lncRNA identification in porcine LD muscle
In order to study the function of lncRNAs in muscle development during porcine embryonic development, we examined the DELs of LD muscles between HN and LW pigs at the three embryonic-development stages. In total, 1.94 billion clean reads were obtained from 18 samples, of which 79.63% could be aligned to the reference porcine genome (Sscrofa10.2), 12.87% were multiply mapped, 66.76% were uniquely mapped, 33.34% mapped to “+”, 33.47% mapped to “–”, 19.24% were splice reads, and 47.52% was nonsplice reads (Additional Table.2). Expression correlation between the three samples in the same treatment was from 0.953 to 0.969 (Additional Fig. 1), indicating that sample selection was reasonable, and experiment results were reliable. Raw sequence data were submitted to the NCBI Sequence Read Archive under succession number SRP243554.
Lncrna Properties In Porcine Ld Muscle
In total, 2057 annotated lncRNAs and 2131 novel lncRNAs were obtained from 18 samples (Fig. 1a), and only lincRNA(89.9%)and intronic lncRNA were found in the novel lncRNAs (Fig. 1b). The average length of the annotated lncRNAs was shorter than that of the novel lncRNAs, but there was no significant difference in exon number and open reading frame (ORF) length (Fig. 1c). For the novel lncRNAs, the average length of antisense lncRNAs was longer than that of lincRNAs (Fig. 1d). Information on the novel lncRNAs is shown in Additional Table 3.
Expression Difference Of Lncrnas Between Hn And Lw Pigs
The fragments per kilobase per million (FPKM) of lncRNAs were lower than those of mRNAs (Fig. 2a), and the FPKM distribution of LW pigs at 78 dpc was higher than that of the others (Fig. 2b). Systematic cluster analysis was performed to compare the relationship between 18 LD muscle libraries, and results indicated that three replicates of the same sample were very conservative (Fig. 2c). The expression differences between the different stages were larger than those of the two breeds. There were 1155 and 751 DELs during muscle development in HN and LW pigs, respectively. In HN pigs, 579 and 809 DELs were identified at 58 vs 38 dpc and 78 vs 58 dpc, and 213 DELs were shared in these two comparisons. In LW pigs, there were 579 and 809 DELs at 58 vs 38 dpc and 78 vs 58 dpc, respectively, and 167 DELs were shared. At 78 vs 58 dpc, there were more DELs in HN than in LW pigs. There were 291, 305, and 683 DELs between these two breeds at 38, 58, and 78 dpc, and 37 DELs were shared by these three stages (Fig. 2d and Additional Fig. 2 and Additional Table 4).
During embryonic muscle development, the GO enrichment of DEL-co-expressed genes in HN showed that skeletal muscle tissue/organ development, muscle organ development, striated muscle tissue development, as well as the response to oxygen-containing compound were upregulated in 58 dpc. In LW pigs, only the response to the oxygen-containing compound upregulated at 58 dpc, and muscle cell differentiation, myofibril assembly only upregulated at 78 dpc; other muscle development-related pathways were continuously upregulated in both breeds. At 58 vs 38 dpc, muscle cell differentiation and myofibril assembly were only upregulated in HN at 58 dpc. Skeletal muscle tissue/organ development, muscle organ development, striated muscle tissue development was only upregulated in LW at 78 dpc (Table 1).
KEGG enrichment of DEL-co-expressed genes shown that the cyclic adenosine monophosphate (cAMP), glyoxylate and dicarboxylated metabolism, metabolic pathways were only upregulated in HN pigs at 58 dpc. Fatty acid degradation and fructose and mannose metabolism was only upregulated in LW pigs at 78 dpc. The adipocytokine pathway was upregulated in HN but downregulated in LW pigs at 58 dpc. The MAPK pathway was upregulated only at 58 dpc in both breeds, and fructose and mannose metabolism was upregulated only at 78 dpc in both breeds. DEL-co-expressed genes related to fatty acid degradation, cAMP, glyoxylate and dicarboxylated metabolism, fatty acid metabolism, metabolic pathways continuously changed only in HN or LW pigs. Genes associated with alanine, aspartate and glutamate metabolism, and Rap1 were higher expressed only in HN pigs at 78 dpc, while genes in protein digestion and absorption were expressed at a higher level in LW pigs at 78 dpc (Table 2 and Additional Fig. 3).
