Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.     

Discovery of MicroRNAs Associated with Myogenesis by Deep Sequencing of Serial Developmental Skeletal Muscles in Pigs

MicroRNAs (miRNAs) are short, single-stranded non-coding RNAs that repress their target genes by binding their 3′ UTRs. These RNAs play critical roles in myogenesis. To gain knowledge about miRNAs involved in the regulation of myogenesis, porcine longissimus muscles were collected from 18 developmental stages (33-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100- and 105-day post-gestation fetuses, 0 and 10-day postnatal piglets and adult pigs) to identify miRNAs using Solexa sequencing technology. We detected 197 known miRNAs and 78 novel miRNAs according to comparison with known miRNAs in the miRBase (release 17.0) database. Moreover, variations in sequence length and single nucleotide polymorphisms were also observed in 110 known miRNAs. Expression analysis of the 11 most abundant miRNAs were conducted using quantitative PCR (qPCR) in eleven tissues (longissimus muscles, leg muscles, heart, liver, spleen, lung, kidney, stomach, small intestine and colon), and the results revealed that ssc-miR-378, ssc-miR-1 and ssc-miR-206 were abundantly expressed in skeletal muscles. During skeletal muscle development, the expression level of ssc-miR-378 was low at 33 days post-coitus (dpc), increased at 65 and 90 dpc, peaked at postnatal day 0, and finally declined and maintained a comparatively stable level. This expression profile suggested that ssc-miR-378 was a new candidate miRNA for myogenesis and participated in skeletal muscle development in pigs. Target prediction and KEGG pathway analysis suggested that bone morphogenetic protein 2 (BMP2) and mitogen-activated protein kinase 1 (MAPK1), both of which were relevant to proliferation and differentiation, might be the potential targets of miR-378. Luciferase activities of report vectors containing the 3′UTR of porcine BMP2 or MAPK1 were downregulated by miR-378, which suggested that miR-378 probably regulated myogenesis though the regulation of these two genes.     

Solexa sequencing identification of conserved and novel microRNAs in backfat of Large White and Chinese Meishan pigs

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.     

miR-425-5p as an exosomal biomarker for metastatic prostate cancer

Prostate cancer-related deaths are mostly caused by metastasis, which indicates the importance of identifying clinical prognostic biomarkers. In this study, we evaluated the expression profile of exosomal microRNAs (miRNAs) derived from metastatic prostate cancer (mPCa) cell lines (LNCaP and PC-3). miRNA signatures in exosomes and cells were evaluated by miRNA microarray analysis. Fourteen miRNAs were identified as candidates for specific noninvasive biomarkers. The expression of five miRNAs was validated using RT-qPCR, which confirmed that miR-205-5p, miR-148a-3p, miR-125b-5p, miR-183-5p, and miR-425-5p were differentially expressed in mPCa exosomes. Bioinformatic analyses showed that miR-425-5p was associated with residual tumor, pathologic T and N stages, and TP53 status in PCa samples. Gene ontology analysis of negatively correlated and predicted targeted genes showed enrichment of genes related to bone development pathways. The LinkedOmics database indicated that the potential target HSPB8 has a significant negative correlation with miR-425-5p. In conclusion, this study identified a panel of exosomal miRNAs with potential value as prognostic biomarkers for prostate cancer.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

     

Discovery of porcine miRNA‐196a/b may influence porcine adipogenesis in longissimus dorsi muscle by miRNA sequencing

Intramuscular fat (IMF) is one of the fat traits that has economic importance in the pork industry. Longissimus dorsi muscle contains IMF and is suitable for studying adipogenesis. To discover further potential regulatory miRNAs that may influence adipogenesis, we analyzed miRNA in the longissimus dorsi muscle of Yorkshire (YY, lean‐type) and Chinese Wannanhua (WH, fatty) pigs using miRNA sequencing (miRNA‐seq). From this dataset, we identified 598 unique miRNAs comprising 325 pre‐miRNAs and 273 novel pre‐miRNAs through comparison with known miRNAs in miRBase version 21. We found 42 miRNAs including nine up‐ and 33 down‐regulated between the YY and WH pigs. Moreover, we found two miRNAs, miR‐196a/b (miR‐196a, miR‐196b‐5p), that had the highest level of expression in WH pigs, and miR‐196a/b may influence porcine adipogenesis in longissimus dorsi muscle through an adipocytokine signaling pathway.     

Profile-based detection of microRNA precursors in animal genomes

MOTIVATION: MicroRNAs (miRNA) are essential 21-22 nt regulatory RNAs produced from larger hairpin-like precursors. Local sequence alignment tools such as BLAST are able to identify new members of known miRNA families, but not all of them. We set out to estimate how many new miRNAs could be recovered using a profile-based strategy such as that implemented in the ERPIN program. RESULTS: We constructed alignments for 18 miRNA families and performed ERPIN searches on animal genomes. Results were compared to those of a WU-BLAST search at the same E-value cutoff. The two combined approaches produced 265 new miRNA candidates that were not found in miRNA databases. About 17% of hits were ERPIN specific. They showed better structural characteristics than BLAST-specific hits and included interesting candidates such as members of the miR-17 cluster in Tetraodon. Profile-based RNA detection will be an important complement of similarity search programs in the completion of miRNA collections.