated.In the previous basic researches of orthopedics,many Lnc RNAs have been found closely related to orthopedic diseases by using chip technology and high-throughput sequencing.Some of the regulatory levels of Lnc RNA are located to transcriptional and post-transcriptional,as well as epigenetic modifications.Moreover,many Lnc RNAs can act on mi RNAs,which inhibit mi RNAs by a sponge-like adsorption function,or function as precursor mi RNAs to exert negative regulatory functions.In conclusion,in the experiments of bone marrow mesenchymal stem cells to perform osteogenic biological functions,Lnc RNAs and mi RNAs,together with the specific transcription factors that interact with them,have gradually become the focus of research due to their important regulatory capabilities.Exploring their roles in new bone formation and clarification of the regulatory mechanism of BMSCs differentiation or related signal transduction pathways,can help to find new therapeutic ideas and theoretical support in the basic research of bone regeneration and repairing.Studies have found that Lnc RNA H19 can promote the differentiation of mouse bone marrow mesenchymal stem cells into osteogenic direction and inhibit the adipogenic process.Another animal studies found that high expression of mi R-188 can increase the risk of osteoporosis.At the same time,it found that the expression of mi R-188 was significantly different in osteogenic or adipogenic differentiation.Although there have been few clear conclusions about the mechanism of Lnc RNAs and mi RNAs in osteogenic differentiation,based on the experimental reports on the regulation of BMSCs in recent years,we suspect that both of them are involved in the osteogenesis processes of bone marrow mesenchymal stem cells.And there might be a relationship between each other.Meanwhile,in vitro-induced adipogenesis experiments,some scholars have reported a nuclear receptor co-repressor which is called ligand-dependent corepressor(LCo R),and revealed that it is a negative adjustment factor in process of adipogenic differentiation.Since such transcription factors are often affected by mi RNAs during the expression of target genes,we suggest that it may have unidirectional or mutual regulation between mi RNAs and LCo Rs during the osteogenic or adipogenic process of BMSCs as well as the expression stages of related genes.However,whether Lnc RNA H19/mi R-188/LCo R has a targeted subordinate relationship in the regulation of bone marrow mesenchymal stem cell differentiation requires multi-level experimental confirmation and interpretation.Objectives:In this study,mouse bone marrow mesenchymal stem cells were used as the cytological basis of in vitro experiments.Lnc RNA H19,mi R-188 and LCo R were selected as the research objects.Combined with the predictions provided by the bioinformatics database,in vitro experiments were used to verify the regulatory relationship between osteogenesis and adipogenic differentiation,and preliminary analysis of Lnc RNA H19/mi R-188/LCo R affecting the subordinate regulation mechanism and related signaling pathways of m BMSCs differentiation.And animal experiments were carried out to verify that the regulation was also available in complex in vivo environments.Our study aim to explore further understanding of bone development and enrich the theoretical system of bone marrow mesenchymal stem cells,also to provide a new idea and theoretical basis for tissue engineering and clinical treatment on bone regeneration and repair.Methods:1.Isolation,culture and identification of mouse bone marrow mesenchymal stem cellsThe m BMSCs were isolated by whole bone marrow adherence method,and the target cells were obtained by subculture.The morphology of the cells was observed under the microscope.The cell proliferation ability was identified by MTT assay.The phenotype was detected by Flow cytometry.The cells after osteogenic and adipogenic induction were detected by Alizarin red and Oil red O staining,and their multi-directional differentiation ability was identified.2.Interaction of Lnc RNA H19,mi R-188 and LCo R in m BMSCsBioinformatics analysis of the expression differences of Lnc RNA,mi RNA and related transcription factors m RNA during the differentiation of m BMSCs was performed.The expression profiles of up-regulated and down-regulated RNAs were obtained by microarray chip screening.m BMSCs were then intervened by transfection of artificial vectors,and the expression levels of Lnc RNA H19,mi R-188 and LCo R were detected by nucleic acid and protein levels using q RT-PCR and Western blot,respectively.Next,a bioinformatics database was used to predict possible binding sequences between the three and verified by luciferase reporter assay.Finally,vectors expressing up-regulated or down-regulated expression were constructed and transfected into m BMSCs,and the expression of Lnc RNA H19,mi R-188 and LCo R was analyzed by q RT-PCR and Western blot to determine the mutual regulation.3.Mechanism research and in vivo study of LCo R in regulating osteogenic and adipogenic differentiation of m BMSCsFirstly,the bioinformatics database was used to analyze the signal pathways related to LCo R regulation,and the GO and KEGG enrichment analysis was carried out.Then,the