MIR-210 modulates mitochondrial respiration in placenta with preeclampsia

Abstract

Preeclampsia (PE) affects 5–8% of all pregnancies and is associated with significant maternal and fetal morbidity and mortality. Placental mitochondrial dysfunction has been reported in PE. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression through mRNA degradation and translational repression. MiR-210 has been previously shown to be upregulated in placentas from pregnancies complicated by PE. We hypothesized that placental mitochondrial dysfunction during PE can be mediated by miR-210. Placentas were collected at term from normotensive pregnancies (CTRL) and those complicated by severe PE (n = 6 each) following c-section (no labor). Villous tissue from PE showed significantly increased levels of HIF-1α compared to CTRL with no change in corresponding mRNA expression but with reduced DNA-binding activity. Mitochondrial complex III was significantly decreased in PE along with significantly reduced protein expression in complex I and IV during PE. Among the four miRNAs tested, miR-210 showed significant up regulation in PE and significant downregulation of its target, ISCU mRNA. To understand the role of miR-210 in PE, loss- and gain-of-function studies were performed using primary trophoblasts. Trophoblasts were transfected with miR-210 inhibitor or pre-miR-210 and mitochondrial function was measured using Seahorse Extracellular Flux Analyzer. Cells transfected with pre-miR-210 showed significant reduction in oxygen consumption. In contrast, transfection of trophoblast with AntagomiR-210 was sufficient to prevent the DFO-mediated respiratory deficiency. These data collectively suggest that miR-210 overexpression during PE could be responsible for placental mitochondria dysfunction.

Introduction

Preeclampsia (PE), a multi-system disorder that affects 5–8% of pregnancies worldwide [1], is manifest as maternal hypertension, proteinuria and edema and is associated with significant fetal and maternal morbidity and mortality. Severe PE is associated with fetal growth restriction, indicating placental dysfunction, and with preterm birth and perinatal death. Although the etiology of PE is unknown, the presence of the placenta is a prerequisite for the development of the syndrome. Current dogma is that preeclampsia is associated with inadequate trophoblast invasion, aberrant spiral arterial remodeling and decreased uteroplacental perfusion leading to a relative placental hypoxia/ischemia and release of mediators that damage the maternal endothelium [2], [3].

Placental development is profoundly influenced by oxygen tension [4]. Human cytotrophoblast proliferate in vitro under low O₂ conditions but differentiate at higher O₂ levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal–fetal circulation in vivo [5]. Hypoxia-inducible factor-1α (HIF) plays a role in the regulation of trophoblast function [6]. HIF-1α is expressed in the villous cytotrophoblast and decreases with gestational age in normal pregnancies [7], however, increased stabilization of HIF-1α is observed in pregnancies complicated by preeclampsia [8]. In addition to hypoxia, inflammatory cytokines, estrogen and reactive oxygen species (ROS) are involved in the stabilization of HIF-1α during normoxia in a number of tissue types including placenta [9].

Normal pregnancy is a state of oxidative stress, which is heightened in preeclampsia [10], with placental mitochondria, an important source of reactive oxygen species (ROS), contributing to generation of oxidative stress [11]. Mitochondria are the major oxygen consuming organelles in the cell and play a crucial role in sensing the cellular oxygen concentration [12]. When dysfunctional, mitochondria generate excessive amounts of ROS which may be involved in the triggering of preeclampsia [13].

Regulation of cell proliferation, mitochondrial metabolism, oxygen sensing and apoptosis in placenta has been recently found to be regulated by small (22 nucleotide)non-coding RNAs-microRNAs (miRNAs) [14]. MiRNAs are highly conserved, regulatory molecules that play an important role in the post-transcriptional regulation of gene expression by promoting RNA instability or translational inhibition [15]. Enquobahire et al. [16] have shown eight differentially expressed placental miRNAs in pregnancies complicated by preeclampsia with miR-34C, 139 and 328 being downregulated and miR-210 upregulated [16]. Up regulation of placental miR-210 has been also described in preterm and severe preeclampsia [17], [18], [19], [20], [21]. Zhang et al. found that elevated miR-210 during preeclampsia suppresses trophoblast cell migration and invasion by targeting Homeobox-A9 (HOXA9) and Erphin-A3 (EFNA3) [22]. MiR-210 is known to be involved in the hypoxic response and has been shown to be over-expressed in a HIF-1α-dependent manner in many types of tumors. It is purported to be involved in the shift of tumor metabolism from oxidative phosphorylation to glycolysis (Warburg effect) [23]. In addition a mechanistic link between miR-210, HIF-1α, mitochondria associated proteins, and mitochondrial function has been identified in cancer cells [24], [25]. Based on these observations, we hypothesized that mitochondrial dysfunction seen in the placenta of pregnancies complicated by preeclampsia is mediated by overexpression of miR-210. We demonstrate here that exogenous overexpression of miR-210 in cultured trophoblasts represses mitochondrial respiration, whereas inhibition of miR-210 was able to protect the mitochondria from oxidative damage.