Analysis of microRNA expression profiles in porcine PBMCs after LPS stimulation

In the present study, we used microRNA (miRNA) sequencing to discover and explore the expression profiles of known and novel miRNAs in 1000 ng/ml LPS stimulated for 8 h vis-à-vis non-stimulated (i.e. control) PBMCs isolated from the blood of healthy pigs. A total of 291 known miRNAs were bio-computationally identified in porcine PBMCs, and 228 novel miRNAs (not enlisted in the swine mirBase) were identified. Among these miRNAs, ssc-miR-148a-3p, ssc-let-7g, ssc-let-7f, 3_8760, ssc-miR-26a, ssc-miR-451, ssc-miR-21, ssc-miR-30d, ssc-miR-99a and ssc-miR-103 were the top 10 most abundant miRNAs in porcine PBMCs. Through miRNA differential analysis combined with quantitative PCR, we found the expressions of ssc-miR-122, ssc-miR-129b, ssc-miR-17-5p and ssc-miR-152 were significantly changed in porcine PBMCs after LPS stimulation. Furthermore, targets prediction and function analysis indicated a significant enrichment in gene ontology functional categories related to diseases, immunity and inflammation. In conclusion, this study on profiling of miRNAs expressed in LPS-stimulated PBMCs provides an important reference point for future studies on regulatory roles of miRNAs in porcine immune system.     

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.     

Comparison of microRNAs in the intramuscular adipose tissue from Jinhua and Landrace pigs

MicroRNA (miRNA) is critically involved in lipogenesis occurring in various body parts of humans and animals. In this study, to further investigate the role and distribution of miRNA in porcine intramuscular adipose tissue, small RNAs were extracted from Jinhua and Landrace pigs to identify the expression profiles of miRNAs. miRNA expression profiles revealed that 558 miRNAs including 287 known and 271 novel miRNAs were identified, and 220 of them showed differential expression in the pigs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggested that the target genes of the differentially expressed miRNAs were involved in fatty metabolism. In conclusion, the current study reveals the active participation of miRNAs in the regulation of adipogenesis in the intramuscular adipose tissue of Jinhua and Landrace pigs.     

MicroRNA Transcriptome Profile Analysis in Porcine Muscle and the Effect of miR-143 on the MYH7 Gene and Protein

Porcine skeletal muscle fibres are classified based on their different physiological and biochemical properties. Muscle fibre phenotype is regulated by several independent signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) signalling pathways. MicroRNAs are non-coding small RNAs that regulate many biological processes. However, their function in muscle fibre type regulation remains unclear. The aim of our study was to identify miRNAs that regulate muscle fibre type during porcine growth to help understand the miRNA regulation mechanism of fibre differentiation. We performed Solexa/Illumina deep sequencing for the microRNAome during 3 muscle growth stages (63, 98 and 161 d). In this study, 271 mature miRNAs and 243 pre-miRNAs were identified. We detected 472 novel miRNAs in the muscle samples. Among the mature miRNAs, there are 23 highest expression miRNAs (over 10000 RPM), account for 85.3% of the total counts of mature miRNAs., including 10 (43.5%) muscle-related miRNAs (ssc-miR-133a-3p, ssc-miR-486, ssc-miR-1, ssc-miR-143-3p, ssc-miR-30a-5p, ssc-miR-181a, ssc-miR-148a-3p, ssc-miR-92a, ssc-miR-21, ssc-miR-126-5p). Particularly, both ssc-miR-1 and ssc-miR-133 belong to the MyomiRs, which control muscle myosin content, myofibre identity and muscle performance. The involvement of these miRNAs in muscle fibre phenotype provides new insight into the mechanism of muscle fibre regulation underlying muscle development. Furthermore, we performed cell transfection experiment. Overexpression/inhibition of ssc-miR-143-3p in porcine skeletal muscle satellite cell induced an/a increase/reduction of the slow muscle fibre gene and protein (MYH7), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate MYH7 through the HDAC4-MEF2 pathway.     

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.     

猪伪狂犬病毒Fa株侵染对PK-15细胞microRNAs表达谱的影响

【目的】分析猪伪狂犬病毒Fa株(PRV-Fa)侵染对猪肾传代细胞PK-15 microRNAs(miRNAs)表达谱的影响。【方法】利用Illumina高通量测序技术,鉴定感染和非感染PRV-Fa的PK-15细胞的miRNAs;筛选并利用实时荧光定量RT-PCR(RT-q PCR)验证差异表达miRNAs;对差异miRNAs进行靶基因预测和Gene ontology(GO)分析。【结果】在感染和未感染PK-15细胞中分别检测到384个和405个miRNAs,其中感染PRV-Fa后差异表达的miRNAs共127个(60个上调,67个下调)。荧光定量结果显示差异miRNAs的表达趋势与高通量测序结果一致。GO分析显示,miRNAs广泛参与信号传导、细胞代谢、免疫反应、基因表达等生物学进程,其中miR-10b、miR-16、miR-18a、miR-19b、miR-20a、miR-145-5p、miR-146a、miR-181a、miR-499-5p等miRNAs与免疫相关。在靶基因调控网络图中,ssc-miR-30a-5p与ssc-miR-30d处于关键位置。研究鉴定出5个新的病毒编码miRNAs,其中PRV-miR-LLT2与PRV-miR-LLT4靶向PRV早期蛋白基因EPO。【结论】伪狂犬病毒Fa株感染对PK-15细胞编码miRNAs有显著影响。     

Identification of host encoded microRNAs interacting with novel swine-origin influenza A (H1N1) virus and swine influenza virus

The discovery of microRNAs (miRNAs) is a remarkable breakthrough in the field of life science, and they are important actors which regulate gene expression in diverse cellular processes. Recently, several reports indicated that miRNAs can also target viruses and regulate virus replication. Here we discovered 36 pig-encoded miRNAs and 22 human-encoded miRNAs which have putative targets in swine influenza virus (SIV) and Swine-Origin 2009 A/H1N1 influenza virus (S-OIV) genes respectively. Interestingly, the putative interactions of ssc-miR-124a, ssc-miR-136 and ssc-miR-145 with their SIV target genes had been found to be maintained almost throughout all of the virus evolution. Enrichment analysis of previously reported miRNA gene expression profiles revealed that three miRNAs are expressed at higher levels in human lung or trachea tissue. The hsa-miR-145 and hsa-miR-92a putatively target the HA gene and hsa-miR-150 putatively targets the PB2 gene. Analysis results based on the location distribution from which virus was isolated and sequence conservation imply that some putative miRNA-mediated host-virus interactions may characterize the location-specificity.     

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 miRNAs and Their Target Genes Using Deep Sequencing and Degradome Analysis in Trifoliate Orange [<Emphasis Type="Italic">Poncirus trifoliate</Emphasis> (L.) Raf]

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.