dc.description.abstract |
The molecular mechanisms that regulate decidualisation are not fully understood. Decidualisation, the differentiation of endometrial stromal cells into decidual cells during the secretory phase of the menstrual cycle is a complex, biological system, which encompasses a network of genes that change expression in a dynamic manner. To coordinate such complexity, epigenetic mechanisms, such as DNA methylation, histone modification and non-coding RNA may be necessary. We hypothesised that DNA methylation regulates decidualisation of human endometrial stromal cells. We treated a human endometrial stromal cell-line (HESC) with 5-aza-2‘-deoxycytidine (AZA) to inhibit the DNA methylation of gene promoters by passive demethylation. AZA transformed HESC morphology to decidual-like and minimally, yet significantly up-regulated markers of decidualisation, which indicated that AZA partially decidualised endometrial stromal cells. The established treatment to induce decidualisation is estradiol/medroxyprogesterone-acetate (MPA)/cAMP (MPA-mix). Analysis of our microarray data (AZA versus MPA-mix versus control) suggested that both AZA and MPA-mix treatments regulated the RhoGTPase family of cytoskeletal reorganisation genes during decidualisation. To initiate decidualisation, the cytoskeleton is reorganised and the cell cycle is inhibited to arrest stromal cell proliferation. The cell cycle of HESC during AZA and MPA-mix treatments was determined by flow cytometry. MPA-mix inhibited HESC cell cycle at G0/G1 phase early and G2/M phase at 18 days, while AZA treatment inhibited HESC at G2/M phase throughout. Important cell cycle regulators of the G2/M phase include the p53 pathway proteins of p53, p21Cip1, cyclinB1 and 14-3-3sigma, which were all increased after MPA-mix and AZA treatments. In a 6hr to 10day time-course, estradiol/MPA and db-cAMP, independently and in combination, consistently downregulated DNMT3B mRNA (DNA methyltransferase), but only transiently down-regulated DNMT1 and DNMT3A mRNA. To potentially inhibit decidualisation, we over-expressed DNMT3B by plasmid transfection, but this was insufficient to inhibit decidualisation although it attenuated IGFBP1, the decidualisation marker, and combined with MPA-mix, it synergistically increased PRB mRNA. So, over-expression of DNMT3B had the opposite effect of PRA on PRB and IGFBP1. In conclusion, DNA methylation is involved in the regulation of decidualisation, possibly by regulating RhoGTPase family cytoskeletal reorganisation, but further work is needed to elucidate the exact molecular mechanisms and to delineate the specific genes regulated by DNA methylation. |
en |