In this experiment, L_PX was used as the control group and L_JN was the experimental group. A total of 265 differentially expressed circRNAs were identified, of which 178 were up-regulated and 87 were down-regulated. Since most circRNAs come from exon regions within protein coding genes, circRNA can inhibit the expression of host genes . Since the circular structure of circRNAs covalently linked is stable, their half-life is longer. Most circRNAs perform biological functions by acting as molecular sponges for miRNAs. Therefore, we will annotate the host gene function of DEcircRNA, the molecular sponge of miRNA, and ceRNA regulation networks and the circRNA-miRNA-mRNA axis will be used to analyze the potential regulatory functions of circRNA.
In this study, we annotated the host genes of circRNA and found that the host genes are mainly enriched in the cellular response to fatty acid, glycogen catabolic process, positive regulation of lipid catabolic process, regulation of fatty acid biosynthetic process, phosphatidylinositol 3-kinase complex, class III, SMAD protein complex and other GO terms related to fat deposition and metabolism. The result of KEGG enrichment analysis showed that host genes were mainly enriched in Fatty acid biosynthesis, TGF-beta signaling pathway, Citrate cycle (TCA cycle), Hippo signaling pathway and other signaling pathways, numerous of studies have shown that these signaling pathways are involved in adipocyte differentiation and lipid metabolism.
SMADs family phosphorylation can activate TGF- β Signal pathway . TGF- β inhibits the differentiation of 3T3-F442A cells into adipocytes through SMAD3 signal pathway . The transforming growth factor β (TGF β) superfamily consists of more than 33 members, including t TGF βs, BMPs, GDFs, activing, and nodule-associated proteins. BMPs and TGF- β bind to the receptor protein to form a complex that specifically activates and phosphorylates SMADs protein. The phosphorylated SMADs protein binds with SMAD4 to form a complex to transfer to the nucleus, thereby regulating gene transcription . TGF- β-SMAD2/3 signal can stimulate the proliferation of preadipocytes and inhibit fat production. It can also mediate bile acid and phospholipid metabolism after hepatotropic acid liver injury . BMP-SMAD1/5/8 signal pathway can not only promote the proliferation of adipocytes but also promote adipogenesis. The host genes of CircRNA may be involved in the above signaling pathways to regulate adipogenic differentiation and fat metabolism.
In addition to the ability of circRNA to regulate the transcription of host genes, more studies have shown that circRNA performs its regulatory function by competitively binding miRNAs. In this study, we predicted the binding relationship between DEcircRNA and miRNA, and found that circRNA_06424 and circRNA_08840 can bind more miRNA (Fig. 4).
CircRNA_06424 can competitively bind with 25 known miRNAs and inhibit the function of miRNAs. In a mouse model of non-alcoholic fatty liver disease induced by a high-fat diet, the expression level of miR-149 was significantly increased , further exploring the functions of miR-149, It was found that miR-149 can competitively bind to FGF-21 to promote fat production in HepG2 cells . MiR-149 is up-regulated in IMF, and may mainly play a role in promoting fat deposition in IMF cells. It can be used as a marker molecule for breeding high intramuscular fat pig breeds without affecting the level of subcutaneous fat deposition.
A total of 43 miRNAs are predicted to bind to circRNA_08840, miR-331-3p, ssc-miR-128, miR-361-3p, miR-143-5p, miR-874 and other miRNAs are differentially expressed in L_PX and L_JN. Chen et al. used Laiwu pig adipocytes as experimental materials to explore the function of miR-331-3p and found that miR-331-3p can inhibit the proliferation of adipose precursor cells by affecting the expression of key cell cycle genes such as CD2, CD3, and CD4. At the same time, it promotes the differentiation of pre-adipocytes and promotes fatty acid synthesis by regulating DLST to regulate the citric acid-pyruvate cycle . CircRNA_08840 may competitively bind miR-331-3p to regulate the proliferation and differentiation of precursor fat and fatty acid deposition. The expression level of miR-128 in the plasma of type 2 diabetic patients is significantly increased . Pan et al. transfected porcine preadipocytes with over-expressing miR-128 plasmid under glucocorticoid treatment(With miR-SC and miR-128 inhibitors as the control group), the results showed that the level of triglycerides was significantly increased, and further analysis found that miR-128 caused lipid accumulation by inhibiting the expression of SIRT1 . The expression of miR-128 is down-regulated in L_JN. Obviously, this is consistent with the results of previous studies. Wang et al. selected Huainan pig and Duroc as experimental materials and constructed a ceRNA regulatory network and found that miR-874 is closely related to adipogenesis . circRNA_08840 may regulate the level of lipid deposition through binding with target miRNAs.
