Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Castration-induced changes in microRNA expression profiles in subcutaneous adipose tissue of male pigs

MicroRNAs (miRNAs) are class of molecular regulators found to participate in numerous biological processes, such as adipogenesis and obesity in mammals. To determine the roles of miRNAs involved in castration-induced body fatness, we investigated the different miRNA expression patterns in subcutaneous adipose tissue between intact and castrated male pigs. Our results showed that castration led to decrease serum testosterone but increase serum Leptin levels (P?<?0.01). Moreover, castration also increased adipocyte size, body fat content and backfat thickness in male pigs (P?<?0.01). Meanwhile, miRNA expression profiles in adipose tissue were changed by castration, and 18 miRNAs were considered as the differentially expressed candidates between intact and castrated male pigs. Furthermore, functional analysis indicated that the differential expressed miRNAs and their target genes are involved in the regulation of fatty acid metabolism. In brief, our present study provides a comprehensive view on how miRNAs works in subcutaneous adipose tissue with castration. These results suggested that miRNAs might play an important role in the castration-induced fat deposition in male pigs.     

Breed difference and regulation of the porcine adipose triglyceride lipase and hormone sensitive lipase by TNFα

Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are major novel triglyceride lipases in animals. The aim of this study was to determine if there are differences in the porcine ATGL ( pATGL ) and HSL genes between Jinhua pigs (a fatty breed) and Landrace pigs (a leaner breed). In addition, the effect of TNFα and pATGL-specific siRNA ( pATGL-siRNA ) on the expression of pATGL and HSL in porcine adipocytes was also examined. Compared with Landrace pigs, the body weight ( BW ) of Jinhua pigs was lower ( P <  0.01), while intramuscular fat content (in the longissimus dorsi muscle), as well as the back fat thickness and body fat content were higher ( P <  0.01). The expression of pATGL and HSL mRNA in Jinhua pigs was lower ( P <  0.01) in subcutaneous adipose tissue, and greater ( P <  0.01) in longissimus dorsi muscle compared with Landrace pigs. In vitro treatment of porcine adipocytes with TNFα decreased ( P <  0.01) the glycerol release and the gene expression of pATGL , HSL and PPARγ in porcine adipocytes. Furthermore, transfection with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL , while it had no effect on the expression of HSL . Treatment with 25 ng/ml TNFα in conjunction with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL and HSL in cultured porcine adipocytes. These results provide useful information to further the understanding of the function of pATGL and HSL in porcine lipid metabolism, which should be applicable to the regulation of fat deposition and improvement of meat quality.     

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.

     

中国两头乌猪品种内源性逆转录病毒基因研究

目的对5个中国两头乌猪品种(通城猪、东山猪、沙子岭猪、赣西两头乌猪和金华猪)及3个国外品种(大白猪、长白猪和杜洛克猪)猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因的3个亚型A、B、C,分别从DNA和RNA水平上进行研究,以发现中国两头乌猪品种在异种器官移植中的资源优势。方法利用PCR方法在DNA水平上对PERV基因的三个亚型进行鉴定,并通过半定量PCR方法在RNA水平上检测通城猪和大白猪PERV各亚型在心、肝、脾、肺、肾、肌肉、脂肪、淋巴和脑组织中的表达谱。结果4个华中两头乌猪种中env-AB型为主要PERV亚型,分别占被测总数的92%～100%。在这4个品种中均没有检测到C亚型,金华猪以及3个国外猪种中均检测到了C亚型,病毒亚型种类也更丰富。半定量PCR实验结果显示gag、pol基因在两个品种9个组织中广泛表达,env-A在通城猪的心、肝、肺、脂肪和淋巴组织中表达量较低,env-B在通城猪的心脏和淋巴组织中表达量较低,而env-B在大白猪的肾脏中表达很低,其他所测8个组织中表达量都较高。结论通城猪、东山猪、赣西两头乌猪和沙子岭猪可以做为较佳的异种移植候选供体,具有良好的应用前景。     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine sub-cutaneous fat thickness(SFT) and intramuscular fat(IMF) content.Obese and lean-type pig breeds show obvious differences in adipose deposition;however, the molecular mechanism underlying this phenotypic variation remains unclear.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages(1―5 months) of Landrace(a leaner, Western breed) and Taihu pigs(a fatty, indigenous, Chinese breed).Variance analysis(ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant(FDR adjusted permutation, P<0.05) among 5 growth stages.Gene class test(GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages(PErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associ-ated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.Clustering analysis revealed a very high level of significance(FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance(FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs.Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs.Based on a dy-namic Bayesian network(DBN) model, gene regulatory networks(GRNs) were reconstructed from time-series data for each pig breed.These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds;from these results, some potential key genes could be identified.Quantitative, real-time RT-PCR(QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages(R=0.874±0.071).These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven

