Differential LncRNA Expression Profiling Of BMSCs In Steroid-Induced Osteonecrosis Of Femoral Head And Mechanisms Research Of LncRNA HOTAIR In Osteogenic And Adipogenic Transdifferentiation Disorders Of BMSCs

【Background】Osteonecrosis of the femoral head(ONFH)is a complex disease with high morbidity which caused by a combination of genetic factors and environmental factors.The incidence of ONFH has been increased in recent decades.About 20000 to 30000 new patients in the United States and 150000 to 200000 new cases in China are diagnosed with ONFH annually.Moreover,the treatment of ONFH,with 8.12 million patients in China,remains a challenge to surgeons.Steroid-and alcohol-induced ONFH are closely associated with lipid metabolism disorder which lead to excessive fat accumulation and the increased serum lipid level in the damaged bone marrow cavity.Excess steroid has been recognized as one of environmental risk factors.Steroid-induced osteonecrosis of the femoral head(SONFH)is considered as a disease of abnormal osteogenic/adipogenic transdifferentiation of bone marrow mesenchymal stem cells(BMSCs).Lesion of intramedullary adipocytes increase the microvessels pressure of the femoral head and cause osteonecrosis.However,the molecular pathogenesis of SONFH remains unclear.Long noncoding RNAs(lnc RNAs)are a newly discovered class of regulatory molecules which affect a variety of biological processes involved in cell differentiation and gene expression regulation.In recent years,several studies have reported that lnc RNAs are involved in osteoarthritis of knee,ankylosing spondylitis,postmenopausal osteoporosis,multiple myeloma and other orthopedic diseases.In addition,several researches have shown that lnc RNAs play an important role in the regulation of osteogenic differentiation,adipogenic differentiation,chondrogenic and differentiation of mesenchymal stem cells.However,the transcriptome expression profiling of SONFH,especially the lnc RNA expression profiling and related regulation of BMSCs during abnormal adipogenesis and osteogenic differentiation have not been reported.【Objective】The abnormal osteogenic and adipogenic differentiation abilities of BMSCs from patients with SONFH were taken as the entry point in this research.The integrated approach of transcriptomics and bioinformatics were both used.The transcriptomic modifications involved in the abnormal BMSCs osteogenesis/adipogenic transdifferentiation and the variation of related signal pathways were analyzed and validated,with the purpose of providing potential diagnostic and therapeutic targets for clinical SONFH prevention and treatment 【Method】1.Isolation,culture and related detection of BMSCs.Bone marrow blood samples from 16 SONFH patients and 16 fracture patients were collected during hip replacement.The BMSCs were obtained by whole bone marrow tissue adherence method and subcultured.Cell surface antigen analysis,cell cycle detection,cell proliferation and osteogenic/ adipogenic differentiation ability were detected.2.Screening of differential expression of lnc RNAs and m RNAs in BMSCs of SONFH.The BMSCs from 3 SONFH patients and 3 fracture patients were screened by high-throughput gene chip to obtain specific lnc RNA and RNA expression profiles.The accuracy of the chip was verified by q RT-PCR.3.Bioinformatics analysis of differential expression of lnc RNAs and m RNAs in BMSCs of SONFH.Through the conjoint analysis of several bioinformatics databases including GO analysis,KEGG database,STRING,Star Base,Lnc Base,Anno Lnc and Targetscan database,the relationship between differentially expressed genes and ssteogenic and adipogenic differentiation ability of BMSCs were explored.4.Regulation and molecular mechanisms of lnc RNA-HOTAIR in osteogenic and adipogenic differentiation of BMSCs.Osteogenic and adipogenic differentiation ability of SONFH-BMSCs were detected by knockdown and overexpression of HOTAIR.The classical "rescure experimental" were used to verify that HOTAIR regulated DKK1 expression by combining with micro RNA-217 in bioinformatics prediction.The detailed method was that the DKK1 expression was detected after knocking down HOTAIR and adding inhibitor of micro RNA-217,or overexpressing HOTAIR and adding homologous analogue of micro RNA-217.Finally,the regulatory effect of HOTAIR on classical Wnt signaling pathway was explored by knockdown and overexpression methods.