Yanzhi Jiang

Evaluation of the Chinese indigenous pig breed Dahe and crossbred Dawu for growth and carcass characteristics, organ weight, meat quality and intramuscular fatty acid and amino acid composition.

The objectives of the experiment were to evaluate growth and carcass characteristics, organ weight, meat quality and intramuscular fatty acid (FA) and amino acid composition between the Chinese indigenous pig breed Dahe and the crossbred Dawu. The Dahe pigs had lower average daily gain (P < 0.001) and a higher feed conversion ratio (P < 0.001) compared with the Dawu pigs. The Dahe pigs contained less lean meat percentage (P < 0.001) and more carcass fat percentage (P < 0.001) compared with the Dawu pigs. For organ weight, the Dahe pigs had lower relative heart weight and small intestine weight, respectively, compared with that of the Dawu pigs (P < 0.001). In addition, the Dahe pigs showed higher pH values (at 45 min and 24 h, P < 0.001 and P < 0.001, respectively), higher Marbling score (P < 0.05), lower Minolta L values (at 45 min and 24 h, P < 0.001 and P < 0.05, respectively) and lower muscle fiber area (P < 0.05) than did the Dawu pigs. C18:1, C16:0, C18:0 and C18:2 were the main FAs and nine essential amino acids were found in the Longissimus dorsi of the two breeds.

MicroRNA transcriptomes relate intermuscular adipose tissue to metabolic risk.

Intermuscular adipose tissue is located between the muscle fiber bundles in skeletal muscles, and has similar metabolic features to visceral adipose tissue, which has been found to be related to a number of obesity-related diseases. Although various miRNAs are known to play crucial roles in adipose deposition and adipogenesis, the microRNA transcriptome of intermuscular adipose tissue has not, until now, been studied. Here, we sequenced the miRNA transcriptomes of porcine intermuscular adipose tissue by small RNA-sequencing and compared it to a representative subcutaneous adipose tissue. We found that the inflammation- and diabetes-related miRNAs were significantly enriched in the intermuscular rather than in the subcutaneous adipose tissue. A functional enrichment analysis of the genes predicted to be targeted by the enriched miRNAs also indicated that intermuscular adipose tissue was associated mainly with immune and inflammation responses. Our results suggest that the intermuscular adipose tissue should be recognized as a potential metabolic risk factor of obesity.

Comprehensive variation discovery and recovery of missing sequence in the pig genome using multiple de novo assemblies

Uncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs and structural variants, as well as 137.02-Mb sequences harboring 1737 protein-coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research.

Transcriptome Analysis of Liangshan Pig Muscle Development at the Growth Curve Inflection Point and Asymptotic Stages Using Digital Gene Expression Profiling

Animal growth curves can provide essential information for animal breeders to optimize feeding and management strategies. However, the genetic mechanism underlying the phenotypic differentiation between the inflection point and asymptotic stages of the growth curve is not well characterized. Here, we employed Liangshan pigs in stages of growth at the inflection point (under inflection point: UIP) and the two asymptotic stages (before the inflection point: BIP, after the inflection point: AIP) as models to survey global gene expression in the longissimus dorsi muscle using digital gene expression (DGE) tag profiling. We found Liangshan pigs reached maximum growth rate (UIP) at 163.6 days of age and a weight of 134.6 kg. The DGE libraries generated 117 million reads of 5.89 gigabases in length. 21,331, 20,996 and 20,139 expressed transcripts were identified BIP, UIP and AIP, respectively. Among them, we identified 757 differentially expressed genes (DEGs) between BIP and UIP, and 271 DEGs between AIP and UIP. An enrichment analysis of DEGs proved the immune system was strengthened in the AIP stage. Energy metabolism rate, global transcriptional activity and bone development intensity were highest UIP. Meat from Liangshan pigs had the highest intramuscular fat content and most favorable fatty acid composition in the AIP. Three hundred eighty (27.70%) specific expression genes were highly enriched in QTL regions for growth and meat quality traits. This study completed a comprehensive analysis of diverse genetic mechanisms underlying the inflection point and asymptotic stages of growth. Our findings will serve as an important resource in the understanding of animal growth and development in indigenous pig breeds.

Methylation of miR-145a-5p promoter mediates adipocytes differentiation.

MicroRNAs (miRNAs, miR) play important roles in adipocyte development. Recent studies showed that the expression of several miRNAs is closely related with promoter methylation. However, it is not known whether miRNA mediates adipocytes differentiation by means of DNA methylation. Here, we showed that miR-145a-5p was poorly expressed in adipose tissue from mice fed a high fat diet (HFD). Overexpression or inhibition of miR-145a-5p was unfavorable or beneficial, respectively, for adipogenesis, and these effects were achieved by regulating adipocyte-specific genes involved in lipogenic transcription, fatty acid synthesis, and fatty acid transportation. Particularly, we first suggested that miR-145a-5p mimics or inhibitors promoted or repressed adipocytes proliferation by regulating p53 and p21, which act as cell cycle regulating factors. Surprisingly, the miR-145a-5p-repressed adipocyte differentiation was enhanced or rescued when cells treated with 5-Aza-dC were transfected with miR-145a-5p mimics or inhibitors, respectively. These data indicated that, as a new mean to positively regulate adipocyte proliferation, the process of miR-145a-5p-inhibited adipogenesis may be regulated by DNA methylation.

Genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA transcriptomes in oxidative and glycolytic skeletal muscles.

The physiological, biochemical and functional differences between oxidative and glycolytic muscles play important roles in human metabolic health and in animal meat quality. To explore these differences, we determined the genome-wide landscape of DNA methylomes and their relationship with the mRNA and miRNA transcriptomes of the oxidative muscle psoas major (PMM) and the glycolytic muscle longissimus dorsi (LDM). We observed the hypo-methylation of sub-telomeric regions. A high mitochondrial content contributed to fast replicative senescence in PMM. The differentially methylated regions (DMRs) in promoters (478) and gene bodies (5,718) were mainly enriched in GTPase regulator activity and signaling cascade-mediated pathways. Integration analysis revealed that the methylation status within gene promoters (or gene bodies) and miRNA promoters was negatively correlated with mRNA and miRNA expression, respectively. Numerous genes were closely related to distinct phenotypic traits between LDM and PMM. For example, the hyper-methylation and down-regulation of HK-2 and PFKFB4 were related to decrease glycolytic potential in PMM. In addition, promoter hypo-methylation and the up-regulation of miR-378 silenced the expression of the target genes and promoted capillary biosynthesis in PMM. Together, these results improve understanding of muscle metabolism and development from genomic and epigenetic perspectives.

MicroRNA-27b Regulates Mitochondria Biogenesis in Myocytes

MicroRNAs (miRNAs) are small, non-coding RNAs that affect the post-transcriptional regulation of various biological pathways. To date, it is not fully understood how miRNAs regulate mitochondrial biogenesis. This study aimed at the identification of the role of miRNA-27b in mitochondria biogenesis. The mitochondria content in C2C12 cells was significantly increased during myogenic differentiation and accompanied by a marked decrease of miRNA-27b expression. Furthermore, the expression of the predicted target gene of miRNA-27b, forkhead box j3 (Foxj3), was also increased during myogenic differentiation. Luciferase activity assays confirmed that miRNA-27b directly targets the 3’-untranslated region (3’-UTR) of Foxj3. Overexpression of miRNA-27b provoked a decrease of mitochondria content and diminished expression of related mitochondrial genes and Foxj3 both at mRNA and protein levels. The expression levels of downstream genes of Foxj3, such as Mef2c, PGC1α, NRF1 and mtTFA, were also decreased in C2C12 cells upon overexpression of miRNA-27b. These results suggested that miRNA-27b may affect mitochondria biogenesis by down-regulation of Foxj3 during myocyte differentiation.

Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation

Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

Tissue expression pattern and polymorphism of G0S2 gene in porcine.

Adipose triglyceride lipase (ATGL), catalyzing the initial step of hydrolysis of triacylglycerol (TAG) in adipocytes, has been known to be inhibited by G0/G1 switch protein 2 (G0S2). In this study, we determined tissue expression pattern and polymorphism of G0S2 gene in porcine. The results showed that the G0S2 transcript levels were very high in the liver and, to a lesser degree, in adipose tissues of greater omentum and suet fat; and low G0S2 transcript levels were observed in other tissues. A comparative study on the transcript levels between ATGL and G0S2 genes showed that ATGL transcript levels were high in all six adipose tissues, but negligible in the liver. Higher transcript levels were obtained for sows in adipose tissues of the inner layer of subcutaneous fat and suet fat, but higher expression values were found for boars in the liver, spleen, and stomach. 19 single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs (g.-307A>T, g.-394C>G, g.-565G>A, and g.-566T>C), were found in porcine G0S2 genomic DNA. Association analyses showed that the g.-565G>A and g.-742T>A SNPs were associated with back fat thickness (BFT). In conclusion, G0S2 mRNAs are abundantly expressed in porcine liver and adipose tissues of greater omentum and suet fat, and sex affects porcine G0S2 tissue transcript levels; meanwhile, the genetic diversity of porcine G0S2 gene is abundant and 2 SNPs are a genetic factor affecting BFT.

DNA methylation landscape of fat deposits and fatty acid composition in obese and lean pigs

Obese and lean type pig breeds exhibit differences in their fat deposits and fatty acid composition. Here, we compared the effect of genome-wide DNA methylation on fatty acid metabolism between Landrace pigs (LP, leaner) and Rongchang pigs (RP, fatty). We found that LP backfat (LBF) had a higher polyunsaturated fatty acid content but a lower adipocyte volume than RP backfat (RBF). LBF exhibited higher global DNA methylation levels at the genome level than RBF. A total of 483 differentially methylated regions (DMRs) were located in promoter regions, mainly affecting olfactory and sensory activity and lipid metabolism. In LBF, the promoters of genes related to ATPase activity had significantly stronger methylation. This fact may suggest lower energy metabolism levels, which may result in less efficient lipid synthesis in LBF. Furthermore, we identified a DMR in the miR-4335 and miR-378 promoters and validated their methylation status by bisulfite sequencing PCR. The hypermethylation of the promoters of miR-4335 and miR-378 in LBF and the resulting silencing of the target genes may result in LBF’s low content in saturated fatty acids and fat deposition capacity. This study provides a solid basis for exploring the epigenetic mechanisms affecting fat deposition and fatty acid composition.