To understand how miRNAs contribute to the molecular phenotype of adipose tissues and related traits, we performed global miRNA expression profiling in subcutaneous abdominal and gluteal adipose tissue of 70 human subjects and characterised which miRNAs were differentially expressed between these tissues. We found that 12% of the miRNAs were significantly differentially expressed between abdominal and gluteal adipose tissue (FDR adjusted p<0.05) in the primary study, of which 59 replicated in a follow-up study of 40 additional subjects. Further, 14 miRNAs were found to be associated with metabolic syndrome case-control status in abdominal tissue and three of these replicated (primary study: FDR adjusted p<0.05, replication: p<0.05 and directionally consistent effect). Genome-wide genotyping was performed in the 70 subjects to enable miRNA expression quantitative trait loci (eQTL) analysis. Candidate miRNA eQTLs were followed-up in the additional 40 subjects and six significant, independent cis-located miRNA eQTLs (primary study: p<0.001; replication: p<0.05 and directionally consistent effect) were identified. Finally, global mRNA expression profiling was performed in both tissues to enable association analysis between miRNA and target mRNA expression levels. We find 22% miRNAs in abdominal and 9% miRNAs in gluteal adipose tissue with expression levels significantly associated with the expression of corresponding target mRNAs (FDR adjusted p<0.05). Taken together, our results indicate a clear difference in the miRNA molecular phenotypic profile of abdominal and gluteal adipose tissue, that the expressions of some miRNAs are influenced by cis-located genetic variants and that miRNAs are associated with expression levels of their predicted mRNA targets.     

Developmental changes of carcass composition, meat quality and organs in the Jinhua pig and Landrace

The present study was aimed to compare the developmental changes of carcass composition, meat quality characteristics and organ weight in pigs of different breeds. Six pigs (sex balance) of each breed were slaughtered at 35, 80 and 125 days of age, respectively. The carcass was chilled and the left carcass side was dissected into bone, lean meat, fat and skin; additionally, organ weight and meat quality parameters were observed. Carcasses of the Jinhua pig were lighter (P < 0.001), contained less lean meat percentage (P < 0.01) and more carcass fat percentage (P < 0.05) than did carcasses of the Landrace. L*-values were lower in Jinhua pigs than in Landrace at 125 days of age (P < 0.05), but the Jinhua pig had higher a*-values compared with Landrace at the age of 80 days (P < 0.01) and 125 days (P < 0.01), respectively. In addition, Jinhua pigs showed lower colour scores (P < 0.05), higher intramuscular fat (IMF) percentage (P < 0.05), less marbling scores (P < 0.05) and lower drip loss (P < 0.05) than Landrace. For organ weight, Jinhua pigs had higher relative heart weight at the age of 80 days (P < 0.05) and 125 days (P < 0.001), and higher relative liver weight at 125 days of age (P < 0.01) than that of Landrace. In addition, the relative kidney weight was heavier (P < 0.001) in the Jinhua pig than in the Landrace during the whole experiment. These results indicated that developmental changes of carcass composition, meat quality parameters and organ weight displayed breed differences. Jinhua pigs were fatter than Landrace but the former had better quality characteristics in the meat.     

