Evaluation of miR-375 and its 5′-shifted isomiRs in MIN6 cells.

<p>Effects of mimics for 5′-reference miR-375, 5′-shifted miR-375+1, and 5′shifted miR-375-1 in MIN6 cells on the mRNA levels of three genes are shown. <i>Mtpn</i> is a known target of 5′-reference miR-375 but not predicted as a target for either of the 5′-shifted miR-375 isomiRs; <i>Atp6v0c</i> is predicted to be preferentially targeted by miR-375+1; and <i>Cdc42</i> is predicted to be preferentially targeted by miR-375-1. The x-axis lists the gene symbols for each of three genes tested. The y-axis depicts the relative quantitative value (RQV; expression determined by RT-qPCR and normalized to <i>Rps9</i>) in response to the miR-375 mimic (gray), miR-375+1 mimic (orange), or miR-375-1 mimic (green) relative to mock transfection. The data shown represent at least two independent experiments, each conducted in triplicate. P-values were calculated based on Student’s t-tests. *, P<0.05; **, P<0.01, ***, P<0.001.</p

Candidate miRNA regulatory hubs in a type 2 diabetes gene network.

<p>(<b>A</b>) Each data point represents a 5′-reference miRNA or a 5′-shifted isomiR from primary human beta cells, and the y-axis shows the negative Log2 of the p-value of the predicted miRNA targeting score among genes in a type 2 diabetes (T2D) network. The dashed red line denotes the significance threshold (empirical P = 0.05). (<b>B</b>) Effects of miR-29 mimic and inhibitor in MIN6 cells on the mRNA levels of four T2D genes are shown. The x-axis lists the gene symbols for each of four predicted miR-29 target genes and the y-axis depicts the relative quantitative value (RQV; expression determined by RT-qPCR and normalized to <i>Rps9</i>) in response to the miR-29 mimic (blue) or the miR-29 inhibitor (red) relative to mock transfection. The data shown represent at least two independent experiments, each conducted in triplicate. P-values were calculated based on Student’s t-tests. *, P<0.05; **, P<0.01.</p

Beta Cell 5′-Shifted isomiRs Are Candidate Regulatory Hubs in Type 2 Diabetes

<div><p>Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA) transcriptome, which is in large part due to the diversity of miRNA sequence variants (“isomiRs”). Changes to a miRNA’s seed sequence (nucleotides 2–8), including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells (widely used as a surrogate for the study of pancreatic beta cells) and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1) the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2) miRNA loci can generate multiple highly expressed isomiRs with different 5′-start positions (5′-isomiRs); (3) isomiRs with shifted start positions (5′-shifted isomiRs) are highly expressed, and can be as abundant as their unshifted counterparts (5′-reference miRNAs). Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D) gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5′-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by <i>in silico</i> target prediction and <i>in vitro</i> transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5′-shifted isomiRs to T2D.</p></div

miRNA and isomiR profiles in MIN6 cells, primary human beta cells and human islet.

<p>(<b>A</b>) A heatmap is shown depicting the Pearson correlation coefficients of miRNA profiles between pairs of samples analyzed in this study. (<b>B</b>) The x-axis depicts highly expressed miRNAs ordered from left to right by decreasing maximal expression across all samples. The y-axis depicts the Log10 of the average read count per million. Each dot represents a miRNA. miRNAs from a homogenous locus (a pre-miRNA that produces only one mature miRNA per arm of the hairpin) are in gray. miRNAs from a heterogeneous locus (a pre-miRNA that produces more than one mature miRNA per arm of the hairpin) are either pink (5′-reference) or blue (5′-shifted).</p

Comparison of 5′-reference miRNA and 5′-shifted isomiR expression levels among MIN6 cells, human beta cells, and human islet.

