Molecular Mechanism Of CircSYK Regulates Adipogenic And Osteogenic Differentiation Of BMSCs In Steroid-inducd Osteonecrosis Of Femoral Head Through MiR-1285-3p/FGF/?-catenin Signal Pathway

Background:Osteonecrosis of the femoral head(ONFH)is a kind of common orthopedic disease.The early onset is hidden and difficult to detect.When symptoms appear,it is mostly in the late stages of femoral head necrosis.If there is no surgical treatment such as total hip arthroplasty,ONFH will cause a limp and the disability rate is extremely high.So,how to diagnose and treat ONFH in the early stage is an urgent problem to be solved in the field of orthopedics.Steroid-induced osteonecrosis of the femoral head(SONFH)patients caused by long-term or large-scale use of glucocorticoids are the main component of the current ONFH patients.The results of a recent epidemiological study on Chinese patients with ONFH indicate that:24.1%of ONFH patients have a history of taking glucocorticoids.Lipid metabolism disorder theory is currently recognized as the core pathogenesis of SONFH.The application of excessive glucocorticoids can cause bone marrow mesenchymal stem cells(BMSCs)in the bone marrow cavity to tend to adipogenic differentiation and lots of fat further deposits in the blood vessels.The blood supply to the femoral head is reduced in the medullary cavity,which inhibits the repair and reconstruction of bone tissue.Circular RNA(circRNA)is a type of non-coding RNA that forms a stable covalent closed-loop structure by reverse splicing.A large number of studies have shown that circRNAs play role in many kinds of skeletal and muscle diseases such as osteosarcoma,osteoarthritis,intervertebral disc degeneration and menopausal osteoporosis,which have important regulatory effects in the occurrence and development of such diseases.However,there are few circRNA research reports on SONFH.How circRNA regulates BMSCs adipogenic and osteogenic differentiation needs further study.Objective:We adopt high-throughput microarray technology to illustrate the differential expression profile of circRNAs and mRNAs in BMSCs of SONFH.Clarify the mechanism of ceRNA through bioinformatics analysis methods and technologies,and identify the circRNAs that regulate the adipogenic and osteogenic differentiation of BMSCs.Use cell and molecular biology experiments to verify the key circRNA/miRNA/mRNA,so as to identify the signaling pathways and their molecular targets for circRNA-driven BMSCs of SONFH adipogenic and osteogenic differentiation.Methods:1.Aseptically collect 10 ml of bone marrow blood from 16 patients with SONFH and 16 patients with femoral neck fracture(FNF)during hip arthroplasty,establish a SONFH BMSCs control system,and use sterile gradient centrifugation to separate BMSCs from bone marrow blood in the laboratory.The morphology of the extracted BMSCs was observed by microscope,the surface antigen and proliferation ability of the extracted BMSCs were further identified by flow cytometry.2.After osteogenic and adipogenic differentiation of BMSCs were induced with osteogenic and adipogenic medium respectively,the osteogenic differentiation ability of BMSCs in SONFH group and control group was analyzed by Alizarin Red staining and cetylpyridinium chloride osteogenic quantitative experiment,oil red O staining and quantitative analysis of lipid extraction were used to analyze the adipogenic differentiation ability of BMSCs.3.We use Arraystar circRNA V2.0 chip and Arraystar IncRNA/mRNA V4.0 chip to detect BMSCs in 3 patients with SONFH and 3 patients in control group,and screen with |Fold Change|?2 and P<0.05 were as the standards for up-regulation and down-regulated differential expression of circRNAs and mRNAs.We further construct the SONFH circRNA and mRNA differential gene expression profile,and verify the obvious up-regulation and down-regulation of the top 5 genes through qRT-PCR,proving the reliability of the microarray analysis results.4.We adopt bioinformatics analysis methods to further analyze the differentially expressed circRNAs and mRNAs in BMSCs of SONFH,and predict the target miRNAs of circRNAs through Arraystar homemade circRNA target gene prediction software.We use TargetScan and miRDB online prediction analysis website to predict the target mRNAs of miRNAs.We apply Venn analyzes