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| The Detection of miRNAs Involved in Thyroid Hormone Regulation in Pig (Sus scrofa) by High Throughput Sequencing |
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Abstract Abstract Pig (Sus scrofa) is an important economic animal. Studies have found that the serum thyroid hormone levels of lean pigs are higher than those of obese pigs. MicroRNAs (miRNAs) play important roles in various biological processes. Despite a significant improvement in the identification of miRNAs in porcine tissues, the coverage of the porcine thyroid miRNAome is still scarce. In this study, castrated Jinhua (obese pigs) and Yorkshire pigs (lean pigs) at age of 120-days-old were used to delineate the expression profiles of miRNAs with high throughput sequencing. The number of raw reads of each transcriptome ranged from 11 to 12 million. After low quality sequences, polyA, sequences shorter than 18 or longer than 30 nucleotides, and repeated sequences were removed, the number of clean reads from each thyroid miRNA transcriptome decreased to 10~11 million. The small RNA length distribution showed that the most abundant species were 21~24 nucleotides, a typical size range for Dicer-derived products. Totally 293 known miRNAs were successfully identified in porcine thyroid gland. Besides, 60 novel microRNAs were predicted by using miREvo and mirdeap2 software. After expression levels of miRNAs were normalized to transcripts per million (TPM), a total of 15 miRNAs were found to be expressed at high levels (＞10 000), including ssc-miR-148a-3p, ssc-miR-30a-5p, ssc-miR-99a, ssc-miR-27b-3p, ssc-let-7f, ssc-miR-26a, ssc-let-7i, ssc-let-7g, ssc-miR-21, ssc-miR-7, ssc-miR-126-3p, ssc-miR-30d, ssc-miR-10a-5p, ssc-miR-143-3p and ssc-let-7a. This set of miRNAs may play important housekeeping roles in thyroid gland. DESeq was used to analyze the differentially expressed miRNAs in 2 pig breeds. In our study, 18 miRNAs were found with differential expression levels between Yorkshire and Jinhua pigs (P＜0.05), 6 miRNAs had a higher expression level in Yorkshire (ssc-miR-532-3p, ssc-miR-708-5p, ssc-miR-129a-5p, ssc-miR-708-3p, novel_7 and ssc-miR-362), whereas 12 miRNAs in Jinhua (ssc-miR-335, ssc-miR-7, novel_51, ssc-miR-153, ssc-miR-10a-3p, novel_36, ssc-miR-221-3p, ssc-miR-369, ssc-miR-452, novel_12, novel_11 and ssc-miR-221-5p). The expression level of 6 differentially expressed miRNAs was selected and verified by qRT-PCR. Consistent with the miRNA sequencing data, the qRT-PCR results of 6 randomly selected miRNAs showed a similar pattern of expression in 2 pig breeds, which further confirmed that our sequencing data was reliable. MiRanda was used to predict target genes of 18 differentially expressed miRNAs, and a total of 14 913 were identified as potential targets. Among them, thyroid peroxidase (TPO), solute carrier family 5 member 5 (SLC5A5), dual oxidase 2 (DUOX2) and dual oxidase maturation factor 2 (DUOXA2) were in involved the thyroid hormone synthesis. Furthermore, the predicted target genes were annotated in KEGG pathways to identify potential pathways that may be regulated by the differentially expressed miRNAs. The results indicated that target genes were involved in pathways like thyroid hormone synthesis pathway, endocytosis, calcium signaling pathway, peroxisome and lysosome which have been reported to be closely related with thyroid hormone regulation. This is the first study about miRNA profiles of porcine thyroid gland. The results provide information about miRNAs that are differentially expressed in thyroid gland of 2 different pig breeds with different levels of fat deposition, providing a better understanding of the possible roles of miRNAs in thyroid hormone regulation and the molecular mechanism of genetic difference between Yorkshire and Jinhua pigs.
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Received: 22 March 2017
Published: 06 August 2017
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