<p>(<b>A</b>) The x-axis lists selected 5′-reference miRNAs in MIN6 (red), human beta cells (green), and human islets (blue). The y-axis depicts the Log10 of the average read count per million for each 5′-reference miRNA in each sample. (<b>B</b>) The x-axis shows the highly expressed 5′-shifted isomiRs ordered from left to right by decreasing fold-difference between primary human beta cells and MIN6 cells. The y-axis depicts the average read count per million for each 5′-shifted isomiR. (<b>C</b>) The number of genes with at least one conserved target site for miR-375 (gray), miR-375+1 (green), and miR-375-1 (orange) is shown. All sets are mutually exclusive: for example, a total of 390 genes have predicted conserved miR-375 target sites (42 unique to miR-375, 3 shared with miR-375+1 only, 337 shared with miR-375-1 only, and 8 common to all three).</p

miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during <i>Schistosome</i> and <i>Leishmania</i>-associated Inflammation

<div><p>A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4⁺Foxp3⁺ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following <i>Leishmania major</i> or <i>Schistosoma mansoni</i> infection, respectively. <i>In-silico</i> analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4⁺Foxp3⁺ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function.</p></div

miR-182 and miR-10a target a significant number of <i>in-silico</i> predicted targets in Foxp3⁺ Treg cells.

<p>CD4⁺Foxp3⁺ cells isolated from naïve mice and transfected with miR-182 mimics or hairpin inhibitors (A) or miR-10a mimics or hairpin inhibitors (B) to identify predicted target gene regulation. RNA was extracted 24 hours post transfection for analysis. One of 3 individual experiments shown, with 3 biological replicates in each experiment. p-value = 0.05. with data expressed as mean ±SEM.</p

Differential gene expression in CD4⁺Foxp3⁺ cells isolated from chronic <i>S. Mansoni</i> or chronic <i>L. Major</i> infected tissue.

<p>(A) Isolation and FACS sorting of CD4⁺Foxp3⁺ cells from the liver of <i>S. Mansoni</i> or ear of <i>L. Major infected</i> mice. (B) Heat map of differential gene expression showing biological replicates <i>(naïve Treg = 7, S. m. Treg = 4, L. m. Treg = 3 biological replicates)</i> for the isolated Foxp3⁺ populations. 3944 array probes were differentially expressed at a false discovery rate (FDR) less than 0.05. The list of 3944 array probes is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003451#ppat.1003451.s009" target="_blank">Table S1</a>. (C) Number of common (overlap) and unique genes that were differentially regulated between the Sm-Foxp3⁺ cells and Lm Foxp3⁺ cells, relative to ‘Naïve’ Foxp3+ cells (FDR<0.1 and fold change >1.5). (D) Immunity-associated genes up-regulated in Lm Foxp3⁺ cells. (E) Immunity-associated genes up-regulated in and Sm Foxp3⁺ cells.</p

Differentially expressed miRNAs and candidate miRNA regulatory hubs in Th1- and Th2-Treg cells.

<p>miRNAs with significantly altered expression following (A) <i>Schistosoma mansoni</i> (Sm) or (C) <i>Leishmania major</i> (Lm) infection (Student's t-test p-value<0.05); y-axis: log (fold-change) of miRNA expression level. Representation of predicted targets for up-regulated miRNAs among down-regulated genes following <i>S. mansoni</i> (Sm) infection (B). Representation of predicted targets for down-regulated miRNAs among up-regulated genes following <i>L. major</i> (Lm) infection (D). Y-axis: −log of the empirical p-value of predicted target site enrichment over background expectation. Orange: miRNAs predicted to target differentially expressed genes significantly more than expected by chance, full details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003451#ppat.1003451.s012" target="_blank">Table S4</a>. Dashed line: p-value = 0.05.</p

Additional file 1: of A survey of microRNA single nucleotide polymorphisms identifies novel breast cancer susceptibility loci in a case-control, population-based study of African-American women

Table S1. Association of the top seven miRNA SNPs from the full analyses with p < 5 × 10−6 in case versus control and case-only subtype analyses. (XLSX 72 kb