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5-Aza-dC treatment induces mesenchymal-to-epithelial transition in 1st trimester trophoblast cell line HTR8/SVneo.

ABSTRACT: Placental trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. Cytotrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytotrophoblasts and extravillous trophoblasts, respectively, we analyzed the gene expression and DNA methylation status of the known epithelial marker genes, E-Cadherin and Cytokeratin7. We found that, in HTR8/SVneo cells, both genes were silenced and their promoters hypermethylated, as compared with the high-level gene expression and promoter hypomethylation observed in BeWo cells. This result suggests that dynamic DNA methylation of epithelial marker genes plays a critical role in the trophoblast EMT process. To verify these results, we treated HTR8/SVneo cells with 5-aza-dC, a known inhibitor of DNA methyltransferase, for three days. Five-Aza-dC treatment significantly increased the expression of epithelial marker genes and slightly decreased the expression of mesenchymal genes, as detected by qRT-PCR, immunocytochemistry and Western blot. Furthermore, 5-aza-dC treated HTR8/SVneo cells changed their morphology from mesenchymal into epithelial phenotype, indicating that 5-aza-dC induced mesenchymal to epithelial transition. Lastly, we examined the effect of 5-aza-dC on trophoblast migration and invasion capacity. We applied 5-aza-dC to HTR8/SVneo cells in trans-well cell migration and invasion assays and found that 5-aza-dC treatment decreased trophoblast migration and invasion capacity. In conclusion, DNA methylation of epithelial marker genes represents a molecular mechanism for the process of trophoblast EMT.

SUBMITTER: Chen Y

PROVIDER: S-EPMC3593992 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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5-Aza-dC treatment induces mesenchymal-to-epithelial transition in 1st trimester trophoblast cell line HTR8/SVneo.

Chen Ying Y Wang Kai K Leach Richard R

Biochemical and biophysical research communications 20130129 1

Placental trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. Cytotrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytotrophoblasts and extravillous trophoblasts, respectively, we analyzed the gene exp ...[more]

PMID: 23376068

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Project description:BackgroundHow nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.MethodsThe viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n = 3, significance level 0.10, D > 0.642) and/or ANOVA (n = 3, p < 0.05).ResultsThe results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.ConclusionsThis comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

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5-Aza-dC treatment induces mesenchymal-to-epithelial transition in 1st trimester trophoblast cell line HTR8/SVneo.

Publications

5-Aza-dC treatment induces mesenchymal-to-epithelial transition in 1st trimester trophoblast cell line HTR8/SVneo.

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