Inhibition of miR-29 has a significant lipid-lowering benefit through suppression of lipogenic programs in liver

MicroRNAs (miRNAs) are important regulators and potential therapeutic targets of metabolic disease. In this study we show by in vivo administration of locked nucleic acid (LNA) inhibitors that suppression of endogenous miR-29 lowers plasma cholesterol levels by ~40%, commensurate with the effect of statins, and reduces fatty acid content in the liver by ~20%. Whole transcriptome sequencing of the liver reveals 883 genes dysregulated (612 down, 271 up) by inhibition of miR-29. The set of 612 down-regulated genes are most significantly over-represented in lipid synthesis pathways. Among the up-regulated genes are the anti-lipogenic deacetylase sirtuin 1 (Sirt1) and the anti-lipogenic transcription factor aryl hydrocarbon receptor (Ahr), the latter of which we demonstrate is a direct target of miR-29. In vitro radiolabeled acetate incorporation assays confirm that pharmacologic inhibition of miR-29 significantly reduces de novo cholesterol and fatty acid synthesis. Our findings indicate that miR-29 controls hepatic lipogenic programs, likely in part through regulation of Ahr and Sirt1, and therefore may represent a candidate therapeutic target for metabolic disorders such as dyslipidemia

Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5′ and 3′ ends of small RNAs. Studies have shown that adapter ligation introduces a significant but widely unappreciated bias in the results of high-throughput small RNA sequencing. We show that due to this bias the two widely used Illumina library preparation protocols produce strikingly different microRNA (miRNA) expression profiles in the same batch of cells. There are 102 highly expressed miRNAs that are >5-fold differentially detected and some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. In an attempt to mitigate this bias, the new Bioo Scientific NEXTflex V2 protocol utilizes a pool of adapters with random nucleotides at the ligation boundary. We show that this protocol is able to detect robustly several miRNAs that evade capture by the Illumina-based methods. While these analyses do not indicate a definitive gold standard for small RNA library preparation, the results of the NEXTflex protocol do correlate best with RT-qPCR. As increasingly more laboratories seek to study small RNAs, researchers should be aware of the extent to which the results may differ with different protocols, and should make an informed decision about the protocol that best fits their study

Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C

Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30–35 nucleotide-long 5′ tRNA-halves (5′ tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5′ tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5′ tRH abundance was reduced, and relative abundance of individual 5′ tRHs was altered. In hepatitis B-associated HCC, 5′ tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer

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

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

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

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