The distribution and deposition of adipose tissue in different parts of the body are the key factors affecting carcass quality and meat flavor. Subcutaneous fat mainly affects carcass quality. Intramuscular fat (IMF) is the material basis of marbling, and an important factor affecting meat flavor. A large number of studies have shown that IMF is directly involved in the formation of meat tenderness, juiciness and flavor [29, 30]. Goat is an indispensable animal in China's agricultural production, and the molecular regulation mechanism of its lipid deposition has not been fully elucidated yet.
LncRNA is a kind of noncoding RNA longer than 200 nt, which has attracted substantial attention in the last few years. Studies have shown that lncRNAs regulate metabolic tissue development and function, including adipogenesis, hepatic lipid metabolism, islet function, and energy balance [31–35]. Despite the fact that many studies have indicated the importance of lncRNAs in different tissues, little is known about their biological function in goat fat deposition, especially in the differentiation of goat intramuscular and subcutaneous preadipocytes. To the best of our knowledge, our study is the first to screen for lncRNAs and mRNAs regulating goat preadipocyte differentiation by sequencing and annotating the transcriptome of intramuscular and subcutaneous preadipocytes. A total of 1,118,110,544 reads were successfully mapped to the goat reference genome assembly. We identified 12,519 lncRNAs. The average sequence length of lncRNAs was shorter than that of mRNAs, and the number of exons was less than that of mRNAs, with the ORF length being shorter than that of mRNAs. Our results indicated that the predicted lncRNAs were shorter with fewer exons than mRNAs, which are in agreement with the those reproted in previous studies [36–38]. The Pearson correlation (R2) of each sample is greater than 0.8, which indicated that our experiment was reliable and the sample selection was reasonable.
LncRNA functions by regulating mRNA. At present, the mechanism of interaction between lncRNA and mRNA is not clear. We predict the biological function of lncRNAs through its co-expression with protein coding genes. Consequently, we found that many target genes of DELs were also differentially expressed in goat intramuscular and subcutaneous preadipocytes. This suggested that lncRNAs may function through complementary target genes, which can play critical roles in the differentiation of goat intramuscular and subcutaneous preadipocytes. For example, SMAD1 is a target gene of the differentially expressed lncRNAs LNC_009792, LNC_007731, LNC_000706, LNC_008467, LNC_006192 and LNC_004878, and it has been reported to regulate the differentiation of preadipocytes [39]. These findings suggest that these lncRNAs might be involved in the differentiation of intramuscular preadipocytes by affecting the expression of SMAD1. However, there is no DEGs in the differentiation process of subcutaneous preadipocytes in our selected fat development related genes, so we speculate that the network regulating intramuscular adipogenesis is more complex than subcutaneous fat. The higher number of DELs, DEMs and DEGs in IMF than that of subcutaneous fat also supported this, which is consistent with the fact that intramuscular preadipocytes have stronger ability to deposit fat than that of subcutaneous preadipocytes [40].
To explore the similarities and differences of different adipocytes, DELs target genes (IMPA vs IMA and SPA vs SA ) were subjected to GO and KEGG pathway enrichment analyses. We found that few common term was found between the IMPA vs IMA and SPA vs SA comparisons. Several pathways involved in preadipocyte differentiation were previously identified, including the TGF-β signaling pathway(IMF and subcutaneous fat) [41], PI3K/AKT signaling pathway (subcutaneous fat) [42], and arachidonic acid metabolism(subcutaneous fat) [43]. For example, the LncRNA GAS5 inhibits lipogenesis in 3T3-L1 cells through the miR-21a-5p / PTEN signaling pathway [44]. FDNCR1 affects porcine lipogenesis by competitively binding miR-204 to regulate the TGF-β pathway [45]. However, for some pathways identified here, their involvement in the goat preadipocyte differentiation process is being reported for the first time. Interestingly, in the pathway analysis, we found that the components of two pathways, fatty acid metabolism(IMF and subcutaneous fat) and fatty acid degradation (subcutaneous fat), which have been reported to be involved in lipid metabolism, and were enriched in the entire process of differentiation of intramuscular and subcutaneous preadipocytes [46, 47]. The common enrichment pathways during differentiation of both adipocytes involve amino acid metabolism, gluconeogenesis and carbohydrate metabolism, suggesting that IMF and subcutaneous fat are largely different in differentiation pathways and lipid metabolism pathways, while there are similarities in communication with other metabolic pathways. In addition to the specific pathways of the two adipocytes, there are also differences in the common components of the two pathways. Just as IMF is enriched in 19 target genes of TGF-β signaling pathway, 8 target genes of fatty acid metabolism, and subcutaneous fat is enriched in 8 and 7 targat genes. Here, we hypothesize that there are differences in the pathways regulating intramuscular and subcutaneous adipose differentiation, and that there are differences in the downstream target genes in the same pathways, which indirectly demonstrates that gene expression is tissue-specific in goats.
To date, many genes have been reported to regulate the differentiation of preadipocytes. However, few studies have been conducted on the roles of lncRNAs in intramuscular and subcutaneous preadipocytes differentiation. The molecular and cellular mechanisms regulating goat preadipocytes differentiation are thus still poorly understood. Here, we constructed a miRNA-lncRNA-mRNA interaction network, and calculated the degree (the number of times each factor interacts with other factors) of each factor through centerscape. We selected the top 20 miRNAs in the dgree, and found that miR-20 [48], miR-194 [49], miR-335 [50], miR-363 [51], miR-200 [52], miR-199 [53], and miR-302 [54] were related to fat development, and visualized them in Cytoscape. We identified a number of highly connected lncRNAs and mRNAs in the three modules, including the two kinds of adipocytes are unique and common. For example, XM_005693834.3(ACLY) [55] and XM_013975359.2 (ANGPT2) [56] are unique to IMF, and XM_018056618.1(MEDAG) [57] and XM_005678357.3(PLPP3) [58] are unique to subcutaneous fat, whereas, XM_018040030.1 (CAPN10) [59] and XM_018050348.1 (TGFβ1) [60] are shared by the two kinds of preadipocytes in differentiation processes. Seven DELs from the IMF vs subcutaneous fat comparison were validated by qRT-PCR technique and the results were in excellent generally agreement with the RNA-seq findings. This suggests that our RNA-seq findings are reliable.
In conclusion, we first generated the expression profiles of lncRNAs from intramuscular and subcutaneous adipocytes of Jianzhou Daer goat (IMA vs IMPA and SA vs SPA) based on RNA-Seq technique. We found that the number of lncRNAs regulating IMF differentiation was more than that of subcutaneous adipocytes. Our results suggest that those lncRNAs might play important roles in meat quality. Collectively, this study takes the first step toward understanding the molecular mechanisms underlying variations in goat meat quality. Also, our results provided a theoretical basis for molecular breeding to improve the meat quality in goats.