to screen out co-expressed genes.Cytoscape was adopted to visualize the circRNA/miRNA/mRNA ceRNA network.GO and KEGG pathway analysis and protein-protein interaction network were also used to analyze differentially expressed mRNA.Finally,the key pathways that potentially regulate the adipogenic and osteogenic differentiation of BMSCs were screened,and the expression of each gene in the two groups of BMSCs was detected.The expression trend and relationship between the regulatory genes were further analyzed.5.We verify circSYK/miR-1285-3p/FGF1 signaling pathway screened after a series of bioinformatics analysis,construct circS YK overexpression plasmid and small interfering RNA(siRNA).Then knockdown/overexpress circS YK in BMSCs and adopt qRT-PCR to detect the expression levels of circS YK,miR-1285-3p,FGF1 and the key regulated target gene ?-catenin.Adipogenic induction medium induces BMSCs which have been knocked down or overexpressed circSYK,then use oil red O for adipogenic staining and quantitative analysis show the adipogenic ability of BMSCs,qRT-PCR was used to detect the expression levels of the three key adipogenic genes of PPARy,C/EBPa and Adipsin in each group.Osteoinduction medium was used to induce knockdown or overexpression of circSYK BMSCs.Alizarin red osteogenic staining and cetylpyridinium chloride quantitative analysis are adopted to detect the osteogenic ability of BMSCs,qRT-PCR to check the expression levels of osteogenic genes such as BMP2,Osterix and RUNX2 in each group.Western Blot method is applied to measure the protein expression levels of the above genes.6.The dual-luciferase reporter gene experiment was used to further verify the binding sites of miR-1285-3p with circSYK and FGF1.Results:1.A large number of SONFH and control group BMSCs for follow-up experiments were isolated and cultured successfully.The steroid-induced osteonecrosis of the femoral head control system was established.Two groups of BMSCs were observed under the microscope to show the morphology of stem cells with cord-like shape.However,the distribution of BMSCs of the SONFH group was disorder.The results of flow cytometry showed that the two groups of BMSCs were in line with the characteristics of the surface antigen of mesenchymal stem cells,and the potential proliferation ability of BMSCs in the SONFH group was weak.2.The results of oil red O staining showed that BMSCs in the SONFH group produced a large number of lipid droplets after induced by the adipogenic induction medium.The results of the extraction and quantitative analysis showed that the adipogenic differentiation ability of BMSCs in the SONFH group was significantly stronger than that of the control group.The Alizarin Red staining results showed that SONFH calcium nodules increased significantly after BMSCs induced by the osteogenic induction medium.The results of the osteogenic quantitative analysis of cetylpyridinium chloride showed that the osteogenic differentiation ability of BMSCs in the SONFH group was significantly weaker than that of the control group.3.After microarray analysis of the circRNA and mRNA of the two groups of BMSCs,a total of 820 differentially expressed circRNAs and 2775 differentially expressed mRNAs were screened,of which there were 460 up-regulated and 360 down-regulated circRNAs,838 up-regulated mRNAs and 1937 down-regulated mRNAs.The qRT-PCR results showed that the top 5 significantly up-regulated and down-regulated circRNA and mRNA expression levels were in line with the expression levels in the microarray analysis.The microarray analysis results in this study have a certain degree of accuracy.4.We combined circRNA and mRNA differential gene expression profiles,and constructed the regulatory network of up-regulated and down-regulated circRNAs according to the target genes prediction results and completed the visual analysis.There are 116 differentially co-expressed genes in the top 5 up-regulated and differentially expressed circRNA,and 446 differentially co-expressed genes of the top 5 down-regulated circRNAs.GO analysis results show that the differentially expressed genes of BMSCs with SONFH are enriched in glucocorticoid response,glycolytic process and gluconeogenesis which are related to the pathogenesis of SONFH biological process items.KEGG pathway analysis results show that these genes are mainly enriched in stem cell pluripotency,HIF-1,p53 and carbon metabolism that regulate stem cells physiological function.PPI network further reflects hub genes such as TMEM196,FGF1 and GP6 located as the core genes to participate in the expression regulation at the protein level.We finally predict circSYK/miR-1285-3p/FGF1/?