The chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression. Inhibition of let-7 miRNAs by RNA binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development therefore we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation, seemingly differentiating solely towards the syncytiotrophoblast sublineage, secreting higher amounts of hCG and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 allowing HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development.

Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Proliferation and Organ Development pathways, while umbilical serum exosomal and placentomes miRNAs were predicted to target cellular development and organismal/embryonic development.

LIN28B is an RNA‐binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let‐7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let‐7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first‐trimester trophoblast cell line, ACH‐3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral‐based vectors. LIN28B knockdown in ACH‐3P cells resulted in significantly decreased levels of AR and increased levels of Let‐7 miRNAs. Moreover, treatment of ACH‐3P cells with Let‐7c mimic, but not Let‐7e or Let‐7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin‐induced syncytialization and Let‐7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW‐1 and a significant decrease in AR in ACH‐3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let‐7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.

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