Effect Of HDAC8on Osteoblast Differentiation Of Bone Marrow Mesenchymal Stem Cells

Background: Bone marrow mesenchymal stem cells (BMSCs) are multipotentprogenitor cells that can differentiate into osteocytes, chondrocytes or adipocytes.Due to their accessibility and lack of immunogenicity, BMSCs hold significantpromise for clinical applications related to bone regeneration in skeletal diseases.While a number of studies have been conducted to examine the regulated control ofhistone deacetylases (HDACs) during osteoblast differentiation. Our researchinvestigated the effect of HDAC8on osteoblast differentiation of BMSCs and furtherexplored the regulatory mechanism of HDAC8to BMSCs during osteoblastdifferentiation, which will provide some strategy to promote bone regeneration byinhibiting HDAC8activity in future.Part I Isolation，culture and biological characterization identification of rat bonemarrow mesenchymal stem cellsObjective: Isolation and culture of rat BMSCs in vitro and identification ofbiological characteristicsMethods: BMSCs were isolated and cultured by whole bone marrow adherent cultureand the nonadherent cells were removed by changing the culture medium after3days cultured. Cells were passaged when the monoclonal colony formed and attachedeach others; Proliferating function of BMSCs was identified by colony formationassays, MTT assays and flow cytometry; Surface antigen were detected by flowcytometry. Oil red stain and alizarin red stain were used to detected the multi-differentiation ability.Results: BMSCs cultured stably were spindle, with great soma and large nucleus, anddistributed like swirling; BMSCs were powerful proliferation and not easydifferentiation; Flow cytometry results showed that its positive for CD29andnegative for CD34, which was hemopoietic stem cell antigen; Undergoing osteoblastsinduction and adipogenic induction, BMSCs could differentiate into osteoblast andadipocyte.Conclusion: This expression obtained BMSCs by whole bone marrow adherentculture. Spindle BMSCs distributed like swirling and had proliferation capacity.Meanwhile, BMSCs with positive stem cell marker had multi-differentiation ability.In conclusion, our BMSCs had stem cell properties and provided basis for the futureexperiments.Part II The effect of HDAC8on osteogenic differentiation of rat bone marrowmesenchymal stem cellsObjective: To detect the expression and role of HDAC8during osteogenicdifferentiation of rat BMSCs.Methods:1、The expression of HDAC8during osteogenic differentiation wasdetected by Western blot and immunocytochemistry; HDAC8overexpressionlentiviral vector was constructed and then transfected BMSCs. Real-time PCR andWestern blot were used to detected the transfection efficiency; The proliferation ofBMSCs was assessed by MTT assays; ALP activity assays, Alizarin red stain,Real-time PCR and Western blot were applied to estimate the osteogenic capacity ofBMSCs with7days osteogenic induction.2、With treatment of1mM VPA, theexpression of HDAC8was detected by Western blot. MTT assays was used toassessed the change of proliferation and Alizarin red stain, Real-time PCR andWestern blot were applied to estimate the change of osteogenic capacity; HDAC8-siRNA transfected BMSCs. Western blot and Real-time PCR detectedtransfection efficiency and the change of osteogenic capacity; In addition, theosteogenic capacity change of BMSCs with HDAC8overexpression which wastreated with1mM VPA was estimated by Real-time PCR and Western blot.Results:1、HDAC8was decreased during osteogenic differentiation of BMSCs; Usedinverted fluorescence microscope to observed transfection efficiency after HDAC8overexpression lentiviral vector transfected BMSCs. Green fluorescein protein wasunobservable and the protein and mRNA level were increase. Moreover, proliferationhad no change after transfection. The expression of osteogenesis-related genes andthe capacity to form calcified nodules were decreased.2、With treatment by1mMVPA, the expression of HDAC8was decreased in24hours. Meantime, theproliferation of BMSCs was decreased. After osteogenic induction7days, theexpression of osteogenesis-related genes and the capacity to form calcified noduleswere increase. The osteogenic capacity had similar change afer BMSCs transfectedHDAC8-siRNA. In addition, the osteogenesis-related genes expression of BMSCswith HDAC8overexpression which was added1mM VPA were increase.Conclusion: HDAC8could suppress osteogenic differentiation of rat BMSCs.Part III Regulatory mechanism of HDAC8on osteogenic differentiation of ratbone marrow mesenchymal stem cellsObjective: To detect the acetylation change of histone H3K9and to research theinteraction of histone H3K9acetylation (H3K9Ac), HDAC8and RUNX2. Further toexplore regulatory mechanism of HDAC8on osteogenic differentiation of ratBMSCs.Methods: The change of H3K9Ac during osteogenic differentiation of BMSCs wasdetected by Western blot and immunocytochemistry; Western blot was used toanalyse the acetylation level of histone H3K9of BMSCs with HDAC8either overexpression or inhibition. The enrichment of H3K9Ac in RUNX2promoter regionwas assessed by Chromatin Immunoprecipitation (ChIP); The interaction betweenHDAC8and RUNX2during osteogenic differentiation was detected byco-immunoprecipitation (co-IP).Results: During osteogenic differentiation of BMSCs, the acetylation level of histoneH3K9was increase and changed in the opposite direction as the change of HDAC8;ChIP results showed that the acetylation level of histone H3K9in RUNX2promoterregion was increase following osteogenic induction. Co-IP results revealed that bothHDAC8and RUNX2expressed in nucleus and the bind of RUNX2and HDAC8wasdecreased.Conclusion: In this study we indicated two pathway that HDAC8regulated BMSCsosteogenic differentiation via ChIP and co-IP researches. On one hand, HDAC8couldinteract with RUNX2in osteoblast differentiation and consequently repressed itstranscriptional activity. On the other hand, HDAC8indirectly regulated theexpression of RUNX2via H3K9acetylation and further repressed BMSCs osteogenicdifferentiation.