【Results】1.The BMSCs in SONFH group and control group were successfully isolated and cultured.The BMSCs from SONFH and control groups were all plastic-adherent and spindle-shaped cells.The BMSCs in both groups had typical mesenchymal stem cells surface markers(positive for CD90,CD73,and CD105 and negative for CD34 and CD45).The cell cycle distribution of BMSCs in both groups was not statistically different.The proliferation of BMSCs in SONFH group was lower than that in the control group,but there was no statistically significant difference.2.The osteogenic differentiation ability of BMSCs in patients with SONFH was reduced,and the adipogenic differentiation ability was enhanced in vitro.The ARS,ALP staining and quantitative analysis of SONFH group showed that the osteogenic differentiation ability was weakened,oil red O staining and quantitative analysis showed that the ability of adipogenic differentiation was enhanced on the contrary.The expression of osteogenic differentiation markers including BMP2,OPG and RUNX2 decreased significantly(P<0.05),while the expression of adipogenic differentiation markers PPARγ,C/EBPα and Adipsin increased significantly(P <0.05).The immunohistochemical staining of RUNX2,Osterix in SONFH group was weaker than that in control group,while the immunohistochemical staining of PPARγ and C/EBPα was stronger than that in control group.3.Differential expression of lnc RNAs and m RNAs in BMSCs of SONFH were screened by high throughput microarray.A total of 3286 lnc RNAs and 2775 m RNAs were differentially expressed(fold change>2.0,P value<0.05).To confirm the reliability of the microarray data,we selected 10 differentially expressed m RNAs and 10 lnc RNAs and measured their expression by q RT-PCR.The results showed that the q RT-PCR and microarray had the same expression trends.4.The differential expression of lnc RNAs and m RNAs in BMSCs of SONFH disease were deeply analyzed by bioinformatics analysis.It was found that the differential expression of lnc RNAs and m RNAs in BMSCs of SONFH disease were closely related to osteogenesis and adipogenesis.Gene ontology(GO)analysis was used to analyze the main functions of the di erentially expressed genes.Single organism process,intermediate filament and growth factor activity were the most significantly up-regulated terms enriched(FDR <0.05,P<0.05).Regulation of nucleobase-containing compound metabolic process,nucleus and DNA binding were the most significantly down-regulated terms enriched(FDR <0.05,P<0.05).The KEGG database was used to analyze the signaling pathways involved in differentially expressed genes.Forty pathways showed significant di erences including 16 pathways involving up-regulated genes,and 24 pathways involving down-regulated genes.The coding-non-coding(CNC)network and ce RNA network were constructed for further analyzation the relationship between differentially expressed lnc RNAs and m RNAs.It was found that lnc RNAs-HOTAIR,RP1-193H18.2,MALAT1 m RNABmi1、DKK1、RECK、CTNNB1、JAK2、FOXO1、APC and DLX5 were closely related to the regulation of osteogenic and adipogenic differentiation of BMSCs.5.Lnc RNA-HOTAIR played an important role in the regulation of osteogenic and adipogenic differentiation in BMSCs of SONFH patients.After knockdown HOTAIR,the osteogenic differentiation ability of SONFH-BMSCs was increased and adipogenic differentiation ability was decreased.Moreover,after overexpressed HOTAIR,the osteogenic differentiation ability of SONFH-BMSCs was decreased and adipogenic differentiation ability was increased.By RNA interference,we found that HOTAIR could regulate DKK1 expression by binding to micro RNA217.As DKK1 was one of the Wnt/β-catenin signaling pathway inhibitor and involved in the regulation of OCT4 and β-catenin expression in Wnt/β-catenin signaling pathway,we speculated that abnormally expression of HOTAIR in SONFH patients could suppress osteogenesis differentiation and promote adipogenic differentiation of BMSCs via Wnt/β-catenin signaling pathway.This study provided key molecular targets and signaling pathways for SONFH disease.【Conclusion】1.The characteristic expression profiles of lnc RNA and RNA in BMSCs of SONFH disease were obtained for the first time.There were 3286 lnc RNA and 2775 differentially expressed genes.2.Conjoint analysis of various bioinformatics databases and tools showed that differentially expressed lnc RNA RP1-193H18.2,HOTAIR and MALT1 played an important role in the abnormal osteogenic/adipogenic transdifferentiation of BMSCs in SONFH.3.Compared with the control group,HOTAIR expression was significantly up-regulated in SONFH group.Knocking down or overexpressing HOTAIR could affect adipogenic and osteogenic differentiation ability.4.HOTAIR promotes adipogenic differentiation of BMSCs and inhibits osteogenic differentiation by regulating DKK1 expression through binding to micro RNA217.HOTAIR is expected to become a potential molecular target for clinical intervention in the pathogenesis of SONFH.Above all,our findings provide new insights in the pathogenic molecular mechanism of SONFH and establish a solid foundation for further study of abnormal osteogenic/adipogenic transdifferentiation of BMSCs in SONFH.