Experimental strategies for microRNA target identification

MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression in most biological processes. They act by guiding the RNAi-induced silencing complex (RISC) to partially complementary sequences in target mRNAs to suppress gene expression by a combination of translation inhibition and mRNA decay. The commonly accepted mechanism of miRNA targeting in animals involves an interaction between the 5'-end of the miRNA called the 'seed region' and the 3' untranslated region (3'-UTR) of the mRNA. Many target prediction algorithms are based around such a model, though increasing evidence demonstrates that targeting can also be mediated through sites other than the 3'-UTR and that seed region base pairing is not always required. The power and validity of such in silico data can be therefore hindered by the simplified rules used to represent targeting interactions. Experimentation is essential to identify genuine miRNA targets, however many experimental modalities exist and their limitations need to be understood. This review summarizes and critiques the existing experimental techniques for miRNA target identification.     

Expression of VEGFA‐regulating miRNAs and mortality in wet AMD

MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age‐related macular degeneration (AMD). Repeated injections with anti‐VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti‐VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR‐34‐5p, miR‐126‐3p, miR‐145‐5p and miR‐205‐5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti‐VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.     

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.     

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.     

Mammalian hyperplastic discs homolog EDD regulates miRNA-mediated gene silencing

MicroRNAs (miRNAs) regulate gene expression through translation repression and mRNA destabilization. However, the molecular mechanisms of miRNA silencing are still not well defined. Using a genetic screen in mouse embryonic stem (ES) cells, we identify mammalian hyperplastic discs protein EDD, a known E3 ubiquitin ligase, as a key component of the miRNA silencing pathway. ES cells deficient for EDD are defective in miRNA function and exhibit growth defects. We demonstrate that E3 ubiquitin ligase activity is dispensable for EDD function in miRNA silencing. Instead, EDD interacts with GW182 family proteins in the Argonaute-miRNA complexes. The PABC domain of EDD is essential for its silencing function. Through the PABC domain, EDD participates in miRNA silencing by recruiting downstream effectors. Among the PABC-interactors, DDX6 and Tob1/2 are both required and sufficient for silencing mRNA targets. Taken together, these data demonstrate a critical function for EDD in miRNA silencing.     

Hypoxia potentiates microRNA-mediated gene silencing through posttranslational modification of Argonaute2

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.     

VEGFA Expression Is Inhibited by Arsenic Trioxide in HUVECs through the Upregulation of Ets-2 and miRNA-126

Arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia. Recently, studies have shown that ATO can induce apoptosis in leukemic cells and blood vessel endothelial cells in a time- and dose-dependent manner through the inhibition of vascular endothelial growth factor A (VEGFA) production. VEGFA is a key factor in angiogenesis initiation. Targeted inhibition of VEGF or VEGFA expression can suppress angiogenesis; however, little is known about the mechanism by which ATO inhibits VEGFA expression. In this study, we investigated the role of miRNA-126 in the mechanism of action of ATO in human umbilical vein endothelial cells (HUVECs). ATO significantly decreased the viability and proliferation of HUVECs and decreased their migration at 48 h. Cell proliferation was inhibited by 50% (IC50) when 5.0 μmol/L ATO was used. ATO treatment induced miR-126 upregulation and HUVEC apoptosis. Transfection with a miR-126 mimic significantly downregulated VEGFA mRNA levels, and transfection with a miR-126 inhibitor significantly upregulated VEGFA mRNA levels. Finally, we showed that ATO treatment upregulated Ets-2 and miR-126 expression in HUVECs. These results demonstrate that ATO inhibits the growth of HUVECs and induces apoptosis by downregulating VEGFA. One mechanism by which this occurs is Ets-2 upregulation, which results in an increase in miR-126 levels and downregulation of VEGFA expression.     

Combined analysis of microRNome and 3'-UTRome reveals a species-specific regulation of progesterone receptor expression in the endometrium of rhesus monkey

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.     

Posttranscriptional regulation of miRNAs harboring conserved terminal loops

We recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, acts as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. Here, we provide the mechanism by which hnRNP A1 regulates this event. We show that hnRNP A1 binds to the loop of pri-miR-18a and induces a relaxation at the stem, creating a more favorable cleavage site for Drosha. We found that approximately 14% of all pri-miRNAs have highly conserved loops, which we predict act as landing pads for trans-acting factors influencing miRNA processing. In agreement, we show that 2'O-methyl oligonucleotides targeting conserved loops (LooptomiRs) abolish miRNA processing in vitro. Furthermore, we present evidence to support an essential role of conserved loops for pri-miRNA processing. Altogether, these data suggest the existence of auxiliary factors for the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA biogenesis.