Table 2
The muscle-related GO terms of DELs co-expressed genes during muscle development stages in Huainan and Large white pigs
GO terms | H58/H38 | LW58/LW38 | H78/H58 | LW78/LW58 | H58/LW58 | H78/LW78 |
muscle cell differentiation | ↑ 9 (0.260) | | ↑ 9 (0.241) | ↑10 (0.062) | ↑ 6 (0.438) | ↓ 6 (0.868) |
contractile fiber | ↑ 8 (0.065) | ↑ 7 (0.180) | ↑ 8 (0.084) | ↑ 8 (0.011) | ↑ 5 (0.361) | |
myofibril | ↑ 7 (0.089) | ↑ 6 (0.268) | ↑ 7 (0.106) | ↑ 7 (0.027) | ↑ 4 (0.415) | |
regulation of glucose metabolic process | ↑ 5 (0.124) | ↑ 5 (0.227) | ↑ 5 (0.163) | ↑ 5 (0.046) | ↑ 3 (0.480) | |
muscle structure development | ↑12 (0.157) | ↑11 (0.278) | ↑12 (0.174) | ↑13 (0.029) | ↑ 8 (0.385) | |
muscle system process | ↑ 9 (0.160) | ↑ 8 (0.278) | ↑ 6 (0.247) | ↑ 9 (0.061) | ↑ 6 (0.385) | |
muscle tissue development | ↑11 (0.148) | ↑10 (0.278) | ↑10 (0.212) | ↑13 (0.011) | | ↓ 7 (0.757) |
skeletal muscle tissue development | ↑ 8 (0.160) | ↑ 8 (0.240) | | ↑10 (0.011) | ↑ 5 (0.415) | ↓ 5 (0.778) |
skeletal muscle organ development | ↑ 8 (0.161) | ↑ 8 (0.260) | | ↑10 (0.011) | ↑ 5 (0.438) | ↓ 5 (0.847) |
muscle organ development | ↑10 (0.180) | ↑ 9 (0.312) | | ↑12 (0.013) | ↑ 6 (0.480) | ↓ 7 (0.608) |
myofibril assembly | ↑ 4 (0.124) | | ↑ 4 (0.163) | ↑ 4 (0.046) | | ↓ 3 (0.544) |
striated muscle tissue development | ↑10 (0.180) | ↑ 9 (0.334) | | ↑12 (0.015) | | ↓ 7 (0.608) |
response to oxygen-containing compound | ↑19 (0.124) | ↑19 (0.179) | | | | |
cell proliferation | | | | ↑20 (0.195) | | ↓16 (0.544) |
The results of GO enrichment of DELs between HN and LW pigs at the same stage showed that no pathway was enriched between these two breeds at 38 dpc. At 58 dpc, genes in muscle cell differentiation and eight other pathways had a higher expression level in HN pigs. At 78 dpc, the genes in seven pathways were downregulated in HN pigs. Compared with LW pigs, the genes associated with muscle cell differentiation, skeletal muscle tissue/organ development, muscle organ development pathways showed, higher expressional level at 58 dpc, but a lower expression level in HN pigs (Table 1 and Additional Table 4).
KEGG analysis indicated that DEL-co-expressed genes involved in insulin, calcium, and the cAMP signaling pathway were continuously highly expressed in HN pigs. Genes in steroid biosynthesis were expressed at a higher level in HN pigs at 58 and 78 dpc. Compared with LW pigs, genes in hypoxia-inducible factor 1 (HIF-1) and adipocytokine were upregulated in LW pigs at 38 and 78 dpc, but downregulated at 58 dpc. Genes involved in insulin secretion and Rap1 were downregulated at 58 dpc and upregulated at 78 dpc in HN pigs. The expression trends of genes in the biosynthesis of amino acids, fatty acid metabolism, and fructose and mannose metabolism were completely opposite (Table 2 and Additional Fig. 3).
Potential Function Of Dels
The expressional level of 37 shared DELs between these two breeds at three stages are shown in Fig. 3a, b. The ceRNA regulatory network of these shared DELs was constructed and visualized using Cytoscape software, including 30 lncRNAs, 27 miRNAs, 27 mRNAs, and 24 pathways (Fig. 3c). LncRNAs had up to 7 interacting miRNAs, such as ALDBSSCT0000006192, and miR-199a-5p had the most target lncRNAs, seventeen target lncRNAs for each miRNA. Considering the abundance and transcript length, IMFlnc1 was selected for subsequent verification among miR-199a-5p target lncRNAs. IMFlnc1 is located on porcine chromosome 7, and includes two exons.