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.     

The novel gene pFAM134B positively regulates fat deposition in the subcutaneous fat of Sus scrofa

In this study, we analyzed the global gene expression profiles in the subcutaneous fat (SAT) of Jinhua pigs and Landrace pigs at 90 d. Several genes were significantly highly expressed in Jinhua pigs, including genes encoding the rate limiting enzymes in the TCA cycle, fatty acid activation, fatty acid synthesis and triglyceride synthesis. We identified a novel gene tagged by the EST sequences as public No. BF702245.1, which was named porcine FAM134B (pFAM134B) and the pFAM134B mRNA levels of SAT was significantly higher in Jinhua pigs than that in Landrace pigs at 90 d (P < 0.01). Then the effects of pFAM134B on lipid accumulation were investigated by using RNAi and gene overexpression in the subcutaneous adipocytes. The results showed that pFAM134B played a significant positive role in regulating lipid deposition by increasing the mRNA levels of PPARγ, lipogenic genes fatty acid synthetase (FAS) and acetyl-CoA carboxylase (ACC) (P < 0.01) and reducing the mRNA levels of adipose triglyceride lipase (ATGL) and lipase, hormone-sensitive (HSL) (P < 0.01). This study implied that pFAM134B might be a positive factor in lipid deposition, providing insight into the control of fat accumulation and lipid-related disorders.     

猪背最长肌中肌肉生长和脂肪沉积相关基因的差异表达和主成分分析

骨骼肌细胞和脂肪细胞在分化生长速度上相对竞争的平衡点是猪肉质和胴体性状的决定因素.利用Oligo功能分类芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在初生、1、2、3、4和5月龄间背最长肌中肌肉生长和脂肪沉积相关基因的动态表达变化.差异表达分析结果显示,在初生至5月龄的品种间分别有15、16、11、13、18和20个基因的表达差异倍数大于2倍.品种内的方差分析表明,长白猪分别有18和22个基因,太湖猪分别有3和7个基因在月龄间的表达差异达极显著(Ｐ<0.01)和显著水平(Ｐ<0.05).主成分分析结果显示,先降后升是两品种内最具代表性的基因表达模式,且长白猪和太湖猪分别有7和6个基因的表达模式明显偏离其他基因,提示其可能受到了重要的调控. 此外,5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势.以上结果筛选出了对于猪肉质和胴体性状可能具有重要影响,值得深入研究的一些候选基因,为深入研究生长发育过程中参与肌纤维生长和脂肪酸合成关键基因的表达变化规律和互作调控机制提供了基础数据.     

Mapping, expression and regulation of the TRα gene in porcine adipose tissue

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRα and TRβ. The TRα isoform plays a critical role in mediating the action of thyroid hormone in adipose tissue. We mapped the porcine TRα gene to chromosome 12 p11-p13, by using the ImpRH panel. We examined tissue-localization of TRα and determined expression patterns of TRα in porcine adipose tissue with quantitative real-time PCR. TRα was expressed in all tissues, including heart, liver, spleen, stomach, pancreas, brain, small intestine, skeletal muscle, and subcutaneous adipose tissue. In the adipose tissue, the expression of TRα decreased postnatally. Compared to Yorkshire pigs, Jinhua pigs had significantly lower expression levels of TRα gene in the subcutaneous fat tissue. The expression levels of β2-AR, HSL and ATGL were also significantly lower in Jinhua pigs than in Yorkshire pigs. However, no significant differences in PPARγ and SREBP-1C expression levels were found between Jinhua and Yorkshire pigs. Incubation of porcine adipose tissue explants with high doses of isoproterenol (100 and 1000 nM) significantly increased the expression levels of TRα. We conclude that there is considerable evidence that TRα plays an important role in fat deposition in porcine adipose tissue.