miR-30d is known to be up-regulated during the acquisition of receptivity in the endometrium. In order to determine the transcriptomic and proteomic changes which occur after transient overexpression of miR-30d in primary endometrial epithelial cells, in vitro cultured human endometrial epithelial cells (hEECs) were studied experimentally. Two different miRNAs (scramble versus mimic; n = 15) were transiently transfected into primary hEECs from four different patients and were evaluated for mRNA and protein expression using Agilent's gene expression microarray and iTRAQ analysis techniques, respectively. A set of differentially expressed mRNAs were validated by qPCR and several differentially expressed proteins were validated by western blot. Finally, methylation differential immunoprecipitation (MeDIP) was used to validate the epigenetic changes in the H19 gene. The results showed that transient transfection with miR-30d miRNA induced the differential mRNA-expression of 176 genes (75 up-regulated and 101 down-regulated). Several of them have been associated with reproductive and endocrine system disorders, tissue development, and are implicated in epithelial cell proliferation. Also, the down-regulation of some genes such as H19 and N-methyltransferase (NNMT) may suggest that epigenetic alterations are induced. Furthermore, upstream effects of genes regulated by the estrogen receptor alpha 1 (ESR1) transcription factor have been predicted. Proteomic analysis identified 2290 proteins, of which 108 were differentially expressed (47 up-regulated and 61 down-regulated). Among these differentially expressed proteins DNA methyl transferase (DNMT)1 was found to be up-regulated; this protein participates in the maintenance of DNA methylation, supporting an epigenetic role for miR-30d. Finally MeDIP showed an increase in methylation in the H19 DMR region. In conclusion transient in vitro overexpression of the receptivity-up-regulated miRNA miR-30d in hEECs seems to activate genes which are associated with hormonal response and the epigenetic status of these cells.
Keywords: endometrium; miR-30d; microRNA; proteomics; transcriptomics.
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