Imflnc1 Inhibits Adipogenesis Of Intramuscular Adipocytes
RT-qPCR showed that IMFlnc1 is expressed at the highest level in the gut and lungs, followed by in intermuscular fat. Its expression level in subcutaneous fat was lower than it was in intermuscular fat, but higher than it was in LD muscle (Fig. 4a). Moreover, time-course analysis showed that the expression level of IMFlnc1 was upregulated during porcine intramuscular preadipocyte differentiation (Fig. 4b).
CPC software showed that IMFlnc1 has very low coding potential, similar to ADNCR [25], a well-known lncRNA (Fig. 4c). To explore the function of IMFlnc1 in adipogenesis, we performed knockdown IMFlnc1 in intramuscular adipocytes by lncRNA smart silencer, which significantly reduced its RNA level by 60%, and the expression of CAV-1 and adipogenic markers PPARgama decreased significantly (Fig. 4d). Oil O staining indicated that the knockdown of IMFlnc1 inhibited adipogenesis (Fig. 4e).
Imflnc1 And Cav-1 Were Target Genes Of Mir-199a-5p
RNA FISH indicated that IMFlnc1 is predominantly localized in the cytoplasm of preadipocytes (Fig. 5a), so it might participate in the regulation of adipogenesis through ceRNA mechanism. The IMFlnc1-miR-199a-5p-CAV-1 pathway was selected from the ceRNA network to verify its function in adipogenesis. Bioinformatics analysis of the RNAhybrid showed that there exists a putative miR-199a-5p binding site in IMFlnc1 and CAV-1 (Fig. 5b), and the binding site in CAV-1 is conservative in different animals (Fig. 5c). RT-qPCR results showed that the expression of IMFlnc1 and CAV-1 has a positive correlation in porcine LD muscle (Fig. 5d, R2 = 0.590).
Luciferase assay indicated that miR-199a-5p significantly reduced the Rluc activity of the miR-199a-5p sensor and psiCHECK2-IMFlnc1 (p < 0.01, Fig. 5e), but the reduction of the miR-199a-5p sensor was higher than the reduction of psiCHECK2-IMFlnc1. However, miR-199a-5p had no effect on the fluorescence activity of psiCHECK2-IMFlnc1-Mut, which indicated that IMFlnc1 was the target gene of miR-199a-5p. Similarly, luciferase assay proved that CAV-1 was the target gene of miR-199a-5p (Fig. 5f).
IMFlnc1 participates in adipogenesis by increasingCAV-1 in miR-199a-5p-dependent manner
To verify whether IMFlnc1 might act as the sponge of miR-199a-5p, the miR-199a-5p sensor was transfected with pcDNA3.1+, pcDNA-miR-199a-5p or pcDNA-miR-199a-5p and pcDNA-IMFlnc1. The Rluc activity of the miR-199a-5p sensor was significantly reduced by pcDNA-miR-199a-5p (P < 0.01), but IMFlnc1 recovered the Rluc activity in a dose-dependent manner (P < 0.01, Fig. 5g). However, pcDNA-IMFlnc1-Mut, in which the binding site of miR-199a-5p was mutated, no longer elicited such an effect. These results indicated that IMFlnc1 act as a ceRNA for miR-199a-5p.
As shown in Fig. 4d, h, the mRNA expression of CAV-1 was reduced during the reduction of IMFlnc1. To further determine whether IMFlnc1 regulated CAV-1 through miR-199a-5p, psiCH2-CAV-1 was co-transfected with pcDNA3.1, pcDNA-IMFlnc1 or pcDNA-IMFlnc1Mut, respectively. The Rluc activity of CAV-1 was improved by the overexpression of IMFlnc1, but the overexpression of IMFlnc1 with the mutated miR-199a-5p binding sites no longer elicited similar effect (Fig. 5h). In summary, these results indicated that IMFlnc1 might promote adipogenesis by acting as a ceRNA for miR-199a-5p to regulate CAV-1 expression.