关键词: 藏猪, 巴马猪, 脂肪细胞, lncRNA, WGCNA

Abstract:

【Objective】Long non-coding RNAs (lncRNAs) are a class of RNA molecules with a length exceeding 200 nucleotides that generally do not encode proteins, and they play important regulatory roles in cold stress-induced adipose thermogenesis. This study aimed to analyze the transcriptomic data of subcutaneous adipose tissue from Tibetan pigs and Bama pigs to identify lncRNAs involved in regulating adipose metabolism under cold stress, thereby laying a foundation for further elucidation of the mechanisms underlying adipose thermogenesis in response to cold stress.【Method】Five-week-old Tibetan and Bama pigs (n = 8 per breed) were assigned to room temperature or 4 ℃ groups (four biological replicates per group). After 4 h, subcutaneous adipose tissue was collected for RNA-seq. Based on the sequencing data, lncRNAs and mRNAs were identified, and differential expression analysis was performed to compare cold stress responses between the two breeds. Target genes of differentially expressed lncRNAs were predicted via cis-regulation, followed by GO/KEGG enrichment analysis using DAVID. WGCNA was used to screen cold stress-related modules and construct co-expression networks of lncRNAs and mRNAs. Finally, RT-qPCR was performed to validate the transcriptome data of differentially expressed lncRNAs.【Result】A total of 2 988 lncRNAs were identified in the subcutaneous adipose tissue of Tibetan pigs and Bama pigs. These lncRNAs generally exhibited short transcript lengths and a small number of exons. Among them, 1 939 lncRNAs were expressed in at least one sample. Following cold stress treatment, 88 and 10 differentially expressed lncRNAs, as well as 360 and 88 differentially expressed mRNAs, were identified in the subcutaneous adipose tissue of Tibetan pigs and Bama pigs, respectively. Enrichment analysis indicated that the differentially expressed mRNAs were mainly enriched in biological processes such as HPV infection, fatty acid metabolism, and antiviral infection, as well as signaling pathways including the phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) pathway, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and the Ras-related protein 1 (Rap1) signaling pathway. Furthermore, pairs of cis-regulated lncRNAs and protein-coding genes were screened. Among these, 108 lncRNAs were located within 10 kb upstream or downstream of 83 protein-coding genes, and 216 differentially expressed lncRNAs were located within 100 kb upstream or downstream of 289 differentially expressed genes. Finally, a co-expression network of lncRNAs and protein-coding genes was constructed using WGCNA, identifying 18 modules. Among these, the yellow module was associated with the cold stress phenotype. Key lncRNAs within this module, such as MSTRG.8042.2 and MSTRG.8043.2, showed significant associations with core lipid metabolism-related genes. It is speculated that these lncRNAs may serve as key regulatory molecules under cold stress, participating coordinately in the host's cold defense and lipid metabolism regulation. The RT-qPCR results confirmed the reliability of the transcriptome data.【Conclusion】This study identified key lncRNAs including MSTRG.8042.2 and MSTRG.8043.2 that affect cold resistance of hosts under cold stress. The results provide a reference for deciphering the molecular mechanism regulating fat metabolism upon cold stress, and offer a novel perspective for elucidating the cold adaptation mechanism in local pig breeds.

Key words: Tibetan pig, Bama pig, adipocyte, lncRNA, WGCNA