results of bioinformatics analysis prediction and related proteins were verified by in vitro detection of the expression levels of the transfected cells.Finally,the mouse femur fracture model was used as the basis of in vivo experiments.The m BMSCs transfected with different expression vectors were transplanted into the fracture area.The influence of LCo R on osteogenesis in vivo was evaluated by X-ray imaging,bone mineral density,biomechanics and histology,to demonstrate the role of LCo R in osteogenic differentiation and fracture healing,and to demonstrate the biological role of the Lnc RNA H19/mi R-188/LCo R targeting axis regulating osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells.Results:1.Bone marrow mesenchymal stem cells with spindle shape and good proliferative activity were successfully isolated and cultured from mouse bone marrow by whole bone marrow adherence method.And through continuous observation,there was no obvious morphological change in cells within 10 passages.The MTT assay detected that cells have good proliferative capacity.The expression of cell surface antigen positive markers was up to 95% by flow cytometry,and the negative markers were less than 2%,which met the identification criteria.After induced by osteogenic and adipogenic culture,specific red-stained calcium nodules or red lipid droplets were observed by Alizarin red staining and Oil red O staining,indicating that m BMSCs possess the corresponding characteristics of osteoblasts or adipocytes,known as multi-directional differentiation potential.2.By integrating the gene chip data in the GEO database and the microarray public database,mi RNAs,Lnc RNAs and m RNAs differentially expressed in osteogenic and adipogenic differentiation of m BMSCs were screened.The volcano plot and heat map were drawn to show that down-regulation of mi R-188 during osteogenic differentiation and down-regulation of Lnc RNA H19 and LCo R were observed during adipogenic differentiation.The results of q RT-PCR and Western blot showed that Lnc RNA H19 and LCo R were up-regulated during osteogenic differentiation of m BMSCs and down-regulated during adipogenic differentiation.However,the expression of mi R-188 was just opposite.The bioinformatics analysis predicted the binding site of Lnc RNA to mi R-188,mi R-188 and LCo R,respectively.Constructed wild-type and mutant artificial vector were transfected into cells and verified by luciferase reporter assay.q RT-PCR was used to detect the expression of nucleic acid in m BMSCs after transfection of different vectors and results showed that the level of mi R-188 was up-regulated after Lnc RNA H19 silencing,while the overexpression of mi R-188 did not cause Lnc RNA H19 change.However,when mi R-188 was overexpressed,LCo R was inhibited,but silencing LCo R did not in turn upregulate mi R-188.Western blot results also showed that mi R-188 had a negative regulatory effect on LCo R expression.3.Through GO and KEGG enrichment analysis,it was found that LCo R may regulate the PPAR pathway downstream to affect the adipogenic differentiation of m BMSCs.GSEA enrichment demonstrates the differential expression of PPAR in the adipogenic differentiation process of m BMSCs.The LCo R over-expressed or silenced vector was constructed and transfected into cells.Western blot analysis revealed that the expression of osteogenic-associated marker protein was up-regulated and the expression of PPAR was down-regulated after overexpression of LCo R.The expression of osteogenic marker protein decreased after silencing LCo R.And PPAR-related protein expression is up-regulated.A mouse femur fracture model was established.Untreated m BMSCs and m BMSCs overexpressed or inhibited by LCo R were transplanted into the fracture area.X-ray imaging,bone mineral density,biomechanical related index analysis and histological observation of fractures were performed.All results demonstrated that overexpression of LCo R could promote fracture healing and inhibition of LCo R can reduce osteogenesis.Conclusion:1.Mouse bone marrow mesenchymal stem cells with uniform morphology and good proliferation viability can be successfully isolated and cultured by whole bone marrow adherence method.Surface antigen markers meet the identification criteria in most literatures.After osteogenic and adipogenic induction,m BMSCs exhibit multipotential differentiation potential.2.Lnc RNA H19,mi R-188 and LCo R are differentially expressed in m BMSCs.During osteogenic differentiation,Lnc RNA H19 and LCo R expression were up-regulated,mi R-188 expression was down-regulated.And mi R-188 expression was up-regulated during adipogenic differentiation,while Lnc RNA H19 and LCo R were down-regulated.3.Lnc RNA H19 targets mi R-188.Silencing Lnc RNA H19 can up-regulate mi R-188 expression.mi R-188 can regulate LCo R,and overexpression of mi R-188 can cause down-regulation of LCo R.4.During the differentiation of m BMSCs,LCo R has a negative regulation on PPAR signaling pathway.When LCo R is silenced,it can up-regulate PPAR expression,while overexpression of LCo R can decrease PPAR expression.5.Overexpression of LCo R in the fracture area promotes bone tissue formation and accelerates fracture healing. |
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