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Wen Wang, Dong-bao Chen, Angiogenesis-Associated MicroRNA-17 Family (17, 20a and 20b) Regulate Trophoblast Differentiation by Targeting Ephrin-b2 and EPHB4., Biology of Reproduction, Volume 87, Issue Suppl_1, 1 August 2012, Page 382, https://doi.org/10.1093/biolreprod/87.s1.382
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MicroRNAs (miRNAs) are a class of noncoding −21–25 nucleotide RNAs that negatively regulate gene expression post-transcriptionally. The expression of angiogenesis-associated miRNA-17 family (miR-17, 20a, and 20b) has been shown to be upregulated in placentas affected by preeclampsia. The putative target genes of these miRNAs include ephrin-B2 and EPHB4 that have been shown to pattern spiral artery remodeling and cytotrophoblast invasion during early human placentation. MiRNA-17 family miRNAs regulate trophoblast differentiation via regulating ephrin B2 and EPHB4 expression. MicroRNA in situ hybridizations (ISH) were performed on formalin-fixed and paraffin embedded (FFPE) placental sections using miRCURY LNATM microRNA ISH Optimization Kit and double digoxignenin (DIG)-labeled LNA probes. Immunofluorescence staining was used to localize ephrin-B2 and EPHB4 expressions in placental sections. MiRNA precursors or antagomirs were transfected into human trophopblast derived HTR-8/SVneo cells. Total RNAs were extracted from the transfected cells for determining the expression of downstream target genes by real-time qPCR. Luciferase reporter assay was performed for verifying that ephrin B2 and EPHB4 were the targets of miRNA-17 family miRNAs using pMIR-REPORT vector containing the 3-UTRs of ephrin B2 and EPHB4. Four days after miRNA transfection, the cells were stained with cytokeratin-7 for assessing syncytialization. In situ hybridization localized miR-20b primarily in villous syncytiotrophoblasts and endothelial cells in first, second trimester and term placenta tissues. Ephrin-B2 and EPHB4 were also primarily expressed in villous syncytiotrophoblasts in placentas. In HTR-8/SVneo cells, overexpression of miR-20b decreased ephrin-B2 mRNA expression and inhibition of miR-20b increased EPHB4 mRNA expression. Transfection with mir-20b also decreased the activities of luciferase reporter genes carrying 3'-UTRs of ephrin B2 or EPHB4. Overexpression of miR-17, 20a and 20b resulted in 40% less syncytiotrophoblast-like multinuclear cells; inhibition of miR-17, 20a and 20b didn't affect cell syncytialization. These data implicate that angiogenesis-associated miRNA-17 famliy miRNAs (miR-17, 20a and 20b) regulate cytotrophoblast differentiation during placental development by targeting ephrin-B2 and EPHB4 (supported by RO1 HL70562 & R21HL98746).
