Molecular Mechanism Of CircRNA CDR1as In Osteogenic And Adipogenic Transdifferentiation Disorders Of Steroid-Induced Osteonecrosis Of Femoral Head BMSCs

Background Osteonecrosis of the femoral head(ONFH)is one of the most common orthopedic diseases,which could be caused by various factors.The blood supply to the femoral head is interrupted,causing bone cells and bone marrow to be necrotic,which leads to bone structural changes and even collapse.In China,due to the large population count,the number of ONFH is about 5,000,000-7,500,000,and the number of new cases is 100,000-200,000 per year.The main factors leading to necrosis of the femoral head include genetic factors and environmental factors.The environmental factors include trauma,extensive use of steroid,long-term alcohol abuse,exposure to radioactive materials,and long-term diving operations.The femoral head necrosis caused by excessive steroid(Steroid-induced osteonecrosis of the femoral head,SONFH)is the main factor of non-traumatic femoral head necrosis.The application of excess steroid can cause lipid metabolism disorder,the accumulation of fat in the medullary cavity,and the damage of the vascular endothelium,which may lead to blockage of the blood vessels in the femoral head.Moreover,mounting evidence suggests that excessive steroid application can cause adipogenic/osteogenic differentiation dysfunction of bone marrow mesenchymal stem cells(BMSCs),which enhances adipogenic differentiation and weakens osteogenic differentiation.It also aggravates the accumulation of fat tissue in the medullary cavity and hinders bone repair and reconstruction.Due to the lack of early diagnosis method,most patients with ONFH often turn to artificial hip replacement surgery to resolve the pain and lameness issues.With the advancement of society and the improvement of people’s living standards,early diagnosis,early treatment,and a deeper understanding of the molecular pathogenesis of ONFH have become an urgent problem to be solved.Circular RNA(circ RNA)is a type of covalently closed non-coding RNA.There is a lot of recent evidence indicate that circ RNA can influence gene expression through different pathways,thereby regulating the occurrence and development of diseases.In the field of orthopedics,many studies have reported differential expression profiles and regulatory functions of circ RNA in the development of orthopedic diseases such as knee osteoarthritis,rheumatoid arthritis,postmenopausal osteoporosis,and osteosarcoma.However,the differential expression of circ RNA in steroid-induced femoral head necrosis BMSCs has not been reported yet,and its regulation mechanism on osteogenesis and adipogenic differentiation of BMSCs is still unclear.Objective In this study,we used high-throughput microarray technology to detect circ RNA and m RNA expression profiles in BMSCs from SONFH patients.Then bioinformatics methods were used to analyze the molecular functions and pathway enrichment of differentially expressed circ RNA and m RNA.Based on the theory of competitive endogenous RNA(ce RNA)regulatory networks,the key circ RNA-mi RNA-m RNA regulatory pathways during osteogenesis and adipogenic transdifferentiation of steroid-induced femoral head necrosis were screened.Validation experiment at the molecular level and cellular function levels were then performed.The aims of our study are to provide new biomarkers and molecular targets for the early diagnosis and treatment of steroid-induced femoral head necrosis.Method1.Isolation and culture of BMSCs: The bone marrow blood of proximal femur were collected from 12 patients with SONFH(experimental group)and 12 patients with femoral neck fracture(control group)during the hip arthroplasty.The BMSCs were isolated by density gradient and then culture-expanded.2.Differential expression profiles of circ RNA and m RNA in BMSCs of SONFH: Three BMSCs specimens from patients with SONFH and three BMSCs specimens from patients with femoral neck fracture were selected,and circ RNA and m RNA expression profiles were obtained by high-throughput gene chip and screened out.The differentially expressed circ RNA and m RNA(|Fold Chang| ≥ 2.0,P<0.05)were verified by the q RT-PCR method.3.Bioinformatics analysis of differentially expressed circ RNA and m RNA in BMSCs of SONFH: The ce RNA regulatory networks of circ RNAs was constructed by Cystoscope based on the site-predicting websites.Gene ontology(GO)and KEGG pathway enrichment were used to analyze the molecular function and pathway enrichments of circ RNA-target m RNAs and differentially expressed m RNAs to further explore their relationship with osteogenesis and adipogenic differentiation of BMSCs.The circ RNA-mi RNA-m RNA regulatory pathways with potential regulatory functions in BMSCs during osteogenic and adipogenic differentiation were screened by combining circ RNA target m RNAs and differentially expressed m RNAs in BMSCs of SONFH.4.The regulatory mechanism of circ RNA CDR1 as in osteogenesis and adipogenic differentiation of BMSCs and signaling pathways.After knocking-down or over-expressing CDR1 as in BMSCs of SONFH,the expression levels of CDR1as、 mi R-7-5p、 WNT5B、 β-catenin were detected.Also,the changes of osteogenic