-catenin based on the results of bioinformatics analysis and previous researches.The signal pathway can potentially regulate the adipogenic and osteogenic differentiation of BMSCs with SONFH.qRT-PCR results showed the expression levels of the above genes in BMSCs are in line with the expression trend of the ceRNA mechanism.5.After transfection of the circSYK overexpression plasmid into BMSCs,the expression of circSYK was significantly up-regulated,the expression of miR-1285-3p was down-regulated and the expression of FGF1 was significantly up-regulated.The expression of ?-catenin,a key signal molecule that regulates the osteogenic and adipogenic Wnt signaling pathway of BMSCs,was inhibited.The results of oil red O staining and the quantitative analysis of lipids showed that the adipogenic differentiation ability of BMSCs was significantly enhanced.The qRT-PCR results showed that the expression of key genes for lipid-forming PPARy,C/EBPa and Adipsin were significantly up-regulated.Alizarin red staining and cetylpyridinium chloride osteogenic quantitative analysis results showed that the osteogenic differentiation ability of BMSCs was significantly suppressed,and the qRT-PCR results showed that the expression of key bone formation genes of BMP2,RUNX2 and Osterix were significantly down-regulated.The protein expression levels of the above genes were consistent with the gene expression trends.After knocking down the circSYK of BMSCs,the results were contrary to the overexpression experiments.The adipogenic differentiation ability of BMSCs was suppressed and the osteogenic differentiation ability was significantly enhanced.6.The fluorescence intensity was significantly inhibited when the wild-type FGF1/circSYK dual-luciferase reporter gene plasmid and miR-1285-3p mimics were co-transfected into 293T functional cells.However,there was no change in fluorescence intensity when the mutant FGF1/circSYK dual-luciferase reporter gene and miR-1285-3p mimics were co-transfected into 293T functional cells.MiR-1285-3p has direct binding sites with FGF1 and circSYK.Conclusions:1.BMSCs were isolated,cultured and identified in this study.BMSCs of SONFH have stronger adipogenic differentiation ability and weaker osteogenic differentiation ability than the control group.2.We established the circRNA and mRNA differential gene expression profile of SONFH,which provides high-throughput data for screening key differentially expressed genes of SONFH.3.Multiple circRNA/miRNA/mRNA signaling pathways that regulate the function of BMSCs are predicted with the combination of bioinformatics analysis methods.These signaling pathways potentially regulate the BMSCs function of SONFH.4.CircSYK regulates the miR-1285-3p/FGF1/?-catenin signaling pathway through a competitive endogenous RNA mechanism,further promotes the adipogenic differentiation of BMSCs and inhibits their osteogenic differentiation,which provides potential diagnostic markers and therapeutic targets for the clinical prevention and treatment of SONFH5.Mir-1285-3p has direct binding sites between circS YK and FGF1.That provides a theoretical basis for the ceRNA mechanism of circS YK.In summary,this study adopted microarray analysis technology to construct the differential gene expression profile of circRNA and mRNA of bone marrow mesenchymal stem cells from steroid-induced osteonecrosis of the femoral head successfully and constructed multiple circRNA/miRNA/mRNA signal pathways that regulated BMSCs biological functions through bioinformatics analysis methods.CircSYK was predicted and verified that can regulate the miR-1285-3p/FGF1/?-catenin signaling pathway through competitive endogenous RNA mechanisms,regulating the adipogenic and osteogenic differentiation of BMSCs.CircSYK has the potential as a molecular target for the early diagnosis and treatment of SONFH.This research provides a new experimental and theoretical basis for further elucidating the mechanism of SONFH.