Coordinated miRNA/mRNA expression profiles for understanding breed-specific metabolic characters of liver between Erhualian and large white pigs

Abstract

MicroRNAs (miRNAs) are involved in the regulation of various metabolic processes in the liver, yet little is known on the breed-specific expression profiles of miRNAs in coordination with those of mRNAs. Here we used two breeds of male newborn piglets with distinct metabolic characteristics, Large White (LW) and Erhualian (EHL), to delineate the hepatic expression profiles of mRNA with microarray and miRNAs with both deep sequencing and microarray, and to analyze the functional relevance of integrated miRNA and mRNA expression in relation to the physiological and biochemical parameters. EHL had significantly lower body weight and liver weight at birth, but showed elevated serum levels of total cholesterol (TCH), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), as well as higher liver content of cholesterol. Higher serum cortisol and lower serum insulin and leptin were also observed in EHL piglets. Compared to LW, 30 up-regulated and 18 down-regulated miRNAs were identified in the liver of EHL, together with 298 up-regulated and 510 down-regulated mRNAs (FDR<10%). RT-PCR validation of some differentially expressed miRNAs (DEMs) further confirmed the high-throughput data analysis. Using a target prediction algorithm, we found significant correlation between the up-regulated miRNAs and down-regulated mRNAs. Moreover, differentially expressed genes (DEGs), which are involved in proteolysis, were predicted to be mediated by DEMs. These findings provide new information on the miRNA and mRNA profiles in porcine liver, which would shed light on the molecular mechanisms underlying the breed-specific traits in the pig, and may serve as a basis for further investigation into the biological functions of miRNAs in porcine liver.

Figure 1. Hierarchical cluster analysis of the differentially expressed mRNAs in liver from Large White (LW) and Erhualian (EHL) piglets.

The figure was drawn by MeV software (version 4.2.6). (A) Differentially expressed mRNAs chosen with FDR <5%; (B) Differentially expressed mRNAs chosen with FDR <10%. Correlation (uncentred) similarity matrix and average linkage algorithms were used in the cluster analysis. Each row represents an individual mRNA, and each column represents a sample. The dendrogram at the left side and the top displays similarity of expression among mRNAs and samples individually. The color legend at the top represents the level of mRNA expression, with red indicating high expression levels and green indicating low expression levels. The codes on the legend are log2-transformed values.

Figure 2. Targets of DEMs among DEGs.

(A) Targets of DEMs included in DEGs list with FDR <5%; (B) Targets of DEMs included in the DEGs list with FDR <10%. Targets of DEMs were predicted by miRanda method. The target genes which were included in the DEGs were plotted. The x axis is log2 transformed fold change of DEMs, while the y axis stands for log2 transformed fold change of DEGs. The p-value was assessed by a two tailed chi-square test.

Figure 3. The targets of each individual DEM included in the lists of DEGs.

(A) DEGs with FDR <5%; (B) DEGs with FDR <10%. A two-tailed Fisher’s Exact Test was used to determine the significance (p<0.05, above red line at 1.3). The negative log of the p-value is plotted on the x-axis for both down-regulated mRNAs (white) and up-regulated genes (black).

Figure 4. Gene ontology analysis of DEGs with FDR <10%.

(A) biological process; (B) molecular function; and (C) cellular component. The GO terms were sorted by the enrichment p-value calculated by MAS 3.0, in an ascending order from bottom to top.

Figure 5. Gene ontology analysis of miRNA targeted DEGs and non-targeted DEGs.

(A) biological process; (B) molecular function; and (C) cellular component. The x axis represents the percentages of genes in total targeted or non-targeted DEGs of each GO term. The p-values assigned to GO terms were calculated by chi-square test, * indicates p-value <0.05.