Differential MiRNA Expression Profiling In Human Bone Marrow-derived Mesenchymal Stem Cells Of Steroid-induced Femoral Head Necrosis And Mechanisms Study Of MiR-23a Which Inhibits Osteogenic Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells

Backgrounds and ObjectivesSteroid-induced femoral head necrosis (SFHN) is a metabolic disorder with long-term use of hormone drugs that causes decreased vascular supply to the femoral head, osteocyte and bone marrow component death, resulting in the change of internal structure of the femoral head, the collapse of the femoral head and hip joint dysfunction. About 75% patient suffer from femoral head collapse within three years from the diagnosis of SFHN, and eventually more than 65% patients have to receive total hip arthroplasty, which brought about great economic burden to patients and society.The studies of the pathogenesis of SFHN at home and abroad have not made a breakthrough. Recently, domestic and foreign researchers have carried an exhaustive study concerning the pathogenesis of SFHN and put forward many theories including high pressure of femoral head, coagulation mechanism change, fat embolism of micrangium, osteoporosis, bone cell apoptosis, membrane corpuscular theory, etc. Up to now, the underlying molecular mechanisms remain elusive and need some further research.Bone marrow mesenchymal stem cells (BMSC) stem from bone marrow which have multilineage differentiation potential and play an important pathophysiological role in the development of a variety of disease processes. It has been reported that in vitro the high dose glucocorticoid could inhibit the proliferation and osteogenic differentiation of BMSC through the glucocorticoid receptor (GR) and activated protein-1 (AP-1) pathway. Related researches have also showed decreased proliferation ability of BMSC in intertrochanteric region of SFHN patients. As important non-coding regulatory molecules, microRNA (miRNA) mediated regulations have been considered as a universal adjusting mode at the posttranscriptional level of multicellular organisms. It has been proved that miRNA played an important role in the process of bone formation. BMSC combined with miRNA is a new perspective to study the pathogenesis of SFHN, which can provide the potential value for early diagnosis and therapy of SFHN.This study screens out related miRNA of SFHN through miRNA microarray, then we further verify the function of the selected target miRNA, and finally we explore the mechanism of target miRNA through dual luciferase report gene system and siRNA technology, in order to clarify the pathogenesis of SFHN.Methods1. Isolation, culture and identification of BMSC. We got bone marrow from 3 healthy people and 5 SFHN patients who received surgical operation, and further obtained BMSC using the density gradient centrifugation method. Then the surface phenotype and osteogenic activity of BMSC were tested.2. Screening of differential miRNA:total RNA were extracted from normal BMSC, SFHN BMSC and high dexamethasone stimulated BMSC in osteogenic differentiation process. The expression of miRNA was detected and differential miRNA was screened out by gene chip.3. Function verification of miR-23a:miR-23a mimics and inhibitor will be transfected into normal BMSC, which simulate the over expression of miR-23a and low expression of miR-23a. The osteogenic activity of BMSC was examinzed by ALP staining, Alizarin red staining, real time PCR, western blot and ALP activity in 3 days,6 days,9 days and 12 days after osteogenic induction to further verify the impact of miR-23a on osteogenesis of BMSC.4. Mechanism of miR-23a-mediated inhibition of osteogenesis:firstly, preliminary target genes of candidate miR-23a were predicted by the target gene professional forecasts site. Secondly, we matched structure validation of target gene and miR-23a through the dual luciferase experiment. Thirdly siNRA interference technology was conducted to further confirm the target gene. Finally, we validated the specific signal transduction pathway of miR-23a through the pathway inhibitors.Results1. BMSC was successfully isolated. The surface phenotype was identified by flow cytometry. Potential capacity for osteogenic differentiation of BMSC was tested through relevant stainings and real time PCR.2. Seven miRNAs(miR-423-5p、miR-193b*、miR-744、miR-214、miR-423-3p、 miR-320a、miR-140-3p) expression decreased and eight miRNAs (miR-152、miR-23a、 miR-425、miR-27a、miR-221、miR-151-5p、miR-22、miR-224*) expression increased in SFHN BMSC and high dexamethasone stimulated BMSC in osteogenic differentiation process.3. The miR-23a was selected for the function test. miR-23a inhibits osteogenic differentiation in the cell level, protein level and mRNA level, which were consistent with the microarray results.4. Eight candidate target genes were identified through the TargetScan and miRBase professional target genes predicted sites. Subsequently LRP5 was determined as the target gene of the miR-23a. Ultimately we found that miR23a inhibited BMSC osteogenic differentiation through the Wnt pathway.Conclusions1. The SFHN specific miRNA expression profile was identified.2. MiR-23a inhibited osteogenic differentiation of BMSC by targeting LRP5 and through the Wnt pathway.