Sequencing And Characterization Of The MRNA,lncRNA And MiRNA In Thyroid Gland Of Yorkshire And Jinhua Pigs

Fat metabolism is a comlex process, and requires the coordinated action of several organs. The thyroid gland regulates the fat metabolism by producing thyroid hormones. The increased thyroid hormones can stimulate both lipogenesis and lipolysis, although lipolysis is influenced more than synthesis. Although previous studies revealed a variety of genes involved in the regulation of thyroid hormones, little is known about the role of non-coding RNAs. Long non-coding RNA (lncRNA) and microRNA are important regulatory non-coding RNAs. The Jinhua pig is a chinses indigenous fat-type pig breed, which has low growth rate, but strong fat deposition. The Yorkshire pig is a lean-type pig breed characterized by high growth rate, but low fat deposition. In this study, castrated Jinhua pigs and Yorkshire pigs of 120 days of age were chosen as experimentanimals. The serum thyroid hormones levels were measured, and RNA-seq and small RNA-seq were employed to explore mRNA, lncRNA and miRNA expression in the thyroid gland of Yorkshire and Jinhua pigs. Differentially expressed genes were filtered, and GO and KEGG analysis were performed. Besides, quantitative real-time PCR was performed to validate the selected differentially expressed genes. At last, we conbined the results of RNA-seq and small RNA-seq.The main results are as follows:(1) Body weight and thyroid gland weight of Yorkshire pigs were significantly higher than those of Jinhua pigs (P< 0.05), yet the thyroid index did not differ between two pig breeds. Yorkshire pigs showed obviously higher serum total thyroxine (TT4) and total triiodothyronine (TT3) levels compared with Jinhua pigs (P < 0.05). Serum free thyroxine (FT4) and free triiodothyronine levels (FT3) of Yorkshire were higher than those of Jinhua pigs, but the difference was not statistically significant (P> 0.05).(2) RNA-seq yielded about 351 million of 125-bp paired-end reads and approximately 83% of reads were ambiguously mapped against the pig reference genome (S. scrofa 10.2). A total of 22,435 known mRNAs were detected in the pig thyroid in this study. Of these identified mRNAs,18,464 expressed in both Jinhua and Yorkshire pigs, while 1,492 and 2,479 were breed-specific in Yorkshire pigs and Jinhua pigs respectively. A total of 1,018 lncRNA were identified the four sequencing libraries, including 760 intergenic,140 intronic and 118 antisense lncRNA. Further, 1189 novel mRNAs were identified. The candidate lncRNAs identified in this study were shorter in both transcript length and ORF length, fewer in exon number and less conserved than mRNAs. In the expression analysis,492 differentially expresssed (DE) mRNAs were identified between Yorkshire and Jinhua pigs. In these DE mRNAs,275 were upregulated in the Yorkshire pigs, while the remaining 217 were upregulated in the Jinhua pigs. The DE miRNAs were involved in the ’calcium signaling pathway’, ’tyrosine metabolism’,’thyroid hormone synthesis pathway’ and so on, and they were significantly enriched in ’cell cycle’ and ’microtubule-based process’. A total of 48 lncRNA were identified to be differentially expressed between Yorkshire pigs and Jinhua pigs. Among these DE lncRNAs,10 were located in nearby differentially expressed coding genes (distance< 100 kb), and 13 miRNA-mRNA pairs were predicted. Four types of alternative splicing events were detected in the porcine thyroid gland. And alternative 5’ first exon and alternative 3’ last exon were the commonest types of alternative splicing event in this study.(3) Small RNA-seq yielded about 44 million of single-end reads and approximately 74% of reads were ambiguously mapped against the pig reference genome (S. scrofa 10.2). A total of 293 known microRNAs were identified in the porcine thyroid gland, corresponding to 266 pre-miRNAs, belonging to 146 miRNA families. Further,60 novel miRNAs were identified. A total of 18 miRNAs were differentially expressed between Yorkshire pigs and Jinhua pigs (P< 0.05), of which 12 miRNAs were down-regulated and 6 were up-regulated in Yorkshire pigs compared to Jinhua pigs. The DE miRNAs were involed in’thyroid hormone synthesis pathway’,’the calcium signaling pathway’,’peroxisome’,’glutathione metabolism’,’lysosome’, and’tyrosine metabolism’.(4) To validate the expression profiles obtained by RNA-Seq and small RNA-seq, quantitative real-time PCR (qRT-PCR) was performed on 18 differentially expressed genes. And the results of qRT-PCR were similar to high-throughput sequencing.(5) In order to investigate the functions of the differentially expressed mRNAs, lncRNA and miRNAs, we conbined the results of RNA-seq and small RNA-seq. Finally, a network including 237 mRNAs,18 miRNAs and 1 lncRNA were predicted.