Roles of miR-1246, miR-146, and miR-155 and Their Targets in Human Trophoblast Differentiation

BACKGROUND: Abnormal placental implantation has been implicated in preeclampsia (PE), a devastating hypertensive disorder of pregnancy that is a leading cause of maternal and neonatal morbidity and mortality. Placental development depends upon fetal cytotrophoblast (CytT) invasion into the maternal decidua and enlargement of the uterine arterioles with increased blood flow and O2 availability to chorionic villi. CytT fuse and differentiate into syncytiotrophoblast (SynT), which mediate gas and nutrient exchange between mother and fetus, and produce several key steroid and polypeptide hormones of pregnancy. Recent studies from our lab suggest that SynT differentiation is regulated by differentially expressed microRNAs (miRNA/miR). By miRNA microarray of RNA from mid-gestation human CyT before and after differentiation to SynT in culture, miR-1246, miR-146, and miR-155 were found to be highly and significantly upregulated. Interestingly, miR-1246 and miR-155 have been predicted and proven, respectively, to target Jarid2, which recruits the polycomb repressive complex (PRC) 2 to developmentally-regulated genes. PRC2 catalyzes methylation of histone H3 on lysine 27. This chromatin mark promotes recruitment of the PRC1 complex, resulting in repressed chromatin. Moreover, miR-1246, miR-155 and miR-146 all are predicted to modulate components of the Wnt signaling pathway, which plays an integral role in placental development. OBJECTIVE: To analyze expression of miR-155, miR-1246, miR-146 and their targets during differentiation of human trophoblasts in culture and effects of hypoxia. METHODS: CytT were isolated from mid-gestation human placenta and placed in monolayer culture for 24, 48 and 72 h, under normoxic (20 O2%) and hypoxic (2% O2) conditions. RNA was isolated and analyzed for expression of Jarid2, miR-1246, -146, and -155 using Taqman-based RT-qPCR and for markers of SynT differentiation using SYBR Green RT-qPCR. RESULTS: As observed in the microarray, miR-1246, -146, and -155 were dramatically upregulated during SynT differentiation. Conversely, Jarid2 decreased markedly with SynT differentiation. Hypoxia had no effect of expression of any of these miRNAs or on Jarid2 mRNA or protein. CONCLUSION: This research establishes the expression patterns of miR-1246, miR-155, miR-146, and Jarid2 during differentiation of human CytT to SynT in culture. Further studies will evaluate effects of overexpression and inhibition of these miRNAs on trophoblast differentiation and expression of targets, and in PE placentas, where differentiation is poorly regulated. Increased understanding of these miRNAs will provide insight into mechanisms that regulate trophoblast differentiation and the pathogenesis of PE.