| Objective:Isolation, culture and identification of bMSCs. We aimed to investigate the effects of glucocorticoid (GC) use on MSCs differentiation, and the differential expression of miRNAs in MSCs of patients with GC-ONFH.Methods:We stipulated specific inclusive criteria for those patients to build comparison relationships between samples. Human MSCs were isolated and cultured as the previously reported. We performed the immunophenotype analysis on MSCs by flow cytometry to identify cell phenotypes. The MSCs were treated in human mesenchymal stem cell osteogenic or adipogenic differentiation mediumfor 14 days, then respectively stained with alizarin red S or oil red O. The images were visualized under a light microscope. The total RNA samples of GCs 1-3 and Con 1-3 were enriched and labeled. Then, they were hybridized on a miR microarray (Affymetrix 3.0). The figure signals were transformed and analyzed with the free miR QC Tool software. Then differentially expressed miRs were selected.Results:We successfully got samples and build groups, isolated and cultured MSCs. Our cells were uniformly positive for CD29 and CD44, and negative for hematopoietic lineage markers, including CD34 and CD4S. The osteogenic differentiation capacity of GC-MSCs was subdued and that the adipogenesis differentiation capacity was enhanced when compared with normal MSCs. We selected two overexpressed miRs (miR-483-5p and miR-708) and six underexpressed miRs (miR-92a-1, miR-20b, miR-25, miR-486-3p, miR-30a, and miR-106b) from the GCs groups by miR microarray analysis for further verification.Conclusion:Our results indicated that the culture cells were human MSCs. Glucocorticoid suppressed osteogenic differentiation and enhanced adipogenesis differentiation capacity of MSCs, and lead to overexpression of miR-483-5p and miR-708, underexpression of miR-92a-1, miR-20b, miR-25, miR-486-3p, miR-30a, and miR-106b.Objective:We aimed to master the techniques of qRT-PCR related to miRNA and detect the expression levels of the eight miRs we selected by microarray analysis. Then we picked out some of the miRs for further target validation and functional verification.Methods:We tested the expression of those eight miRs (miR-483-5p, miR-708, miR-92a-1, miR-20b, miR-25, miR-486-3p, miR-30a and miR-106b) between GCs Group and Control Group in MSCs samples from patients GCs 4-6 and Con 4-6 by qRT-PCR. Dexamethasone was tested in vitro at gradient concentrations (0 M,10-8M, 10-7M, and 10-6M) and the cells were harvested after 7 days of Dex intervention for qRT-PCR to observe the expression level changes of those eight miRs.Results:RT-PCR results showed that miR-25, miR-92a-1, miR-708 and miR-483-5p were significantly differentially expressed (p<0.05) between GCs Groups and Con Groups. After treatment by Dex, the expression changes of miR-708 and miR-483-5p in MSCs were in agreement with the previous results of the microarray analysis and qRT-PCR verification, and Dex induced the expression of miR-708 significantly in a dose-dependent manner. Furthermore, the expression of miR-25 and miR-20b were decreased at a Dex concentration of 10"6 M.Conclusion:Considering all these results above, we demonstrated that only miR-708 was highly expressed in vivo and in vitro by GCs. Therefore, the miR-708 was considered as the chosen miR for further target validation and functional verification because of its stable and significant expression change in all the experiments above.Objective:We aimed to master the techniques of bioinformatics, miR transfection and luciferase reporter assay.We tried to search for the target gene of miR-708 and explain the mechanism and relationship between GC use and the osteogenic differentiation dysfunction of MSC in GC-ONFH.Methods:We used computational predictions to identify possible targets by online software. Then those predicted target lists were analyzed for association with Gene Ontology (GO) terms and KEGG pathway analysis using online software MAS 3.0, and tried to organize those possible target genes into hierarchical categories and identify their potential regulatory network. We generated luciferase reporter to determine whether miR-708 could directly regulate the 3’-UTR of SMAD3. Spontaneously for target verification, we used RT-PCR and Western blot analysis to test the expression levels of downstream genes after the transfection of miR-708 mimic in normal MSCs and miR-708 inhibitor in GC-MSCs.Results:SMAD3 and SMAD4 were predicted as the target of miR-708. The effects of our miR transfection were eligible. The transfection of miR-708 mimic markedly decreased the expression levels of SMAD3 and RUNX2 in MSCs. And according to the result of luciferase reporter, miR-708 directly targeted the 3’-UTR of SMAD3. Furthermore, miR-708 inhibitor recovered the osteogenic differentiation capability and the expression level of SMAD3 in GC-treated MSCs.Conclusion:These data suggested that the GC use led to overexpression of miR-708 in human MSCs. The upregulated miR-708 suppressed the expression of its target gene, SMAD3. We inferred that SMAD3 played a major role as the induction of RUNX2 expression in TGF-beta-related signaling pathway, and regulated osteogenic differentiation, which finally resulted in the occurrence and development of GC-ONFH. |
PDF Full Text Request