Comprehensive Analysis of Differentially Expressed mRNA, lncRNA and circRNA and Their ceRNA Networks in the Longissimus Dorsi Muscle of Two Different Pig Breeds.

Circular RNA (circRNA) and long non-coding RNA (lncRNA) are known to participate in adipogenesis and myogenic differentiation, but their impact on porcine muscle traits is not well understood. We compared their expressional profiles in the longissimus dorsi muscle of Chinese Huainan pigs (HN, the fat type) and Western commercial Duroc×(Landrace×Yorkshire) (DLY, the thin type) pigs, and 854 mRNAs, 233 lncRNAs, and 66 circRNAs (p < 0.05 and ｜log2FoldChange｜>1) were found to be differentially expressed. The differentially expressed mRNA and circRNA parental genes were enriched in the Wnt signaling pathway (adipogenesis), the transition between fast and slow fibers (myogenic differentiation), and alanine, aspartate and glutamate metabolism (pork flavor). The potential lncRNAs/circRNAs-miRNAs-mRNAs regulatory networks shared MYOD1, PPARD, miR-423-5p and miR-874, which were associated with skeletal muscle muscular proliferation, differentiation/regeneration and adipogenesis. Taken together, these differentially expressed non-coding RNAs may be involved in the molecular basis of muscle traits, acting as the competing endogenous RNA (ceRNA) for miRNAs.

Identification and characterization of long non-coding RNAs in subcutaneous adipose tissue from castrated and intact full-sib pair Huainan male pigs.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate adipose tissue metabolism, however, their function on testosterone deficiency related obesity in humans is less understood. For this research, intact and castrated male pigs are the best model animal because of their similar proportional organ sizes, cardiovascular systems and metabolic features. RESULTS: We identified lncRNAs in subcutaneous adipose tissue by deep RNA-sequencing using the intact and castrated Huainan male pigs. The results showed that castration reduced serum testosterone but increased body fatness-related traits (serum triglyceride levels, backfat thickness, intramuscular fat content, and adipocyte size). Meanwhile, 343 lncRNAs from subcutaneous adipose tissue were identified, including 223 intergenic lncRNAs (lincRNAs), 68 anti-sense lncRNAs, and 52 intronic lncRNAs. It was predicted that there were 416 recognition sites for C/EBPα in the 303 lncRNA promoter region, and 13 adipogenesis-promoting miRNAs and five adipogenesis-depressing miRNAs target these lncRNAs. Eighteen lncRNAs, including nine up- and nine down-regulated had more than 2-fold differential expression between the castrated and intact male pigs (q-value < 0.05). Functional analysis indicated that these 18 lncRNAs and their target genes were involved in fatty acid, insulin, and the adipocytokine signaling pathway. We further analyzed the features of a conserved mouse lncRNA gene ENSMUST00000189966 and found it mainly expressed in the cell nucleus and target the Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) gene. In 3 T3-L1 cells, differentiation down-regulated their expression, but dihydrotestosterone (DHT) significantly up-regulated their expression in a concentration-dependent manner (P < 0.05). CONCLUSIONS: These results suggested that lncRNAs and their target genes might participated in the castration-induced fat deposition and provide a new therapeutic target for combatting testosterone deficiency-related obesity.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Effects of Saccharomyces cerevisiae on alleviating cytotoxicity of porcine jejunal epithelia cells induced by deoxynivalenol.

Deoxynivalenol (DON) is one of the mycotoxins most frequently encountering in cereal-based foods throughout the world. Saccharomyces cerevisiae was used to alleviate porcine jejunal epithelia cell (IPEC-J2) injury induced by DON in this study. The results indicated that cell viability and proliferation rates were significantly decreased when DON concentrations were increased from 0 to 64 µM after 24 h incubation (p < 0.05). The longer incubation time and higher DON concentrations would cause more serious effects on cell viability. S. cerevisiae could significantly degrade DON and decrease lactic dehydrogenase (LDH) release in the cells induced by DON (p < 0.05). DON (4 µM) could increase necrotic and apoptotic cell rates as well as decrease viable cell rates, compared with the control group (p < 0.05). However, S. cerevisiae addition in the DON group could decrease necrotic, late apoptotic and early apoptotic cell rates by 38.05%, 46.37% and 44.78% respectively, increase viable cell rates by 2.35%, compared with the single DON group (p < 0.05). In addition, S. cerevisiae addition could up-regulate mRNA abundances of IL-6, IL-8 and IL-10 in IPEC-J2 cells (p < 0.05), but down-regulate mRNA abundances of tight junction proteins (TJP-1) and occludin by 36.13% and 50.18% at 1 µM of DON (p < 0.05). It could be concluded that S. cerevisiae was able to alleviate IPEC-J2 cell damage exposed to DON.

Long non-coding RNA analysis of muscular responses to testosterone deficiency in Huainan male pigs.

Long non-coding RNAs (lncRNAs) participated in growth and development of skeletal muscle; however, little is known about their response to testosterone deficiency in porcine skeletal muscle. We compared lean mass related carcass traits and lncRNAs expression files in Longissimus dorsi (LD) muscle between intact and castrated Huainan male pigs. The results showed that castration significantly reduced eye muscle area and lean meat percentage (P < 0.05), but increased the fat mass weight (P < 0.05). Meanwhile, 8946 lncRNAs, including 6743 intergenic lncRNAs (lincRNAs), 498 anti-sense lncRNAs, and 1705 intronic lncRNAs, were identified in porcine LD, among which, 385 lncRNAs were considered as the differentially expressed candidates between intact groups and castrated groups (q-value < 0.05). Functional analysis indicated that these differently expressed lncRNAs and their target genes were involved in the estrogen receptor signaling pathway and skeletal and muscular system development and function. We first detect porcine muscular lncRNA response to castration, and the results suggested that lncRNAs and their target genes participated in the regulation of testosterone deficiency-related skeletal muscle growth.

Castration-induced changes in the expression profiles and promoter methylation of the GHR gene in Huainan male pigs.

Castration plays a regulatory role in growth and carcass traits, particularly in fat deposition, but its molecular mechanisms are still not clear. The present study showed that castration significantly reduced the serum growth hormone and the responses of the growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-I), IGF-IR and peroxisome proliferator-activated receptor gamma (PPARγ) to castration were similar in different adipose tissues. However, the GHR expression trends were opposite between the liver and the adipose tissues; bisulfite sequencing PCR (BSP) showed that its methylation in these two tissues was different. In particular, the GHR methylation rate in the liver of castrated and intact pigs were 93.33% and 0, respectively, which was consistent with its higher expression level in the intact group. It was predicted that there were potential binding sites for 11 transcription factors in the ninth CpG site (which was methylated and demethylated in subcutaneous adipose tissue of the intact and castrated groups, respectively), including androgen receptor (AR), CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPß, all of which are important factors in lipid metabolism. These results indicate that DNA methylation may participate in castration-induced fat deposition.