Effect Of H3K27 Methylation On Osteogenic Differentiation Of BMSCs In Diabetic Environment

Background: There are more and more adult patients seeking for orthodontic treatments in clinic,and a considerable number of these patients are diabetics.At present,it is generally accepted that the orthodontic treatments for diabetics should be cautious.Diabetes Mellitus(DM)is a common metabolic disease,and its complication in skeletal system is usually Diabetic Osteoporosis(DO).The biological basis of orthodontic tooth movement is bone modeling.The balance of osteoblasts and osteoclasts is crucial for the stability of the skeletal system,and osteoblasts are mainly derived from BMSCs.The pathogenesis of the abnormal bone metabolism is complex,which may be related to the unbalance of osteoblasts and osteoclasts caused by diabetic environment.It has been shown that bone formation is reduced in diabetic rats,and cells cultured with high glucose and the advanced glycation end products(AGEs)in vitro can also inhibit its osteogenic differentiation.Epigenetics is the study of heritable alteration in gene expression in response to stimulation absence of changes of underlying DNA sequence,mainly including histone modification,DNA methylation,and non-coding RNAs etc.Histone modification mainly includes methylation,phosphorylation,acetylation and ubiquitination,which plays a key role in regulating gene transcription,occurrence of disease,the growth and development of the body and some other biological processes.H3K27 is the 27 th Lysine in histone H3 peptide chain,and its level of methylation can be regulated by EZH2 and KDM6B,so as to influence gene transcription.It has been proved that KDM6B and EZH2 can regulate the level of H3K27Me3 at the promoter of certain osteogenic genes to regulate its osteogenic differentiation under different conditions.Purpose: We simulate the biological effects of diabetes conditions by diabetic rats model,culturing cells with high glucose and AGE in vitro,so as to verify the effect of diabetic condition on osteogenetic differentiation.We also study the influence of diabetic environment on the expression KDM6B,EZH2 and H3K27Me3,in order to explore the mechanism of the effect of H3K27 methylation on osteogenetic differentiation in diabetic condition.Methods: Part ?: Diabetic rat model was constructed by high-fat and high-sugar foods and STZ.BMSCs derived from diabetic and normal rats were isolated and cultured with method of whole bone marrow adherent.Cells were divided into 6 groups and osteoblastic induced for 7 days,including diabetic group(BMSCs derived from diabetic rats),normal group(BMSCs derived from normal rats),high glucose group(with 30 mmol/L glucose),normal glucose group(with 5.5 mmol/L glucose),AGE group(with 200 ?g/mL AGE)and BSA group(with 200 ?g/mL BSA).ALP activity was detected by ALP staining.The expression of RAGE,OCN,OPN,ALP,Runx2,KDM6B and EZH2 mRNA was tested by realtime-PCR.Expression of EZH2 protein was detected by western-blot and and its subcellular localization by immunofluorescence staining.Part ?: The cells were divided into 6 groups as part I.The expression of H3K27Me3 protein was detected by western-blot and subcellular localization by immunofluorescence staining.Chromatin Immunoprecipitation assay was used to detecte the expression of H3K27Me3 at promoters of BMP2,BMP4,HOXC6 and Runx2 genes under diabetic condition.Results: Part ?: After BMSCs was osteoblastic induced for 7 days,ALP staining showed ALP activity decreased in diabetic groups,and Realtime-PCR indicated RAGE expression increased,OCN,OPN,ALP and Runx2 expression decreased,KDM6B expression decreased,and EZH2 increased expression except for AGE group.The trend of western-blot was consistent with Realtime-PCR,and the immunofluorescence staining shows that the EZH2 protein express at cell nucleus.Part ?: after BMSCs subgroup osteogenic induction for 7 days,the expression level of H3K27Me3 protein in each group was increased by western-blot analysis,and location of H3K27Me3 protein was in the nucleus detected by immunofluorescence staining.Chromatin immunoprecipitation showed that the H3K27Me3 level at the promoter of BMP4 gene in the diabetic group was significantly higher than that in the control group.Conclusion: 1.The osteogenic differentiation of BMSCs was inhibited and the expression of osteogenic genes OCN,OPN,ALP and Runx2 decreased under diabetic condition.2.The expression of histone methylase EZH2 increased in BMSCs,while the expression of histone demethylase KDM6B decreased under diabetic condition.3.The level of H3K27Me3 protein in BMSCs increased under diabetic condition,among which,the most significant increase was at the promoter of BMP4 gene,suggesting that the inhibition of osteogenic differentiation in diabetic condition may be related to the transcriptional inhibition of BMP4 gene.