Integrative Analysis Of Gene Expression Profile And DNA Methylation Profile And The Role Of NOTCH Signaling Pathway In Malignant Transformation Of BMSCs

Backgroud and Objective:Mesenchymal stromal cells(MSCs)are multipotent stem cells with self-renewal and multiple lineage differentiation potential,which can differentiate into bone,cartilage,fat,myocardium,liver,nerve,skin and other tissue cells.In addition,MSCs also have strong immune regulatory capacity,and have broad prospects in clinical applications such as organ tissue regeneration and repair,transplantation,cancer treatment.An important prerequisite for the application of MSCs in vivo is safe and efficient amplification in vitro.The changes of biological characteristics and safety of MSCs after long-term culture in vitro deserve attention.It has been found that MSCs can be spontaneously transformed into malignant cells after long-term culture in vitro.And MSCs are also involved in the formation of tumors.However,the molecular mechanisms of MSCs transformation is not very clear.In this study,we first studied the morphology,karyotype analysis,clone forming in soft agarose,tumor-related gene expression and tumorigenesis in mice of porcine bone marrow MSCs(BMSCs)cultured in vitro.Secondly,the epigenetic modulated differentially expressed genes from transformed BMSCs were screened by integrating the result of whole genome expression data and whole genome promoter methylation data.Finally,the possible role of NOTCH signaling pathway in inducing transformation of BMSCs was explored.Methods:1.Bone marrows were aspirated from the medullary cavity of the proximal tibia in 6 pigs.The MSCs were isolated by Ficoll density gradient centrifugation combined with adherent culture method.The transformation of MSCs was tested by several methods including cell morphology observation,karyotype analysis,clone forming in soft agarose,serum requirement assay,and tumor forming in mice.2.The Agilent Pig 4x44 k Gene Expression Microarray was used to investigate the differentially expressed mRNA.The methylated genes expression profile was performed using customized pig methylation chip.The gene expression and DNAmethylation profiles were integrated to find out the epigenetic modulated differentially expressed genes,and to complete the bioinformatic analysis.3.Mouse BMSCs were divided into control,vector,NOTCH1-siRNA,?-secretase inhibitor,and NOTCH1-siRNA+?-secretase inhibitor groups.siRNANOTCH1,?-secretase inhibitor,and NOTCH1-siRNA + ?-secretase inhibitor groups were treated with Notch1 siRNA and/or ?-secretase inhibitor.After treatment,cell proliferation was evaluated by Cell Counting Kit-8.Tumor-related factors,including TGF-?1,c-Myc and p53,were detected by real-time PCR and western blotting.BMSC osteogenic differentiation was induced.Calcium deposit was observed and quantified by Alizarin red staining method at 14 and 21 days.And.Alkaline phosphatase activity(AKP)was also evaluated.Results:1.During cultivation in vitro,the shape of BMSCs assumed significant change from the initial spindle to small fusiformis shape.After additional passages,MSCs gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth(soft-agar colony formation),high cell viability in low serum,chromosomal abnormality,down-regulation of p53 and Fas gene expression and up-regulation of c-Myc,NOTCH1 and Hes5 gene expression,and tumor-like tissue formation in mice.2.The Gene chip analysis demonstrated that 257 genes were upregulated and315 genes were downregulated during long-term cultures as well as multiple signal pathways transduction involved,such as cell cycle,ECM-receptor interaction,Focal adhesion,Regulation of actin cytoskeleton,Pathways in cancer and p53.The analysis from methylation chip of coding genes suggested epigenetic regulation was involved in MSCs spontaneous transformation and play a important role on it.962 genes were hypermethylated and 1219 genes were hypomethylated,which were involved in the biological process of cellular metabolic,structure and tumor generation.Inaddition,17 hypomethylated miRNAs and 42 hypermethylated miRNAs were found.The combined analysis from DNA methylation and gene chip demonstrated that several genes related with cancer generation were regulated bymethylation during spontaneous transformation.Hypomethylation of CDKN3 gene involved in cell cycle may lead to cell transformation.3.siRNA-Notch1 and ?-secretase inhibitor both reduced BMSC proliferation.siRNA Notch1 and ?-secretase inhibitor treatment significantly reduced expression of TGF-?1 and c-Myc,and increased expression of p53 at the gene and protein level.Interestingly,combined siRNA-Notch1 and ?-secretase inhibitor treatment did not reduce cell viability to a greater extent.After induction of osteogenesis,calcium deposit and alkaline phosphatase activity were found to be significantly greater in cells with siRNA-Notch1 and ?-secretase inhibitor.Conclusion:1.Compared with early normal BMSCs,long-term cultivated BMSCs have spontaneous malignant transformation and tumorigenicity.2.Methylation modification can be involved in BMSCs spontaneous transformation and play a important role on it.The results deepen our understanding of the crucial role of coding genes and miRNA methylation in BMSCs transformation,and may provide not only new approach to establish safe criteria for MSCs clinical applications and transformation prevention,but also new clues for studying the origin and evolution of cancer stem cells.3.NOTCH1 inhibition reduces BMSCs proliferation,up-regulate the expression of c-Myc and down-regulate the expression of p53,and promotes the osteogenic differentiation,which indicate that NOTCH signaling pathway can promote the proliferation of BMSCs and may paly some role in cell transformation.notch signal may promote cell proliferation and paly an important role in cell transformation.