The study results found that some miRNAs, such as miR-328, miR-296-5p, miR-365-5p, miR-339-5p, and miR-774, can simultaneously bind to circRNA_08840 and circRNA_06424. Recently, it has been found that there will be metabolic disorders in cancer patients, especially lipid metabolism. Therefore, some scholars believe that abnormally metabolized lipids are markers of cancer . Numerous of studies have shown that miR-328 regulates the anti-tumor effect of cancer by regulating target genes or downstream pathways [48, 49]. Overexpression of miR-328-3p aggravates oxidative stress injury of endothelial cells in the arterial valve, while inhibition of miR-328-3p expression can reduce oxidative stress injury of endothelial cells . MiR-328 can improve the inflammation, apoptosis, and oxidative stress response of endothelial cells induced by oxidized low-density lipoprotein (Ox-LDL) by specifically binding to HMGB1. MiR-328 overexpression PI3K-Akt signal pathway by acting on the target gene PTEN , the activity of PI3K-Akt signal pathway is decreased, and glucose absorption is decreased, which in turn regulates lipid metabolism. Overexpression of miR-328 can inhibit Bace1 translation, increase glucose metabolism, promote lipid accumulation in muscle progenitor cells, and promote the differentiation of brown adipocytes and brown adipocytes . In human adipose hepatocytes, miR-296-5p can target the 3 'UTR of PUMA and reduce the expression of PUMA, thus reducing lipotoxicity and adipocyte apoptosis . Belarbi compared the activity of EBF1 and differentially expressed miRNA in the white fat (WAT) of obese women and non-obese women, and found that mi-365-5p was expressed at a high level in obese women’s WAT and could target and regulate BEF1 and overexpress miR-365-5p can reduce the activity of EBF1 and cause white fat hypertrophy .
In this study, we performed functional annotation analysis on the ceRNA network centered on circRNA_06424 and circRNA_08840. The results showed that the target genes of circRNA_06424 were mainly enriched in the positive regulation of phosphatidylinositol 3-kinase signaling, phosphatase inhibitor activity, fatty acid binding, and other related GO items related to lipogenesis and metabolism. The results of KEGG showed that the target genes of circRNA_06424 are mainly involved in signal pathways such as Biosynthesis of unsaturated fatty acids, PPAR signaling pathway, and Wnt signaling pathway. These signaling pathways are also classic signaling pathways that regulate fat deposition metabolism. The functional annotation analysis of the ceRNA regulatory network with circRNA_08840 also obtained similar results. Wnt signal pathway is divided into classical Wnt signal pathway and atypical Wnt signal pathway. Typical Wnt signaling pathway enhances preadipocyte proliferation and inhibits the differentiation of preadipocytes into adipocytes. Atypical Wnt signaling pathway inhibits PPAR transcription in bone marrow mesenchymal stem cells, thereby inhibiting the differentiation of mesenchymal stem cells into adipocytes. However, some studies have shown that atypical Wnt signal pathway ligand WNT5B can inhibit the typical Wnt signal pathway when adipogenic precursors are stimulated by adipogenesis, thus promoting adipose differentiation.
Previously, we verified the function of ceRNA network and found that plays an important regulatory role in lipogenesis and metabolism. Similarly, we found that the target gene of novel circle RNA is at a key position in the PPI network. We found that genes regulated by circRNA_06424 can have more interactions, and these differential genes may play a more critical role in regulating fat. We select the circRNA_06424-ssc-miR-339-5p-PPARD axis for discussion.
Osmotic proliferator-activated receptor family (PPARs) are lipid-activating factors that play a variety of roles in fat development and metabolism. The main function of PPARD is to directly or activate related genes to regulate adipogenesis and lipid metabolism. In mice, PPARD agonists can alleviate obesity caused by high-fat diet. In vitro experiments, activating PPARD in fat cells can promote fatty acid oxidation and utilization . PPARD is more expressed in brown fat and consumes more lipid droplets , it can also induce the expression of UCP1 (a marker gene of brown fat). PPARD can also participate in fatty acid metabolism in skeletal muscle [62, 63]. Mouse myoblasts overexpressing porcine PPARD can inhibit the formation of myotubes under the conditions of in vitro culture. When PPARD and PPARG are co-expressed, they can promote the expression of fatty acid-binding protein (aP2) and other adipogenic genes in the cell, and triglycerides Large accumulation . Cytokines secreted by adipocytes can induce PPARD expression, which in turn induces alternate activation of macrophages . Adipocytes and macrophages use mitochondrial transfer between cells to participate in the metabolic homeostasis of the body, including promoting the utilization of glucose by adipocytes, regulating lipid storage and increasing energy consumption . PPARD can activate MMP9, break down insulin-like growth factor binding protein (IGFBP-3), IGF-1 receptor is activated, and promote the differentiation of umbilical vein endothelial cells (EPCS) and C2C12 grade cells . And EPCS and C2C12 are cultured in vitro under certain specific conditions and have the potential to differentiate into intramuscular fat [68, 69]. MMP9 can degrade the basement membrane , which may promote the invasion of macrophages into fat cells. In addition, the expression of MMP9 is also correlated with the level of low-density lipoprotein sterol , lipid metabolism and lipid uptake , visceral obesity , atherosclerotic lesions , diabetes and tumors , and other lipogenesis and metabolism diseases. With the continuous deepening of research on PPARD, it is found that the single nucleotide polymorphism (SNP) of PPARD has an important influence on its function [76, 77], for example, RS6902123 is an SNP significantly associated with type 2 diabetes . In this study, PPARD has a higher level of expression both in IMF and SCF, comparatively; PPARD expression is higher in SCF. The amount of IMF and SCF in Laiwu pigs is significantly higher than that of other breeds of pigs. PPARD has different mechanisms of action on fat deposition in different parts of Laiwu pigs, which may be related to the interaction of related genes and SNP. Still, the exact mechanism of action is not sure. We found that PPARD plays an important role in fat deposition, which is consistent with the results of previous studies. CircRNA_06424 can regulate the expression of PPARD by competitively binding miRNA with the mRNA of PPARD. This may also be the reason for the different regulatory mechanisms of PPARD in different adipose tissues. CircRNA_06424 can regulate the expression of PPARD by competitively binding miRNA with the mRNA of PPARD. This may also be the reason for the different regulatory mechanisms of PPARD in different adipose tissues.