Genome-wide identification and functional prediction of long non-coding RNAs in Sprague-Dawley rats during heat stress

Abstract

Background: Heat stress (HS) is a major stress event in the life of an animal, with detrimental upshots in production and health. Long-non-coding RNAs (lncRNAs) play an important role in many biological processes by transcriptional regulation. However, no research has been reported on the characterization and functionality of lncRNAs in heat-stressed rats.

Results: We studied expression levels of lncRNAs in rats during HS, using strand-specific RNA sequencing. Six rats, three in each of Control (22 ± 1 °C) and H120 (42 °C for 120 min) experimental groups, were used to screen for lncRNAs in their liver and adrenal glands. Totally, 4498 and 7627 putative lncRNAs were identified in liver and adrenal glands of the Control and H120 groups, respectively. The majority of lncRNAs were relatively shorter and contained fewer exons than protein-coding transcripts. In total, 482 (174 up-regulated and 308 down-regulated) and 271 (126 up-regulated and 145 down-regulated) differentially-expressed lncRNAs (DElncRNAs, P < 0.05) were identified in the liver and adrenal glands of the Control and H120 groups, respectively. Furthermore, 1274, 121, and 73 target differentially-expressed genes (DEGs) in the liver were predicted to interact with DElncRNAs based on trans-/cis- and sequence similarity regulatory modes. Functional annotation analyses indicated that these DEGs were mostly significantly enriched in insulin signalling, myeloid leukaemia, and glucagon signalling pathways. Similarly, 437, 73 and 41 target DEGs in the adrenal glands were mostly significantly enriched in the cell cycle (trans-prediction) and lysosome pathways (cis-prediction). The DElncRNAs interacting with DEGs that encode heat shock proteins (HSPs) may play an important role in HS response, which include Hsf4, Dnaja1, Dnajb4, Hsph1 and Hspb1 in the liver, and Dnajb13 and Hspb8 in the adrenal glands. The strand-specific RNA sequencing findings were also further verified through RT-qPCR.

Conclusions: This study is the first to provide a detailed characterization and functional analysis of expression levels of lncRNAs in liver and adrenal glands of heat-stressed rats, which provides basis for further studies on the biological functions of lncRNAs under heat stress in rats and other mammalian species.

Keywords: Adrenal glands; DEGs; Heat shock protein; Heat stress response; Liver; LncRNAs.

Fig. 1

The detailed schematic pipeline of long-non-coding RNA (lncRNA) transcripts identification. Control was kept at room temperature (22 ± 1 °C, relative humidity [RH] (%): 50%); H120 were subjected to 42 °C and RH 50% for 120 min. NR: (National Center for Biotechnology Information) NCBI non-redundant (NR) protein database; UniProt: Universal Protein Resource; CNCI: coding-non-coding index; CPAT: the coding potential assessment tool; PLEK: predictor of lncRNAs and mRNAs based on the k-mer scheme

Fig. 2

The Venn diagram for prediction of coding potential of non-coding transcripts in liver and adrenal glands. The > 2 SAMPLES means that only transcripts identified in at least two samples were retained for further analyses

Fig. 3

The classification and characterization of lncRNAs identified in liver and adrenal glands. a Number of lncRNAs in different categories. b Transcript lengths of protein-coding transcripts and lncRNAs. c Number of exons per transcript for protein-coding transcripts and lncRNAs. Left panel depicts results for liver and right panel for adrenal glands

Fig. 4

Hierarchical clustering and validation analysis of the specific differentially-expressed lncRNAs (DElncRNAs). a The Pheatmap of the top20 DElncRNAs in liver and adrenal glands. b The Pheatmap of commonly identified DElncRNAs in liver and adrenal glands. c The comparative analysis of the expression level of randomly selected lncRNAs in liver and adrenal glands using RNA-seq and RT-qPCR. The log _(10 + 1)-transformed FPKM values of DElncRNAs (rows) are clustered using hierarchical clustering, and the samples are grouped according to the similarity of expression profiles of DElncRNAs

Fig. 5

Statistical analysis of co-expression and the classification of the correlation frequency between DElncRNAs and differentially-expressed genes (DEGs). a Pearson correlation coefficient (PCC) analyses between DElncRNAs and DEGs in liver and adrenal glands. b Statistical analysis of the number of DEGs related to the same DElncRNAs

Fig. 6

Functional annotation of DEGs encoding proteins of the heat shock protein (HSP) family and their potential interactions with DElncRNAs. a and b Functional interaction networks among various DEGs that encode heat shock proteins (HSPs) identified in liver (a) and adrenal gland (b) tissues. c and d Interaction networks among different HSP-encoding DEGs and DElncRNAs. Interactions between DEGs and DElncRNAs were predicted by trans-, cis- and sequencing similarity regulatory module. The interaction network was visualized by Cytoscape software. For the nodes, the red ovals mean DElncRNAs, blue rectangles mean DEGs; for the lines, the dark orange lines mean positive correlation between DEGs and DElncRNAs (trans prediction), the blue lines mean negative correlation between DEGs and DElncRNAs (trans prediction), the black lines mean DElncRNAs regulated DEGs by cis-action and the green lines mean DElncRNAs regulated DEGs based on the sequence similarity