Construction Of Different Cell Culture Models Of Mda-mb-231 Cells And DNA Methylation Analysis

Background and objective:Breast cancer is the highest incidence of cancer in Chinese women. Each year the number of new cases in China accounted for 12.2% of the world, the number of deaths accounted for 9.6%, Breast cancer is the sixth cause of cancer death in Chinese women, following lung cancer, stomach cancer, liver cancer, esophageal cancer, colorectal cancer. Tumor is under induction of multi-factor in vivo and gradually evolved in complex environment. The accumulation of mutations is considered as the main reason for tumorigenesis, but epigenetic is proposed and a growing number of studies have shown that genetic and epigenetic changes can cause abnormal expression of genes. Epigenetic changes is very important to tumor development and progression. Studies have shown that biological behavior of tumor cells is different under the different cell culture method, and that the cells can increase or decrease the methylation of promoter to influence the function of gene, which control the characters and malignant of tumor cells. But whether the changes of DNA methylation status is affected by cell culture method is unclear. By constructing 2D, 3D cell culture and tumor orthotopic transplantation model, we observed the differences of tumor cell growth pattern in all three culture models, and explored the differences of gene DNA methylation status and related cellular pathways of MDA-MB-231 cells in different cell culture methods. The results are further discussed that promoter methylation status of genes infulence on occurrence and development of tumor.Methods:We construct the two dimensional(2D) or traditional cell culture, three dimensional(3D) cell culture model and mouse orthotopic transplantation model(Tissue, Ti).The MDA-MB-231 cells were cultured with these three culture models. We observed the growth pattern of the cells under the three models, and collected the cells, then extracted DNA by DNA extraction kit. We detected, calculate and analysis each genome methylation sites of each gene in MDA-MB-231 cells after cultured with three different culture methods by DNA 450 K methylation chip and genomestudio analyzing software, using β value, Diff Score value, Delta Beta to represent respectively. We compared genomic methylation status of MDA-MB-231 cells to screen differential methylated genes between the two models, and performed GO and Pathway analysis by using DAVID online analysis tool.Results:1、The breast cancer cell MDA-MB-231 in 2D cell culture were grown as monolayers, spindle, only existing the gaps between the cells. The single MDA-MB-231 cell in 3D cell culture were growed as perfect spherical structures, existing cell-to-cell and cell-matrix and cell connections. The MDA-MB-231 cells transplanted to mice were grown as tumor mass in the subcutaneous, similar to the real tumor tissue.2 、 All 480 genes of the MDA-MB-231 cells showed significant differences in the degree of methylation in 3D and 2D models(P<0.05);86448 genes in 3D and Ti models(P<0.05); 90005 genes in Ti and 2D models(P<0.05).3、Differential methylation genes in 3D and 2D,3D and Ti,and Ti and 2D models had the same enrichment of Gene Ontology on the part terms, such as the multicellular organismal development term(P<0.05) and cell differentiation term(P < 0.05). These differential methylation genes between the three models also exists some overlap in enrichment of cell pathway, such as MAPK signaling pathway(P<0.05),cell adhesion molecules(CAMs)(P<0.05),and regulation of actin cytoskeleton(P<0.05). Differential methylation genes in 3D and Ti,and Ti and 2D models were significantly enriched on transmembrane transport, Calcium ion binding, cytokine and cytokine receptors interaction pathways(P <0.05), but no difference in 3D vs 2D.Conclusion:1、Biological behavior of tumor cells in different cell culture method is different.2、There are differences in DNA methylation status of MDA-MB-231 cells cultured in 2D,3D cell culture and mouse orthotopic transplantation models, which may be related to the growth environment.3 、 3D cell culture models has certain advantages in research of biological behavior, such as cell to cell interaction and cell polarity,compared to 2D cell culture and tumor cell orthotopic transplantation model.