MicroRNA-210: a unique and pleiotropic hypoxamir

Abstract

Inadequate oxygen availability or hypoxia induces a complex and still incompletely understood set of adaptations that influence cellular survival and function. Many of these adaptations are directly controlled by a master transcription factor, hypoxia inducible factor-alpha (HIF-α). In response to hypoxia, HIF-α levels increase and directly induce the transcription of > 100 genes, influencing functions ranging from metabolism, survival, proliferation, migration, to angiogenesis, among others. Recently, it has been demonstrated that a specific set of microRNA molecules are upregulated by hypoxia, which we denote here as "hypoxamirs." In particular, the HIF-responsive hypoxamir microRNA-210 (miR-210) is a unique microRNA that is evolutionarily conserved and ubiquitously expressed in hypoxic cell and tissue types. A number of direct targets of miR-210 have been identified by in silico, transcriptional, and biochemical methods, a subset of which have been extensively validated. As a result, miR-210 has been mechanistically linked to the control of a wide range of cellular responses known to influence normal developmental physiology as well as a number of hypoxia-dependent disease states, including tissue ischemia, inflammation, and tumorigenesis. Thus, reflecting the pleiotropic actions of HIF-α, miR-210 appears to function as a "master microRNA" relevant for the control of diverse functions in the hypoxic state.

Figure 1

MiR-210 directly represses multiple transcripts associated with diverse cellular functions. To date, at least 35 unique transcripts (denoted by their official gene symbol) have been identified and further verified as direct targets of repression by miR-210. These transcripts are mechanistically linked to a wide array of fundamental cellular processes (denoted in the gray ovals) that are critical to hypoxic survival and adaptation of the mammalian cell. Here, verified targets of miR-210 are listed if previously found to be downregulated by miR-210, either through gain-of-function (forced expression of miR-210 during normoxia) or loss-of-function (inhibition of endogenous miR-210 during hypoxia) assays, coupled with either confirmation of specific recognition of the predicted target sequence by miR-210 (i.e., transactivation luciferase assays) or biochemical immunoprecipitation of the target transcript by miR-210-enriched AGO2/RISC. Notably, additional in silico-predicted transcripts have been further implicated as possible targets of miR-210 through either miR-210-dependent immunoprecipitation of AGO2/RISC or transcriptional/proteomic high-throughput analysis after manipulation of miR-210 expression. We expect that a significant percentage of these candidates will be confirmed as targets in the future, and as a result, the number of associated cellular processes controlled by this versatile hypoxamir will continue to expand.

Figure 2

HIF-dependent gene pathways coincide with miR-210-dependent gene pathways. Pathways of gene regulation in endothelial cells by miR-210 as assessed by transcriptional and proteomic analyses intersect with a majority of well-characterized gene expression changes induced by HIF-1α during hypoxic exposure. While some HIF-dependent processes have not been definitively implicated in the actions of miR-210 (i.e., direct erythropoiesis), it is still possible that a subset of these actions may be linked to miR-210 in separate cell-specific or environmental contexts. Furthermore, while HIF-1α appears to play a predominant role in the induction of miR-210, baseline, endogenous levels of miR-210 are higher in certain cell types and may carry some biological activity during normoxia. Thus, HIF-independent effects of miR-210 are possible and await further clarification.

Figure 3

MiR-210 inhibits mitochondrial metabolism via repression of ISCU1/2 and iron-sulfur cluster-dependent function. (A) Louis Pasteur first described a fundamental cellular response to hypoxia, resulting in a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis. The molecular mechanisms underlying this complex event have been incompletely characterized to date. (B) Iron-sulfur cluster assembly proteins ISCU1/2 are essential for iron-sulfur cluster biogenesis. These moieties are incorporated into a wide variety of proteins, many of which are involved in mitochondrial metabolism and aerobic energy production. While the function of iron-sulfur clusters has been more extensively studied in bacteria and yeast, less is known regarding their dynamic regulation and function in hypoxic mammalian cells. (C) MiR-210 regulates iron-sulfur cluster-dependent metabolic function during hypoxia via direct repression of ISCU1/2, leading to downregulate iron-sulfur cluster biogenesis and iron-sulfur-dependent metabolic enzyme activity. In doing so, miR-210 disrupts mitochondrial respiration and potentially other iron-sulfur cluster (ISC)-dependent functions such as iron metabolism, among others. As a result, miR-210 modulates a unique constellation of essential metabolic functions that predominates in the Pasteur effect, and influences cellular adaptation to hypoxia in the mammalian cell.

Figure 4

Complex control of cellular survival and proliferation by miR-210. (A) MiR-210 increases cell survival during hypoxia by modulating multiple direct targets involved in apoptosis and cell death. By repressing CASP8AP2 and directly modulating caspase-8 activity, miR-210 improves survival of mesenchymal stem cells after engraftment into infarcted cardiac tissue. Furthermore, by repressing ISCU1/2, miR-210 appears to decrease ROS levels and thereby improve survival of human pulmonary vascular endothelial cells exposed to hypoxia. There likely exist additional direct targets of miR-210 that further contribute to its pro-survival and adaptive functions during hypoxic stress. (B) MiR-210 may induce cell type-specific control of proliferation. In certain transformed cells, inhibition of direct target MNT and perhaps E2F3b and ACVR1b by miR-210 leads to an activation of G₁/S cell cycle progression and increased cellular proliferation. However, in other transformed and tumor cells, inhibition of FGFRL1, HOXA3, and, perhaps, HOXA9 and E2F3a appear to reduce cellular proliferation. It is unclear how to reconcile these seemingly paradoxical findings. However, it is reasonable to hypothesize that a given cell type or basal milieu may carry different subsets of the total complement of targets of miR-210 and, thus, differentially respond to the control of cellular proliferation based on those distinct targets.