and adipogenic differentiation ablity of BMSCs were also detected by alizarin red or oil red O staining,and key osteogenic and adipogenic gene markers were also detected via the q RT-PCR method.5.The luciferase reporter gene assay was used to further verify the binding site of mi R-7-5p to circ RNA CDR1 as and WNT5 B.Results1.The BMSCs were successfully isolated and cultured.The BMSCs in the experimental group and the control group showed the morphology of the spindle-shaped adherent cells,and they all had typical surface markers of mesenchymal stem cells.2.The differential expression profiles of circ RNA and m RNA of SONFH were detected via high-throughput microarray.The results showed that there were 820 differentially expressed circ RNAs,including 460 up-and 360 down-regulated circ RNAs.2775 differentially expressed m RNA,including 838 up-and 1937 down-regulated m RNA,were also detected in BMSCs of SONFH.Ten differentially expressed circ RNAs and ten m RNAs were selected and their expression levels were validated in 12 SONFH and 12 control BMSCs via q RT-PCR,which showed the same trend as the chip results and proved that the chip results were accurate.3.The ce RNA regulatory networks were constructed,and the number of target m RNAs of 5 most significantly up-and down-regulated circ RNAs were 202 and 99,respectively.The pathway enrichment analysis of these target m RNAs indicates that these up-regulated circ RNA-targeted m RNAs were mainly enriched in the m TOR signaling pathway,Notch signaling pathway,stem cell pluripotency and autophagy.These down-regulated circ RNA-targeted m RNAs were mainly enriched in glyceride metabolism,biotin metabolism,and p53 signaling pathways.Similarly,the up-regulated m RNAs in m RNA expression profiles were mainly enriched in the HIF-1 signaling pathway,TNF signaling pathway,regulation of stem cell pluripotency pathway,carbon metabolism and other signaling pathways,while down-regulated m RNAs were mainly enriched in RNA transport,RNA degradation,p53 signaling,and signaling pathways that regulate stem cell pluripotency.These enriched signaling pathways play important roles in the differentiation of BMSCs.The results of Venn analysis of circ RNA-target m RNAs and differentially expressed m RNAs showed that 31 of these circ RNA-target m RNAs were also in the m RNA differential expression profile,indicating that these 31 target m RNAs did differentially express in SONFH.Via analyzing the function of these m RNAs,we predicted that CDR1as-mi R-7-5p-WNT5 B regulatory pathway plays an important role in the osteogenic and adipogenic differentiation of SONFH BMSCs.4.Circular RNA CDR1 as regulates the osteogenic and adipogenic differentiation of BMSCs in patients with SONFH.Via RNA interference experiments,it was found that the expression of mi R-7-5p was increased after knockdown of CDR1 as,while the expression of it’s downstream target m RNA WNT5 B was decreased.The decrease of WNT5 B expression promoted the expression of β-catenin.The results of alizarin red or oil red O staining and key osteogenic and adipogenic gene markers expression levels showed that the osteogenic differentiation ability of BMSCs was enhanced,while the ability of adipogenic differentiation was decreased.However,in the CDR1 as overexpression experiment,the opposite results were obtained.After overexpression of CDR1 as,the osteogenic differentiation ability of BMSCs was decreased and the adipogenic differentiation ability was enhanced.5.The binding sites of mi R-7-5p to CDR1 as and WNT5 B was validated.After co-transfecting p GL6-mi R-CDR1as(WT)or p GL6-mi R-WNT5B-3’UTR(WT)and mi R-7-5p mimics,the fluorescence of 293 T cells was significantly reduced,which fully demonstrated the binding site between mi R-7-5p and CDR1 as or WNT5 B.Conclusion1.The differential expression profiles of circ RNA and m RNA in SONFH were obtained.A total of 820 circ RNAs and 2775 m RNAs were differentially expressed in BMSCs of SONFH.2.Bioinformatic analysis predicted that CDR1 as can promote the expression of WNT5 B by sponging mi R-7-5p,and then cause osteogeneic and adipogenic transdifferentiation disorder of BMSCs by regulating Wnt/β-catenin signaling pathway.3.After knocking down CDR1 as,the osteogenic differentiation ability of BMSCs was enhanced,while the ability of adipogenic differentiation was decreased.After over-expressing CDR1 as,the osteogenic differentiation ability of BMSCs was decreased,while the adipogenic differentiation ability was enhanced.4.The luciferase reporter gene assay verified the binding site between mi R-7-5p and CDR1 as or WNT5 B.In conclusion,CDR1 as,which is highly expressed in BMSCs of SONFH,promotes the expression of WNT5 B by increasing the “sponging” of mi R-7-5p.Then the high level of WNT5 B could inhibit the expression of β-catenin,a key factor in the Wnt/β-catenin signaling pathway.Finally,the osteogenic differentiation ability of SONFH BMSCs is decreased,while the the adipogenic differentiation ability was enhanced.Our results provide new insights into the molecular mechanisms of osteogenic and adipogenic transdifferentiation disorders in SONFH and provide new biomarkers and molecular targets for the diagnosis and treatment of SONFH.