Overexpression of miR-155 in the Liver of Transgenic Mice Alters the Expression Profiling of Hepatic Genes Associated with Lipid Metabolism

<div><p>Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.</p></div

Microarray revealed the altered hepatic lipid metabolism genes in the liver of Rm155LG/Alb-Cre mice.

<p>(<b>A</b>) Class comparison and hierarchical clustering of differentially expressed hepatic lipid metabolism-related genes between Rm155LG/Alb-Cre and control mouse liver. A cluster heat map for hepatic lipid metabolism-related genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s010" target="_blank">S6 Table</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s011" target="_blank">S7 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s012" target="_blank">S8 Table</a>) is shown. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s002" target="_blank">S2 Fig</a>. (<b>B-C</b>) Gene ontology (GO) (B) and KEGG pathway (C) analyses of up- and down-regulated genes between Rm155LG/Alb-Cre and control mouse liver. Genes with expression changes of greater than 2-fold with P values below 0.05 were identified and classified using GO categories.</p

Enforced expression of miR-155 in the liver of Rm155LG/Alb-Cre mice improved HFD-induced hepatic steatosis.

<p><b>(A)</b> Body weight of Rm155LG/Alb-Cre mice and controls fed normal chow diet or HFD. <b>(B)</b> Liver weight of Rm155LG/Alb-Cre mice vs. controls fed normal chow diet or HFD. <b>(C)</b> H&E staining and ORO staining of liver sections from control and Rm155LG/Alb-Cre mice. <b>(D-G)</b> Quantification of TC and TG in the serum and liver of control and Rm155LG/Alb-Cre mice fed either chow diet or HFD. Data are mean±SD (n = 6–8). Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.g003" target="_blank">Fig. 3</a>.</p

Rm155LG/Alb-Cre mice improved lipid metabolism in liver.

<p>(<b>A</b>) Body weight of Rm155LG/Alb-Cre mice and controls at different ages. (<b>B</b>) Relative liver weight of Rm155LG/Alb-Cre mice vs. controls. (<b>C</b>) Adult Rm155LG/Alb-Cre mouse (right) and control (left) fed a normal diet. (<b>D</b>) Gross morphology of Rm155LG/Alb-Cre mouse (right) and control (left) livers. <b>(E)</b> H&E staining and Oil red O (ORO) staining of liver sections from control and Rm155LG/Alb-Cre mice. (<b>F</b>) Quantification of TC, TG, HDL and FFA storage in the liver of control and Rm155LG/Alb-Cre mice. TC, total cholesterols; TG, triglycerides; HDL, high-density lipoprotein; FFA, free fatty acids. Data are mean±SD (n = 9–10). Statistical significance was determined by two-tailed student t-test.</p

Blood profile for control and Rm155LG/Alb-Cre mice.

<p>Cre control mice were littermates of the Rm155LG/Alb-Cre mice. Aspartate aminotransferase (AST), alanine transaminase (ALT), cholesterol and triglyceride values were determined in serum. WAT, white adipose tissue; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Data are mean ± SD. Statistical significance was determined by two-tailed student t-test.</p><p>*, <i>P</i><0.05.</p><p>Blood profile for control and Rm155LG/Alb-Cre mice.</p

Identification of Ces3/TGH as a miR-155 target gene.

<p><b>(A)</b> Ces3 is a target gene of miR-155. The luciferase reporter assay was performed using Hepa1–6 cells as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#sec002" target="_blank">Materials and methods</a> section. <b>(B)</b> Microarray revealed the reduced expression of Ces3 in the liver of Rm155LG/Alb-Cre mice. A cluster heat map for 9 hepatic lipid metabolism-related genes is shown. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s002" target="_blank">S2 Fig</a>. <b>(C)</b> qRT-PCR analysis of Ces3 expression in Rm155LG/Alb-Cre mouse liver. <b>(D-E)</b> qRT-PCR assay for Ces3 expression in vector- and miR-155-expressing 7402 (D) and 7404 (E) cells. <b>(F)</b> qRT-PCR analysis of Ces3 expression in miR-155 knockout mouse liver. <b>(G)</b> Schematic diagram indicating the pathway of miR-155-mediated downregulation of Ces3 expression leading to reduced hepatic and blood TG levels.</p

A proposed model on miR-155 overexpression reducing hepatic and blood lipid profiles.

<p>The enforced expression of miR-155 in the liver of transgenic mice is able to induce a general downward trend in the expression profile of hepatic genes involved lipogenesis, fatty acid metabolism, triacylglycerol metabolism, cholesterol metabolismn and bile acid biosynthesis, etc, thereby causing the decreased hepatic lipid content by decreasing adipogenic and lipogenic gene expression in liver, which reduces blood lipid concentration. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s010" target="_blank">S6 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s011" target="_blank">S7 Table</a>.</p

cDNA microarray and qRT-PCR revealed a general downward trend in the expression of hepatic cholesterol, triacylglycerol and fatty acid synthesis-related genes in Rm155LG/Alb-Cre transgenic mouse liver.

<p>Graph illustrating the fold change in gene expression of representative differentially hepatic lipid metabolism-related genes between Rm155LG/Alb-Cre and control mouse liver. qRT-PCR validated microarray-derived data on the increased or decreased mRNA expression of hepatic lipid metabolism-related genes in Rm155LG/Alb-Cre transgenic mouse liver. Additionally, a cluster heat map for hepatic lipid metabolism-related genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s010" target="_blank">S6 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s011" target="_blank">S7 Table</a>) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.g004" target="_blank">Fig. 4A</a>. Other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118417#pone.0118417.s002" target="_blank">S2 Fig</a>.</p

MiR-155 Enhances Insulin Sensitivity by Coordinated Regulation of Multiple Genes in Mice

<div><p>miR-155 plays critical roles in numerous physiological and pathological processes, however, its function in the regulation of blood glucose homeostasis and insulin sensitivity and underlying mechanisms remain unknown. Here, we reveal that miR-155 levels are downregulated in serum from type 2 diabetes (T2D) patients, suggesting that miR-155 might be involved in blood glucose control and diabetes. Gain-of-function and loss-of-function studies in mice demonstrate that miR-155 has no effects on the pancreatic β-cell proliferation and function. Global transgenic overexpression of miR-155 in mice leads to hypoglycaemia, improved glucose tolerance and insulin sensitivity. Conversely, miR-155 deficiency in mice causes hyperglycemia, impaired glucose tolerance and insulin resistance. In addition, consistent with a positive regulatory role of miR-155 in glucose metabolism, miR-155 positively modulates glucose uptake in all cell types examined, while mice overexpressing miR-155 transgene show enhanced glycolysis, and insulin-stimulated AKT and IRS-1 phosphorylation in liver, adipose tissue or skeletal muscle. Furthermore, we reveal these aforementioned phenomena occur, at least partially, through miR-155-mediated repression of important negative regulators (i.e. C/EBPβ, HDAC4 and SOCS1) of insulin signaling. Taken together, these findings demonstrate, for the first time, that miR-155 is a positive regulator of insulin sensitivity with potential applications for diabetes treatment.</p></div

A proposed model on the positive roles of miR-155 in glucose metabolism by coordinated regulation of multiple genes.

<p>A proposed model on the positive roles of miR-155 in glucose metabolism by coordinated regulation of multiple genes.</p