miR-210

miR-210 is a microRNA involved in the regulation of mitochondrial function and hypoxia response. It has been well characterized, in placentas as well as in di fferent cancer and tissue types [263]. Most of the knowledge on the regulatory pathways that involve miR-210 comes from oncology research. miR-210 has been soon identified as one of the early hypoxia-response miRs, being directly regulated by the Hypoxia inducible factor 1 α (HIF-1 α) [264]. Under hypoxic conditions, miR-210 alters mitochondrial function promoting a metabolic switch to glycolysis. This is achieved by negative targeting of genes involved in the electron-transport chain, namely iron- sulfur cluster sca ffold homolog (ISCU) and cytochrome C oxidase assembly protein (COX10). As a result, miR-210 also increases the levels of Reactive Oxygen Species (ROS) [265]. Under hypoxia, miR-210 and HIF-1 α establish a positive feedback regulation that maintains expression of both factors. This is achieved by miR-210 downregulation of the mRNA of Glycerol-3-Phosphate Dehydrogenase 1-Like, which would otherwise contribute to targeting HIF1 α to the proteasome for degradation. Conversely, stabilized HIF1 α directly activates miR-210 expression [266].

In endothelial cells, miR-210 is involved in regulating angiogenesis and vascularization which are fundamental processes in placenta development. Hypoxia causes miR-210 activation which protects endothelial cells from apoptosis and stimulates chemotaxis driven by VEGF, migration and tube formation [267]. In preeclampsia, miR-210 was first identified as upregulated in placenta samples by Pineles and collaborators (2007) using qPCR. In the first comprehensive study carried out with microarray technologies, miR-210 was consistently found upregulated in placenta of severe preeclamptic women, however in mild preeclampsia it was found to be downregulated, which might sugges<sup>t</sup> di fferent mechanisms at play or rather di fferent metabolic states of the placenta, with a more pronounced ischemia in severe preeclamptic placentas [256]. Moreover, subsequent analyses identified significantly upregulated miR-210 in plasma samples from patients with preeclampsia [181]. In the context of PE miR-210 is involved in the mitochondrial dysfunction observed, which causes metabolic imbalance, excessive ROS production and cell damage. Similarly to what happens in cancer, miR-210 negatively regulates ISCU which is downregulated in preeclampsia samples, directly a ffecting mitochondrial architecture and functionality [268–270]. The deregulation of miR-210 was also found in the placentas of mice from a preeclamptic model [134].

miR-210 is also an important modulator of trophoblast phisiology. In vitro studies using isolated primary trophoblasts and trophoblast cell line JAR, proved how hypoxia induces an increase in miR-210 levels. Artificial overexpression of miR-210 in JAR cells caused a significant downregulation of migration and invasion. In trophoblast cells, hypoxia and ROS can activate HIF1 α but more importantly NFκ-B p50—which si found upregulated in preeclamptic placenta tissues. NFκ-B p50 binds a consensus sequence in the miR-210 promoter, activating its expression. In trophoblasts, miR-210 interacts with a perfect match with the 3-UTR of the transcription factor homeobox-A9 (HOXA9), causing both degradation of the mRNA and downregulation of translation. Another direct target is Ephrin-A3 (EFNA3), a ligand of the Ephrin binding receptors, in this case miR-210 binds the 3UTR of the gene with an imperfect match, causing only translational downregulation. These two transcription factors activate expression profiles involved in migration, invasion and vascularisation [181]. Therefore, in trophoblast, miR-210 expression correlates with a negative regulation of migration and invasion, mediated by downregulation of EFNA3 and HOXA9, in response to hypoxia, ROS and activated NFκ-B signaling.

Further studies have identified additional downstream targets of miR-210 in preeclampsia, which are downregulated in preeclamptic samples and whose expression is altered upon miR-210 activation in cell models. A few examples are inflammation related molecules STAT6 and IL-4 [271], potassium channel modulatory factor 1 (KCMF1) [272], thrombospondin type I domain containing 7A (THSD7A) [273].

This mounting body of evidence highlights a key role of miR-210 in the development and maintainance of a preeclamptic phenotype. However it is still not clear which is the triggering event. It is possible that complications during implantation trigger an immune response which would create a pro-inflammatory environment, activating NFκ-B signaling, causing aberrant expression of miR-210 and all consequent downstream cascades. Recently, Chen and collaborator (2019) analysed the inflammatory profile of preeclamptic women, compared to patients which experienced healthy pregnancie [274]. The concentrations of proinflammatory cytokines (IL-6, IL-17) were higher in plasma samples from peripheral blood in the preeclampsia group. Moreover, Transforming Growth Factor β1 (TGF β1) levels were higher as well. TGF β1 has the function of promoting the prevalence of a subset of regulatory T cells (Tregs) that maintain immunotolerance, allowing a successful implantation and avoiding an immune response against the foetal tissues. These Tregs are characterised by expression of the fork-head box p3 (Foxp3) transcription factor, which promotes an immunotolerant phenotype [275]. However, proinflammatory signals such as IL-6 cause the activation of T cells at the expense of Foxp3-positive Tregs, causing an activation of inflammatory responses. Zhao and coworkers showed that miR210 was upregulated in preeclamptic placentas and Foxp3 mRNA and protein levels were found downregulated, previous studies had shown evidence of direct regulation of Fox3p by miR210, suggesting a pivotal role of this microRNA in regulating the threshold of immunotolerance by altering the balance of Foxp3+ Tregs/activated Tcells [276].
