Comparative analyses of long non-coding RNA in lean and obese pig

Abstract

Objectives: Current studies have revealed that long non-coding RNA plays a crucial role in fat metabolism. However, the difference of lncRNA between lean (Duroc) and obese (Luchuan) pig remain undefined. Here, we investigated the expressional profile of lncRNA in these two pigs and discussed the relationship between lncRNA and fat deposition.

Materials and methods: The Chinese Luchuan pig has a dramatic differences in backfat thickness as compared with Duroc pig. In this study, 4868 lncRNA transcripts (including 3235 novel transcripts) were identified. We determined that patterns of differently expressed lncRNAs and mRNAs are strongly tissue-specific. The differentially expressed lncRNAs in adipose tissue have 794 potential target genes, which are involved in adipocytokine signaling pathways, the PI3k-Akt signaling pathway, and calcium signaling pathways. In addition, differentially expressed lncRNAs were located to 13 adipose-related quantitative trait loci which include 65 QTL_ID. Subsequently, lncRNA and mRNA in the same QTL_ID were analyzed and their co-expression in two QTL_ID were confirmed by qPCR.

Conclusions: Our study provides an insight into mechanism behind the fat metabolic differences between the two breeds and lays an important groundwork for further research regarding the regulatory role of lncRNA in obesity development.

Keywords: QTL; lncRNA; obesity; pig.

Figure 1. Overview of lncRNA sequencing data

(A) The distribution of mapped reads on mRNA (5′–3′). Data in reflects the percentage of mapped reads assigned to all regions of mRNA. The location of the normalized mRNA is on the horizontal (x) axis; the percentage of reads as compared to total mapped reads for the position is on the vertical (y) axis. As the reference mRNA is different in length, each mRNA is divided into 100 intervals by length. (B) Reads mapped to different regions of the genome. (C) Venn diagrams show the result of four computational approaches. 4868 candidate lncRNAs were identified from a intersection results from the CNCI (coding-non-coding index), CPC (coding potential calculator), Pfam (protein folding domain database), and CPAT (coding potential assessing tool). (D) The type and number of predicted long non-coding RNAs (lncRNAs). Intronic lncRNA: lncRNA transcript from the intron region of gene; Antisense lncRNA: lncRNA has opposite transcriptional direction compared to adjacent mRNA; Sense lncRNA: lncRNA has the same transcriptional direction as the adjacent mRNA; Intergenic lncRNA: lncRNA transcribed from a position between two genes.

Figure 2. Features of lncRNAs and mRNAs in the genome

(A) LncRNAs distribution by chromosome. (B) Alternatively spliced isoforms per lncRNA and mRNA molecule. (C&D) Distribution of lncRNA and mRNA molecules by length.

Figure 3. Differential expression of lncRNAs and mRNAs by tissue

(A) The number of differentially expressed lncRNAs and mRNAs by tissue. Red bar represents up-regulated transcripts and the green bar represent down-regulate transcripts. (B) Expression profiles of lncRNA and mRNA in each tissue category. We used log10(FPKM) as the final data to indicate expression level. (C, D) Tissue-specific expression of lncRNAs and mRNA.

Figure 4. Heat-map of differently expression lncRNAs and mRNAs

(A) Cluster heat-map of differentially expressed lncRNAs from each sample. (B) Cluster heat-map of differentially expressed mRNAs from each sample.

Figure 5. GO and KEGG analysis of target genes in adipose tissue

(A) Gene Ontology analysis of target genes of differentially expressed lncRNAs from adipose tissue (L-fat vs D-fat). DEG Unigene: differentially expressed genes number in all annotation Biological Process GO term. All Unigene: Unigene number in all annotation Biological Process GO term. (B) KEGG pathway enrichment analysis of target genes of differentially expressed lncRNAs from adipose tissue (L-fat vs D-fat).

Figure 6. Co-expression of transcripts validation via quantitative real-time PCR

(A) IGV diagram indicates the location of co-expressed transcripts in the same QTL_ID. (B) Quantitative real-time PCR validation of lncRNAs and genes in the same QTL_ID region.