Abstract
The domestic pig (Sus scrofa), an important species in animal production industry, is a right model for studying adipogenesis and fat deposition. In order to expand the repertoire of porcine miRNAs and further explore potential regulatory miRNAs which have influence on adipogenesis, high-throughput Solexa sequencing approach was adopted to identify miRNAs in backfat of Large White (lean type pig) and Meishan pigs (Chinese indigenous fatty pig). We identified 215 unique miRNAs comprising 75 known pre-miRNAs, of which 49 miRNA*s were first identified in our study, 73 miRNAs were overlapped in both libraries, and 140 were novelly predicted miRNAs, and 215 unique miRNAs were collectively corresponding to 235 independent genomic loci. Furthermore, we analyzed the sequence variations, seed edits and phylogenetic development of the miRNAs. 17 miRNAs were widely conserved from vertebrates to invertebrates, suggesting that these miRNAs may serve as potential evolutional biomarkers. 9 conserved miRNAs with significantly differential expressions were determined. The expression of miR-215, miR-135, miR-224 and miR-146b was higher in Large White pigs, opposite to the patterns shown by miR-1a, miR-133a, miR-122, miR-204 and miR-183. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411 and miR-2483 which had homologs in Bos taurus, among which ssc-miR-1343, miR-2320, miR-2411 and miR-2483 were validated in backfat tissue by stem-loop qPCR. Our results displayed a high level of concordance between the qPCR and Solexa sequencing method in 9 of 10 miRNAs comparisons except for miR-1a. Moreover, we found 2 miRNAs, miR-135 and miR-183, may exert impacts on porcine backfat development through WNT signaling pathway. In conclusion, our research develops porcine miRNAs and should be beneficial to study the adipogenesis and fat deposition of different pig breeds based on miRNAs.
Highlights
MicroRNAs, the small non-coding RNAs, were firstly discovered in C. elegans [1] and play important roles in regulating post-transcriptional translation [2]
Small RNAs (18–30 nt) were obtained from the total RNA, 59 and 39 RNA adaptors were ligated to the RNA pool, and the adaptor-ligated small RNAs were subsequently subjected to RT-PCR to produce sequencing libraries
The clean reads in the small RNA library of Large White was more than 19 million, and it was the same for Meishan small RNA library
Summary
MicroRNAs (miRNAs), the small non-coding RNAs (typically 19–23 nucleotides), were firstly discovered in C. elegans [1] and play important roles in regulating post-transcriptional translation [2]. Note: (1) LW-std: The result of Solexa sequencing exhibited, presented the normalized expression level of miRNA in Large White library. To enrich the repertoire of miRNAs in pigs, we constructed and sequenced two small RNA libraries prepared from the backfat tissue of 150-day-old Large White (LW) and Meishan (MS) pigs.
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