[Integrative analysis of gene expression profile and DNA methylation profile of long-term cultivated porcine bone marrow mesenchymal stem cells].

Objective: To integrate the result of whole genome expression data and whole genome promoter CpG island methylation data, to screen the epigenetic modulated differentially expressed genes from transformed porcine bone marrow mesenchymal stem cells (BMSCs) after long-term cultivation.

Methods: Bone marrow from 6 landrace pigs, 3-month-old about 50 kg weight, was aspirated from the medullary cavity of the proximal tibia. The BMSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The transfor mation of BMSCs was tested by several methods including cell morphology observation, karyotype analysis, clone forming in soft agarose, serum requirement assay, and tumor forming in mice. The Agilent Pig 4x44k 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 DNA methylation profiles were integrated to find out the epigenetic modulated differentially expressed genes, and to complete the bioinformatic analysis.

Results: BMSCs showed a change in appearance, from the initial spindle shape to a more flatted morphology then to small contact shape. After additional passages, BMSCs gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth, chromosomal abnormality, and tumor formation in nude mice. The gene chip analysis demonstrated that 257 genes were upregulated and 315 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 BMSCs 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. The combined analysis of genes regulated by methylation in the transformation process of BMSCs found that the methylation changes of the 35 genes were contrary to the direction of expression change (correlation coefficient r =-0.686, P =0.000); in which the methylation level of 21 genes promoter regions were increased while the gene expression decreased, and the methylation level of the 14 genes promoter regions decreased and the gene expression increased. At the same time, KEGG enrichment analysis revealed multiple genes regulated by methylation, involved in stem cell differentiation and multiple cell signaling pathways. Among the 14 down-regulated genes, many of them have the role of regulating the interaction of tumor and immunization, and the change of the methylation status of the CDKN3 promoter region may be closely related to the cell oncology.

Conclusion: The results deepen our understanding of the crucial role of coding genes methylation modification in BMSCs transformation, and may provide new approach to establish safe criteria for BMSCs clinical applications and transformation prevention.