Transcriptome sequencing data analysis
Through circRNA sequencing analysis, raw data quality control and filtering, six cDNA libraries were constructed and a total of 11,187,520,262 to 13,382,8336 clean reads were obtained and all samples had a Q20 value of more than 98%, a Q30 value of more than 94% and a GC content of 47% to 52% (Table 2). The above results indicate that the sequencing data are of high quality and can be used for subsequent experiments.
circRNA prediction
30299 circRNAs were obtained by CIRI2 software analysis, of which 1880 circRNAs originated from exon shearing, 6650 were intron shearing and 3769 were intergenic_region shearing (Fig 1).
Analysis of differentially expressed circRNAs
The transcriptome sequencing data of G24 and F24 subcutaneous adipose tissue were analyzed by DESeq software and the results showed that a total of 628 differentially expressed circ RNAs were screened compared with G24, of which 219 circRNAs were up-regulated and 409 circRNAs were down-regulated (Fig 2).
Source gene GO annotation analysis and KEGG enrichment analysis
The 98 source genes of differentially expressed circRNAs were analyzed using GO function enrichment and the top 20 significantly enriched GO entries were mainly annotated to cellular components, biological processes and molecular functions, which were mainly related to molecular constructs, cellular parts, organs, bioregulation, cellular processes,
etc (Fig 3).
KEGG enrichment analysis of differentially expressed circ RNA source genes was performed using KEGG, in which 98 source genes could be KEGG-enriched again with 80 and the top 20 significantly enriched KEGG pathways were mainly phagosomes, AGE-RAGE signaling pathway in diabetic complications, protein digestion and absorption, cellular adhesion molecules, natural killer cell-mediated cytotoxicity and human T-cell leukemia virus infection (Fig 4, Table 3).
Analysis of KEGG-enriched pathways revealed nine pathways associated with adipose: Glycosaminoglycan biosynthesis-heparan sulfate / heparin, Fat digestion and absorption, Glycerolipid metabolism,Glycerophospholipid metabolism, Cholesterol metabolism, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway and Wnt signaling pathway (Table 4).
Differential expression of circRNA qRT-PCR validation
In order to verify the accuracy of the sequencing results, 19_30428869_30429917, 23_47332020_47332905 and 8_46700648_467014 were randomly selected for qRT-PCR validation, as shown in the figure and the results of the qRT-PCR assay were in agreement with the results of transcriptome sequencing (Fig 5).
Animal meat provides people with rich protein and fat and occupies an important part of their lives. Yak meat is a natural green food, which is characterized by high protein, rich in fatty acids and low fat and provides local residents with daily life needs as well as economic income
(Li et al., 2023). Due to the high altitude and long dry grass period in Qinghai and other places, after entering the dry grass period, the temperature is low and yaks will consume the fat stored in the previous period to provide the energy they need, so reducing fat deposition and fat conversion is an important challenge for yak industry. Animal fat deposition is regulated by a variety of factors, including age, breed and environment,
etc. At the molecular level, most studies have focused on genes, mRNAs and miRNAs. In recent years, researchers have found that circRNAs exert some influence on lipid metabolism
(Yu et al., 2021).
As transcriptome sequencing continues to develop, there are more and more studies on the widespread presence of circRNAs in various organisms, including humans
(Tan et al., 2017), mice
(Gruner et al., 2016), cattle
(Wei et al., 2017), sheep
(Zhai et al., 2022) and pigs
(Li et al., 2018). There are few studies on circRNAs in yak.
(Zhang et al., 2020) performed RNA-Seq sequencing on yak adipocytes at 0 d and 12 d of differentiation and identified a total of 136 differential circRNAs.
Huang et al., (2021) screened 1057 differential circRNAs in bovine-yak and yak dorsal longest muscle using RNA-Seq sequencing technology. The above studies suggest that circRNAs may play important roles in yak cells and tissues. The study of differential circRNAs in subcutaneous fat tissue of plateau-type yaks has not been reported yet. In this study, six samples of yak subcutaneous fat tissue under different feeding conditions were collected for RNA-Seq sequencing and a total of 628 differentially expressed circRNAs were screened, which could help to reveal the changes of circRNAs expression in yak subcutaneous fat tissue under different feeding conditions at the molecular level and further proved that a large number of circRNAs widely existed in the tissues of living organisms and might play a crucial role. In order to investigate the role of circRNAs in subcutaneous fat tissue of yaks under different feeding conditions, the present study was conducted to analyze the functionality of genes originating from differential circRNAs. GO functional annotation analysis showed that the genes originating from differential circRNAs were annotated in cellular components, biological processes and molecular functions, suggesting that they were involved in these processes. KEGG pathway analysis showed that differential circRNA-derived genes were enriched to a total of 9 pathways related to adiposity. Among them,
CD36 was enriched into 4 pathways,
DGKZ was enriched into 2 pathways and
PNPLA3 and
COL1A2 were enriched into 1 pathway. Cluster of differentiation 36 (
CD36) is a class of single-chain transmembrane proteins that act as receptors for the transport of extracellular fatty acids into the intracellular compartment, provide docking sites for fatty acid enzymes such as fatty acid-binding protein 4 (
FABP4) and promote fatty acid transport in adipocytes, skeletal muscle cells and smooth muscle cells. cells and smooth muscle cells (
Abumrad and Goldberg 2016;
Kim and Dyck 2016).
PNPLA3 is patatin like phospholipase domain containing 3 also known as lipocalin.
PNPLA3 is expressed in a variety of human tissues, with the highest expression in liver tissues
(Huang et al., 2010; Pirazzi et al., 2014). And the highest expression was found in white and brown adipose tissues in mice
(Lake et al., 2005). It was found that overexpression of the human mutant
PNOLA3 in the livers of mice fed with high sucrose caused hepatic steatosis in mice
(Li et al., 2012; BasuRay et al., 2019). The heterotrimer composed of two COL1A1 subunits and one
COL1A2 subunit is type I collagen, which belongs to the major structural proteins that make up skin, bone,
etc (Viguet-Carrin et al., 2006). It was found that
COL1A1 and
COL1A2 were differentially expressed in very high intramuscular adiposity and low intramuscular adiposity in pigs
(Pan et al., 2023) and in morphological changes in the structural organization of the bovine fetal ovary
(Franchi et al., 2020), then
COL1A2 may be related to adiposity regulation and the regulatory mechanism of
COL1A2 can be investigated subsequently. NFATC1-5 constitutes a family of nuclear factor of activated T cells (NFAT) transcription factors and it has been found that NFATC1-5 regulates T cell activation and differentiation and also participates in regulating the expression of genes such as cell cycle and death
(Shaw et al., 1988). NFATC1-5 constitutes a family of nuclear factor of activated T cells (NFAT) transcription factors and it has been found that NFATC1-5 regulates T cell activation and differentiation and is also involved in regulating the expression of genes such as cell cycle and death
(Shaw et al., 1988; Park et al., 2020; Li et al., 2023). More and more studies have shown that circRNAs can act as ceRNAs and indirectly regulate the expression of mRNAs by competitively binding to miRNAs
(Liu et al., 2018) to perform biological functions. GO function and KEGG pathway enrichment analyses of their source genes were performed to screen circRNAs related to fat metabolism and the targeting relationship between circRNA, but the verification has yet to be tested.
