Date of Award
5-1-2024
Degree Name
Doctor of Philosophy
Department
Molecular, Cellular, and Systemic Physiology
First Advisor
Bany, Brent
Abstract
In the secretory phase of the menstrual cycle, a notable transformation occurs within the uterine endometrium. Specifically, the elongated fibroblast-like mesenchymal cells change and take a different form, resembling polygonal epithelioid-like cells. This transformation is referred to as "decidualization" and provides the immune-privileged nutritive support required for establishing pregnancy and placental development. Though it is evident that decidualization is under the control of the hormone progesterone and estrogen and is a complex interplay of various protein-coding genes and long noncoding RNA’s (lncRNA’s), precise mechanisms involved during decidualization are not fully understood. This study was carried out to explore the roles of various protein-coding genes and long noncoding RNAs (lncRNAs) in human endometrial stromal fibroblast (hESF) decidualization. RNA sequencing of in vitro decidualization of hESF cells isolated from term placenta was compared with RNA sequencing analysis of in vitro decidualization from cells isolated from non-pregnant human endometrial stromal cells (HESC). The NR4A2, NR4A3, and ATOH8 genes, along with 17 selected lncRNAs, were chosen from differential expression analysis from RNA-Seq. All of them were significantly upregulated ( except LncAL121578.3 and LINC01605, which were downregulated) during hESF decidualization. The RNA-Seq data were confirmed with RT-qPCR analysis. NR4A2, NR4A3, and ATOH8 protein abundance were also significantly upregulated during hESF decidualization. Modulating ATOH8 expression influenced the decidualization markers genes, cell size, and shape morphology. In addition, ATOH8 displayed a potential role in EMT/MET transition, glucose metabolism, FST/INHBB meditated IL4 regulation, cell cycle, and apoptosis. Lentivirus-mediated knockdown and overexpression of ATOH8 reveals ATOH8 to be an upstream regulator of the WNT/FZD-FOXO1 pathway, previously shown to be critical for hESF decidualization. We also explored possible regulators of ATOH8 expression during hESF decidualization. BMP2 significantly enhanced ATOH8 mRNA and protein expression when cells were stimulated to undergo decidualization. DHM1, an ALK2/3 inhibitor, significantly reduced ATOH8 mRNA and protein expression. In addition, the cAMP analog alone represented the primary effect of ATOH8 expression when cells were stimulated to undergo decidualization.Interestingly, targeting NR4A2 and NR4A3 with lentivirus-mediated shRNA gene delivery reduced the expression of ATOH8 mRNA, indicating that NR4A2 and NR4A3 are somehow involved in the upstream regulation of ATOH8. In addition, targeting LncAC027288.3 with lentivirus altered the decidualization marker genes and reduced the expression of ATOH8 mRNA expression levels, indicating that LncAC027288.3 acts as an upstream regulator of ATOH8 expression. Our research suggests decidualization is a complicated yet coordinated interplay of events involving transcription factors, bHLH transcription factors, and LncRNAs.Keywords: ATOH8, Decidualization, LncRNA.
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