The Study Of Changed Biological Behavior And Aberrantly Expressed Transcriptome In BMSCs In Steroid-induced Osteonecrosis

Background Osteonecrosis of femoral head is a common orthopaedic disease leading to joint disability.The number of patients undergo total hip arthoplasty due to ONFH accounts for 30%of all patients,bring great burden to the society.In China,usage of steroid is one of the major reason of ONFH,and over 35%of ONFH patients is steroid-induced ONFH.However,the mechanism of pathogenesis of steroid-induced ONFH remains elusive,thus limiting the early diagnosis and interference of ONFH.The decreased cell number and changed biological behavior of bone marrow stem cells(BMSCs)were considered related to the occurrence of steroid-induced ONFH.However,no consensus was reached on the particular behavior change of BMSCs.In previous studies,we confirmed aberrantly expressed miRNA in BMSCs of steroid-induced ONFH.However,miRNA alone could not fully elucidate the molecular mechanisms underlying steroid-induced ONFH.Multiple non-coding RNAs could competitively interact with each other and regulated gene expression.As a result,revealing the transcriptome profile may facilitate us to further explore the molecular mechanism of steroid-induced ONFH.ObjectivesIn this study,we first compare the cell function between BMSCs from steroid-induced ONFH patients and healthy controls.Then we use high throughput sequencing to profile the expression of transcriptome,and explore the interaction among different RNAs by bioinformatics analysis.Methods1.Isolation and culture of BMSCs:we obtained bone marrow tissue from 7 steroid-induced ONFH patients and 7 controls,isolated BMSCs and identified the phenotype by flow cytometry.2.We compared the proliferation,apoptosis between two groups of BMSCs using CCK-8,Edu proliferation assay and Annexin V-PI assay;we further induced the osteogenic and adipogenic differentiation in the two groups of BMSCs and compare their differentiation capacity using ALP staining,alizarin red staining and oil o staining,as well as monitoring the osteogenic and adipogenic markers.3.High throughput transcriptome sequencing were admitted to 3 samples of BMSCs in each group to acquire the differentially expressed mRNA,miRNA,lncRNA and circRNA.4.We performed GO and KEGG analysis of the differentially expressed genes and co-expression analysis of IncRNA-mRNA and circRNA-miRNA to reveal the potential mechanisms.5.We further enhanced/silenced the expression of lncRNA-RP11-154D6,lncRNA-RP3-331H24.6 and lncRNA-RP3-331H24.7 in BMSCs.Then observed their functions in altering the proliferation and apoptosis of BMSCs and further preliminarily explored their potential interaction with miR-30a-5p and miR-30c-5p.Results1.We isolated the BMSCs and they were positive for CD29,CD44,CD73,CD90 and CD 105,while negative for CD31 and CD45.2.Cultured in vitro for 24h,48h and 72h,the OD value of the BMSCs from patient group in CCK-8 assay were much less than control group(24h:0.35 ±0.02 vs 0.39 ±0.04,P=0.02;48h:0.54±0.06vs0.68±0.08,P=0.004:72h:0.92±0.04vs1.15±0.08,P=0.0006)。after 24h culture,the positive rate of Edu in BMSCs from patient group was 26.67%± 10.85%,which was much lower than 66.03%±8.71%in control group(p=0.0021),indicating an impairment in proliferation.The total apoptotic rate of the BMSCs from patient group was 24.64±3.68%,which is significantly higher than control group(17.48±4.05%,p=0.0041).3.Measured by ALP staining,alizarin red staining and oil o staining,as well as the osteogenic markers(ALP,OCN,RUNX2 and BSP)and adipogenic markers(PPARγ,LPL and FABP4),we found a significantly lowered osteogenic capacity and an enhanced adipogenic capacity in BMSCs from patient group.4.High throughput transcriptome sequencing revealed 3114 differentially expressed mRNAs,572 differentially expressed IncRNAs,224 differentially expressed miRNAs and 231 differentially expressed circRNAs.5.Bioinformatics analysis revealed that these differentially expressed genes could potentially interact with each other and form a regulatory network to regulate the progression of steroid-induced ONFH.6.The expression of miR-30a-5p and miR-30c-5p in BMSCs were found to be positively related to the expression of lncRNA-RP11-154D6,while negativly related to the expression lncRNA-RP3-331H24.6 and lncRNA-RP3-331H24.7.Besides,lncRNA-RP11-154D6 was found to impair the proliferation and promote apoptosis in BMSCs.LncRNA-RP3-331H24.6 and lncRNA-RP3-331H24.7 were detected to facilitate proliferation while inhibit apoptosis in BMSCs.Conclusion1.The proliferation,apoptosis and differentiation capacity of BMSCs in patients with steroid-induced ONFH were aberrantly changed,which could possibly lead to steroid-induced ONFH.2.Aberrantly expressed mRNAs,miRNAs,lncRNAs and circRNAs were detected in BMSCs in patients with steroid-induced ONFH.And these differentially expressed genes could potentially interact with each other and regulate cell function,leading to the occurrence of steroid-induced ONFH.3.lncRNA-RP11-154D6,lncRNA-RP3-331H24.6 and lncRNA-RP3-331H24.7 could regulate the proliferative capacity and apoptosis potentially through interaction with miR-30a-5p and miR-30c-5p or their encoding genes.