MicroRNA Expression Profiling Of Bone Marrow Mesenchymal Stem Cells In Steroid-induced Osteonecrosis Of The Femoral Head And Function Exploration

Background:In 1950 s,corticosteroid drugs were reported to have the side effects to induce ischemic necrosis of the femoral head for the first time.Presently,long-term or mass glucocorticoid(GC)application has become the first pathogenesis of osteonecrosis of the femoral head(ONFH).Steroid-induced osteonecrosis of the femoral head(SONFH)is a metabolic orthopedic disorder that occurs due to the application of glucocorticoid drugs,leading to impaired blood supply to the femoral head and death of osteocytes and bone marrow composition,which in turn lead to structural change of the trabeculae,collapse of the femoral head,and articular dysfunction.Eventually,the majority of the patients require total hip arthroplasty surgery,which severely impairs the human health and life quality,and results in heavy economic burden to family and society.Over the years,to explore the mechanism of SONFH deeply,many theories have been proposed,including the lipid metabolism disorder theory,the bone marrow mesenchymal stem cells decreased osteogenic potential theory,the insufficient blood supply theory,the inflammation and apoptosis theory,the gene polymorphism and non-coding RNA theory and so on.However,the exact pathological process of SONFH is not completely clear,and further research is still needed.Therefore,exploring the pathogenesis of SONFH is of great significance to the prevention,early diagnosis and treatment of the disease.Previous studies discovered that the bone marrow mesenchymal stem cell(BMSC)from SONFH patients declares decreased proliferation ability and weakened osteogenic differentiation ability,while the ability of adipogenic differentiation is enhanced.BMSC is a kind of pluripotent stem cell in bone marrow,that can differentiated into a variety of bone stromal cells.BMSC plays an important pathophysiological role in the development of many orthopedic disorders.It is reported that the dysfunction of BMSC is related to the occurrence and development of multiple metabolic bone diseases,such as osteoporosis,osteoarthritis,ankylosing spondylitis,and osteosarcoma.The research about the BMSC in metabolic orthopedic disorder has become a hot spot in recent years.Micro RNA(mi RNA)is a family of small single-stranded endogenous non-coding RNAs(18-24 nt).They negatively regulate gene expression by binding to the 3'-untranslated regions(3'-UTRs)of their target m RNAs to inhibit their translation and/or promote degradation.It has been reported that mi RNAs regulates multiple physiological processes,including cell proliferation,differentiation,metabolism and apoptosis.However,changes in mi RNAs of BMSC induced by GC remain to be fully demonstrated,and exploring the mi RNA expression profile in BMSC of SONFH patients is of great meaning for the early diagnosis and treatment of SONFH.In our study,mi RNA microarry was utilized to screen the differentially expressed mi RNAs in the BMSC isolated from SONFH and femoral neck fracture(FNF)patients.And bioinformatics database analysis was utilized to predict the target genes and signaling pathways of the differentially expressed mi RNAs.Finally,mi RNA expression variation tendency was detected to define the correlation between BMSC osteogenic/adipogenic differentiation and the differentially expressed mi RNAs Methods:1?BMSC isolation,culture and identification.Bone marrow from proximal femur of 3 patients with FNF and 3 patients with SONFH who underwent total hip arthroplasty.BMSC was isolated by density gradient centrifugation and cultured in vitro.Then the cell surface phenotype and osteogenic/adipogenic ability of BMSC were preliminarily identified.2?MiRNA microarry profiling of the differentially expressed mi RNA.Total RNA was extracted of the BMSC from SONFH and FNF patients.MiRNA microarray was utilized to detect the differentially expressed mi RNAs,and q RT-PCR was used to verify the microarray result.3?Bioinformatics analysis of differentially expressed mi RNAs.MiRNA target prediction,GO and KEGG analysis was performed to predict the cellular processes and signaling pathways involved in mi RNA regulation.4?MiRNA expression variation tendency detection during BMSC osteogenic/adipogenic differentiation.BMSC was respectively induced osteogenic and adipogenic differentiation in vitro,and total RNA was extracted according to the time point of induction.Then,q RT-PCR was utilized to detect the mi RNA expression variation tendency during osteogenic/adipogenic differentiation,aiming at define the correlation between BMSC osteogenic/adipogenic differentiation and the differentially expressed mi RNAs.Results:1.BMSC was isolated successfully and cuntured in vitro.The cell surface phenotype was identified by flow cytometry,CD29+(98.86%),CD90+(96.42%),CD34-(9.12%).Osteogenic and adipogenic induced differentiation demonstrated the osteogenic and adipogenic ability of BMSC.2.The result of mi RNA microarray revealed that seventeen mi RNA(mi R-4298,mi R-4425,mi R-601,mi R-452-3p,mi R-582-5p,mi R-647,mi R-5006-3p,mi R-516b-5p,mi R-668-3p,mi R-513b-5p,mi R-3687,mi R-181a-3p,mi R-1246,mi R-4436b-3p,mi R-3976,mi R-4530,mi R-4421)were up-regulated and five mi RNA(mi R-127-5p,mi R-887-3p,mi R-519e-3p,mi R-933,mi R-122-3p)were down-regulated.3.The q RT-PCR verification result revealed that mi R-601,mi R-452-3p,mi R-647,mi R-516b-5p,mi R-122-3p were significantly differential expressed and in accordance with the mi RNA microarray result.4.The up-regulated mi RNAs(mi R-601,mi R-452-3p,mi R-647,mi R-516b-5p,mi R-127-5p)in BMSC from SONFH patients declared a downward trend during osteogenic differentiation,while a upward trend observed during adipogenic differentiation.And mi R-122-3p which is down-regulated in BMSC from SONFH patients showed upward trend during osteogenic differentiation and opposite tendency during adipogenic differentiation.5.Bioinformatics analysis of differentially expressed mi RNAs revealed that the targets of the differentially expressed mi RNAs(mi R-601,mi R-452-3p,mi R-647,mi R-516b-5p,mi R-127-5p,mi R-122-3p)mainly enriched in the signaling pathways including ubiquitin mediated proteolysis,choline metabolism in cancer,glycolysis/ gluconeogenesis et al.Conclusions:1?The specific mi RNA expression profile of SONFH BMSC was identified.2?The differentially expressed mi RNAs including mi R-601,mi R-452-3p,mi R-647,mi R-516b-5p,mi R-127-5p,mi R-122-3p regulate the process of proliferation,differentiation and apoptosis of BMSC by regulating the signaling pathways including ubiquitin mediated proteolysis,choline metabolism in cancer,glycolysis/gluconeogenesis et al.3?MiR-601,mi R-452-3p,mi R-647,mi R-516b-5p,mi R-127-5p may be negative regulatory factors of BMSC osteogenesis that inhibit osteogenesis and promote adipogenic differentiation.MiR-122-3p may be a positive regulator of BMSC osteogenesis that promotes osteogenesis and inhibits adipogenic differentiation.