Potential Function Of Super Enhancers In Human Bone Marrow Mesenchymal Stem Cells During Osteogenic Differentiation

Objective:super-enhancers(SEs)are large clusters containing transcriptional activity enhancers,which drive and control expression of cell identity genes,as well as differentiation of specific cell types.SEs have great application potential in studies of pathogenic mechanisms involved in developmental biology,cancer,and other diseases.However,the potential function and regulatory mechanism of SEs in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)has not been studied.Therefore,this study investigated the potential function of SEs in osteogenic differentiation of hBMSCs and their target genes.Methods:Three groups of primary bone marrow mesenchymal stem cells were purchased and amplified.Cells from human bone marrow mesenchymal stem cells after 14 days of osteogenic induction and differentiation were used in osteogenic medium as the experimental group(denoted as D14 group),while cells from human bone marrow mesenchymal stem cells before osteogenic induction and differentiation were used as the control group(denoted as D0 group).The osteogenic differentiation was identified by alizarin red staining.Total RNA from D14 and DO cells was extracted and the expression of osteogenic specific transcription factors RUNX2,OPN and COL1A1 was detected by RT-qPCR.Respectively to extract DNA from D14 and D0 group,by Chromatin Immunoprecipitation-sequencing(ChIP-seq),in view of the histone acetylation lysine 27(H3K27ac)cell markers for high-throughput DNA sequencing of extraction,the data obtained by sequencing ROSE(Rank Ordering of super-enhancers)algorithm analysis,to identify D14 and D0 group Super enhancer,and its target genes were obtained through the database information.In addition,two super enhancer target genes were selected from cells in group D14 and group D0 for rt-qpcr to verify the accuracy of sequencing results.The selected super enhancer target genes were analyzed by bioinformatics.Results:Alizarin red staining was performed on D0 cells,and the results were negative.However,after alizarin red staining,the cells in group D14 showed significant red calcium salt deposition,indicating that the alizarin red staining result was positive.Rt-qpcr detected significant differences in the expression levels of RUNX2,OPN and COL1A1 in group D0 and group D14,with significantly increased expression in group D14.The results of ChIP-seq indicated that there were 1680 super-enhancers in group D0.There are 342 super-enhancers in group D14.There were 1,380 unique super-enhancers in group D0,42 unique super-enhancers in group D14,and 300 common super-enhancers in the two groups.SEs in the D0 group regulated 1680 genes,including 1094 with protein coding function and 551 non-coding genes.SEs in group D14 regulated 342 genes,among which 223 had protein coding function and 116 non-coding genes.KEGG analysis of SEs target genes before and after osteogenic differentiation showed that the TGF-?,PI3K-Akt,and ECM receptor signaling pathways were highly enriched and closely related to osteogenic differentiation,especially the TGF-? signaling pathway.Further,RT-qPCR results of four selected target genes confirmed the sequencing results.Conclusion:osteogenic differentiation of hBMSCs involves changes in multiple cytokines and cell pathways,and SEs may regulate osteogenic differentiation of hBMSCs by regulating expression of target genes.