HIF-1α Stabilization Increases miR-210 Eliciting First Trimester Extravillous Trophoblast Mitochondrial Dysfunction

Abstract

Preeclampsia is associated with first trimester placental dysfunction. miR-210, a small non-coding RNA, is increased in the preeclamptic placenta. The effects of elevated miR-210 on placental function remain unclear. The objectives of this study were to identify targets of miR-210 in first trimester primary extravillous trophoblasts (EVTs) and to investigate functional pathways altered by elevated placental miR-210 during early pregnancy. EVTs isolated from first trimester placentas were exposed to cobalt chloride (CoCl₂), a HIF-1α stabilizer and hypoxia mimetic, and miR-210 expression by qPCR, HIF1α protein levels by western blot and cell invasion were assessed. A custom TruSeq RNA array, including all known/predicted miR-210 targets, was run using miR-210 and miR-negative control transfected EVTs. Mitochondrial function was assessed by high resolution respirometry in transfected EVTs. EVTs exposed to CoCl₂ showed a dose and time-dependent increase in miR-210 and HIF1α and reductions in cell invasion. The TruSeq array identified 49 altered genes in miR-210 transfected EVTs with 27 genes repressed and 22 enhanced. Three of the top six significantly repressed genes, NDUFA4, SDHD, and ISCU, are associated with mitochondrial function. miR-210 transfected EVTs had decreased maximal, complex II and complex I+II mitochondrial respiration. This study suggests that miR-210 alters first trimester trophoblast function. miR-210 overexpression alters EVT mitochondrial function in early pregnancy. Mitochondrial dysfunction may lead to increased reactive oxygen species, trophoblast cell damage and likely contributes to the pathogenesis of preeclampsia.

Keywords: ISCU; NDUFA4; SDHD; extravillous trophoblast; miR-210; miRNA; mitochondrial respiration; preeclampsia.

FIGURE 1

CoCl₂ regulates EVT miR-210 expression and invasion. miR-210 expression was measured in EVT cells treated with the indicated doses of CoCl₂ by qPCR (A). Time course of miR-210 mRNA (B) and HIF-1α protein (C) expression in EVTs treated with CoCl₂ (100 μM) was performed between 0 and 24 h of exposure. An invasion assay was performed on EVTs treated with 100 μM CoCl₂ (D). Values are mean ± SEM. n = 4 for (A,B), n = 3 for (D), ^∗p ≤ 0.05, ^∗∗p < 0.01 compared to non-treated controls.

FIGURE 2

Repression of miR-210 direct target genes. Analysis of the custom TruSeq Targeted RNA array revealed 49 altered (27 repressed and 22 enhanced) genes. The top six significantly repressed genes after EVT transfection of miR-210 are NDUFA4 (A) (adj. p = 1.40E-24), DIMT1 (B) (adj. p = 9.26E-18), CNRIP1 (C) (adj. p = 9.26E-18), SDHD (D) (adj. p = 1.79E-16), ISCU (E) (adj. p = 2.12E-09), and NFIC (F) (adj. p = 1.04E-07). Raw counts per target for 20 samples (n = 10 per group) were normalized and analyzed for differential expression (versus miR-negative transfected controls) using DESeq2. The adjusted p-value was calculated by DESeq2 for FDR (False Detection Rate) by the method of Benjamini and Hochberg.

FIGURE 3

Positive qPCR validation of a subset of genes identified by the TruSeq Targeted RNA array. Three genes identified by the TruSeq Targeted RNA array were chosen for validation by qPCR based on their mitochondria-associated functional annotation. NDUFA4 (A), SDHD (B), and ISCU (C) mRNA expression was reduced after miR-210 transfection of EVTs. Values are mean ± SEM. n = 10 representing EVT cells from 10 independent placentas. ^∗p < 0.05 compared to miR-negative (miR-neg) transfected control.

FIGURE 4

Elevated miR-210 decreases EVT mitochondrial respiration. Using high resolution respirometry (Oroboros Instruments), EVTs transfected with miR-210 (green line) compared to miR-negative controls (miR-neg, red line) showed significant reductions in mitochondrial respiration as evidenced by the representative experimental tracing of oxygen consumption (A). Elevated miR-210 expression significantly reduced maximal respiration (C) and respiration from complex II (E) and complex I+II (F). No changes were seen in basal respiration (B) or complex I (D). Levels of O2 flux were normalized to miR-negative controls. Values are mean ± SEM. n = 7 representing EVT cells from seven independent placentas. ^∗p < 0.05 compared to miR-negative (miR-neg) transfected control.