Tipping the balance toward stemness in trophoblast: Metabolic programming by Cox6B2.

Metabolic effector(s) driving cell fate is an emerging concept in stem cell biology. Here we showed that Cytochrome C Oxidase Subunit 6B2 (Cox6B2) is essential to maintain the stemness of trophoblast stem (TS) cells. RNA interference of Cox6b2 resulted in decreased mitochondrial Complex IV activity, ATP production, and oxygen consumption rate in TS cells. Furthermore, depletion of Cox6b2 in TS cells led to decreased self-renewal capacity indicated by compromised BrdU incorporation, Ki67 staining, and decreased expression of TS cell genetic markers. As expected, the consequence of Cox6b2 knockdown was the induction of differentiation. TS cell stemness factor CDX2 transactivates Cox6b2 promoter in TS cells. In differentiated cells, Cox6b2 is post-transcriptionally regulated by two microRNAs, miR-322-5p and miR-503-5p, leading to its downregulation as demonstrated by the gain-in or loss of function of these miRNAs. Cox6b2 transcripts gradually rise in placental trophoblast gestation progresses in both mice and rats with predominant expression in labyrinthine trophoblast. Cox6b2 expression is compromised in the growth-restricted placenta of rats with reciprocal up-regulation of miR-322-5p and miR-503-5p. These data highlight the importance of Cox6B2 in the regulation of TS cell state and uncompromised placental growth.

MicroRNA regulation of murine trophoblast stem cell self-renewal and differentiation.

Proper placentation is fundamental to successful pregnancy. Placenta arises from differentiation of trophoblast stem (TS) cells during development. Despite being recognized as the counterpart of ES cells in placental development, the role of regulatory miRNAs in TS cell differentiation remains inadequately explored. Here, we have identified complete repertoire of microRNAs present in mouse trophoblast cells in proliferative and differentiated state. We demonstrated that two miRNA clusters, -290 and -322, displayed reciprocal expression during trophoblast differentiation. Loss of miR-290 cluster members or gain in miR-322 cluster members led to differentiation of TS cells. The trophoblast stemness factor, CDX2, transactivated the miR-290 cluster and Cyclin D1 MiR-290 cluster members repressed cell cycle repressors, P21, P27, WEE1, RBL2, and E2F7, in TS cells. MiR-322 cluster members repressed the cell cycle activators, CYCLIN D1, CYCLIN E1, CDC25B, and CDX2, to induce differentiation. Taken together, our findings highlight the importance of posttranscriptional regulation by conserved miRNA clusters that form a regulatory network with CDX2, cell cycle activators, and repressors in equipoising TS cell self-renewal and differentiation.

Dexamethasone-induced Intra-Uterine Growth Restriction impacts NOSTRIN and its downstream effector genes in the rat mesometrial uterus.

Intra-Uterine Growth Restriction (IUGR) is a major cause of fetal and neonatal mortality. Understanding the impact of IUGR on utero-placental gene expression is key to developing effective therapy. In this report we elucidated the impact of IUGR on NOSTRIN and its downstream effector gene expression in the utero-placental compartments. We showed here that induction of IUGR by maternal dexamethasone administration in rats led to up-regulation of NOSTRIN transcript and protein in the mesometrial triangle of the uterus (MG) and not in other utero-placental compartments as compared to control. This was associated with down-regulation of twelve genes and four cytokines that were known to be regulated by NOSTRIN and also required for maintenance of pregnancy. Interestingly, there was remarkable decrease in phosphorylation of RelA transcription factor in the MG during IUGR in line with the fact that the down regulated genes harbour RelA transcription activation domain in their promoters. Furthermore, HIF-1α level was reciprocal to NOSTRIN expression pattern in the mesometrial compartment during IUGR and also in CoCl2 treated endothelial cells. Over-expression of HIF-1α led to a decrease in NOSTRIN levels suggesting inhibition of Nostrin transcription by HIF-1α. Our findings highlight the importance of NOSTRIN in uterine pathophysiology during IUGR.

MicroRNA regulation of Transthyretin in trophoblast differentiation and Intra-Uterine Growth Restriction.

Placental trophoblast cells produce various cytokines, transporters vital to normal embryogenesis. Transthyretin (TTR) aids trans-placental passage of maternal thyroxin (TH) to fetal circulation. Inadequate TH delivery leads to developmental abnormality. Regulation of TTR biosynthesis in placenta is critical for normal embryo development. We showed here that TTR transcripts were expressed more in fetal placenta. Using bioinformatic analysis and confirmation with dual-luciferase reporter assays, we found that miR-200a-3p and miR-141-3p inhibited TTR expression by directly binding to the 3'UTR of TTR, which is reversed by mutation in the microRNA binding site. Differentiation of human trophoblast BeWo cells was associated with decreased TTR transcript and protein levels with concomitant increase in the levels of both microRNAs. Interestingly, ectopic overexpression of the microRNA mimics abrogated thyroxin uptake by BeWo cells, which was reversed by the corresponding inhibitors. Furthermore, in a rat model of intra-uterine growth restriction (IUGR), TTR expression decreased significantly in placenta with reciprocal rise in miR-141-3p but not 200a-3p. In human IUGR placenta, TTR transcript and protein levels were significantly lower associated with high expression of miR-141-3p but not 200a-3p. These data provides new insight into physiological role of miR-141-3p in regulating TTR during trophoblast differentiation and IUGR.

MicroRNA-141-3p and miR-200a-3p regulate insulin-like growth factor 2 during mouse placental development.

Insulin-like growth factor 2 (IGF2) plays a vital role in fetal and placental development throughout gestation. Placental expression of IGF2 decreases substantially in intra-uterine growth restriction (IUGR) and Igf2 null mice develop small placentas. In this report, we examined the role of microRNAs in regulating Igf2 gene expression during mouse placental development. Using bioinformatic analysis, we have identified microRNAs that have conserved binding sites in the 3'-UTR of Igf2. Using luciferase reporter assay, we demonstrated that miR141-3p and miR-200a-3p mimics substantially down regulated relative luciferase activity by binding to 3'-UTR of Igf2, which was reversed by using miR141-3p and miR-200a-3p inhibitors. Furthermore, in a similar assay, use of Igf2 3'-UTR that lacked the binding site for the microRNAs did not have any effect on luceiferase activity. Interestingly, the expression of miR141-3p and miR-200a-3p were inversely and temporally correlated to the expression of IGF2 during mouse placental development. Overexpression of miR141-3p and miR-200a-3p in mouse trophoblast stem cells suppressed endogenous expression of IGF2. Consequently, IGF2 silencing by miR141-3p and miR-200a-3p diminished Akt activation in mouse trophoblast stem cells. Our study provides evidence for regulation of Igf2 by microRNAs and further elucidates the role of miR141-3p and miR-200a-3p in the mouse placental development.