HMGA2 is regulated by LIN28 and BRCA1 in human placental cells†

Abstract

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.

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

“…Furthermore, knockdown of LIN28B resulted in significantly increased levels of let‐7c, e,f, and g . This is similar to West et al (), showing let‐7c was higher in LIN28B KD cells, although it was not statistically significant. To determine if AR possibly is regulated by let‐7′s, ACH‐3P cells were transfected with synthetic let‐7 mimics.…”

“…Specifically, knockdown of LIN28A in mouse trophoblast stem cells leads to their differentiation into trophoblast giant cells, whereas LIN28A knockdown in ACH‐3P cells leads to increased syncytialization (Seabrook et al, ). Because then, we found that LIN28B in fact is the predominant LIN28 homolog expressed in human first‐trimester placenta, localized to the cytotrophoblast cells, and present in commonly used human trophoblast cell lines (West et al, ). This was also recently confirmed by Canfield et al ().…”

“…Similar to LIN28B, AR is not present in the differentiated syncytiotrophoblast layer, which is positive for hCG, but appears to localize to trophoblast cells right beneath the syncytiotrophoblast layer. These possibly are cytotrophoblast cells, based on a similar localization pattern as LIN28B (West et al, ). Furthermore, AR is localized to the stromal cells confirming previous reports describing AR localization to the decidua and villous stromal cells (Horie et al, ; Yoshida, Ikeda, & Aihara, ).…”

“…Knockdown of LIN28B in ACH‐3P cells leads to differentiation towards syncytiotrophoblast, with increased levels of ERVW‐1 and secretion of hCG (West et al, ). This study revealed that knockdown of LIN28B leads to significantly lower levels of AR mRNA and protein, suggesting LIN28B either directly or through let‐7 miRNAs regulates AR abundance in trophoblast cells.…”

“…This is contrary to our previous observation when LIN28A was knocked down in ACH‐3P cells, which revealed an increase in LGALS13 (Seabrook et al, ). However, complete abolishment of both LIN28A and LIN28B in ACH‐3P cells also leads to a decrease in LGALS13 (West et al, ), suggesting regulation of this gene is more complex, which is not surprising considering its localization in not only syncytiotrophoblast but also extravillous trophoblast.…”