LncRNA IMFlnc1 promotes porcine intramuscular adipocyte differentiation by sponging miR-199a-5p to up-regulate CAV-1

Background Local Chinese local pig breeds have better meat quality, such as thinner muscle ber and higher intramuscular-fat (IMF) content. However, its molecular regulation mechanism has not been discussed in-depth. Studies indicated that long non coding RNAs (lncRNAs) participate in the regulation of muscle and fat development. The expressional differences of lncRNAs in the longissimus dorsi (LD) muscle were identied between Huainan pigs (local Chinese pigs, fat-type, HN) and Large White pigs (lean-type, LW) at 38, 58, and 78 days post conception (dpc). Results In total, 2131 novel lncRNAs were identied in 18 samples, and 291, 305, and 683 differentially expressed lncRNAs (DELs) were found between these two breeds at three stages, respectively. GO and KEGG analysis of the DEL co expressed mRNAs showed that muscle development and energy metabolism were more active at 58 dpc in HN, but at 78 dpc in LW pigs. Muscle cell differentiation and myobril assembly might contribute to earlier myogenesis and primary-muscle-ber assembly in HN, and cell proliferation, insulin, and the mitogen-activated protein kinase (MAPK) pathway might contribute to longer proliferation in LW pigs. The PI3K/Akt and cAMP pathways were associated with higher IMF deposition in HN. IMFlnc1 was selected for functional verication, and results indicated that it regulated the expressional level of caveolin-1 (CAV-1) as a competing endogenous RNA (ceRNA) for miR-199a-5p. Conclusion Our data contributed to understanding lncRNAs in porcine-muscle development and IMF deposition, and provided valuable information for improving pig-meat quality.

. Results were helpful to analyze the lncRNAs' regulation mechanism on differences of meat quality between fat-and lean-type pigs during the embryonic period, and provide basic materials for improving meat quality traits for breeding.

Animals and tissue collection
Five HN and ve LW sows (in their second or third parity) with the same genetic background were arti cially inseminated with semen from the boars of the same breed. One sow from each breed was slaughtered at 38, 58, or 78 days of pregnancy. Fetuses were immediately taken out of the uteri, and three males and three females were selected. For 38 dpc, all of the fetuses were selected, and their sex were subsequently identi ed by the SRY gene. LD muscle tissue from the same area was obtained from the embryos and snap-frozen in liquid nitrogen. All pigs were raised under the same feeding and management practices [17] at Henan Xing Rui Agricultural and Animal Husbandry Technology Co., Ltd., Henan province, China.

Rna Isolation, Library Preparation, And Sequencing
Total RNA from LD muscle tissue was extracted by using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocols. The NanoDrop spectrophotometer (Nano-Drop Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA) were used to detect the integrity, purity, and quality of the isolated RNA. Samples with an RNA integrity number (RIN) value larger than eight were used for library construction. DNase (QIAGEN, USA) and Ribo-Zero™ rRNA Removal Kit (Epicentre, USA) were used to remove residual genomic DNA and ribosomal RNA, respectively. Illumina TruSeq™ RNA Sample Prep Kit was used to construct the RNA sequencing library. Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to analyze the libraries' quality. The libraries were sequenced on an Illumina Hiseq 2500 platform, and 125 bp paired-end reads were generated. TopHat2 software was used to map the clean data to the porcine reference genome (Sscrofa 10.2, ftp://ftp.ensembl.org/pub/release-84/fasta/sus_scrofa/dna/) [18].

Identi cation Of Lncrnas And Differential Expression Analysis
The identi cation of lncRNAs followed these ve steps: (1) transcripts that contained a single exon were removed; (2) transcripts shorter than 200 bp were removed; (3) annotated lncRNAs in the database that overlapped with the transcripts in this research were left as annotation lncRNAs for subsequent analysis (Cuffcompare software); (4) transcripts with FPKM ≤ 0.5 were removed;(5) the coding potential of the transcripts was analyzed by Coding Potential Calculator (CPC, score < 0), Coding-Non-Coding-Index (CNCI, Page 5/33 score < 0), Pfam (E value < 0.001), and k-mer scheme (PLEK, score < 0) software. Transcripts that passed all these software tests were considered lncRNAs.

Validation Of Gene Expression In Rna-seq
To verify the accuracy of high-throughput sequencing results, ten lncRNAs were selected from the 37 shared DELs, and their expressional trends at 78 dpc between HN and LW pigs were validated by RT-qPCR. The RNA used in RT-qPCR was the same as that used in Illumina sequencing. PrimeScript RT reagent Kit with the gDNA Eraser (TaKaRa, China) was used to convert total RNA to cDNA [21]. Then, qPCR was performed using the SYBR Green PCR kit (TaKaRa, China) according the manufacturer's instructions. Each qPCR reaction was performed in a 20 µL reaction mixture, including 20 ng template cDNA, 10 µL 2 × SYBR Premix Ex Taq™, and 10 µM forward and reverses primers (Additional Table 1). PCR ampli cation included an initial denaturation step (95 o C for 30 s), and 40 cycles of 5 s at 95 o C and 34 s at 60 o C. Each qPCR experiment was performed in triplicate, and the relative RNA expression values were calculated using the 2 −△△Ct method [22], data are presented as fold changes in expression.

Construction Of Lncrna-mirna-mrna-pathway Regulatory Network
The interaction between the 37 shared DELs and miRNAs was analyzed by the miRanda software. MiRNAs came from the LD muscle of HN and LW pigs at 38, 58, and 78 dpc, and these data were not published.
The construction of the network included three steps, and the methods were the same as those previously described [16].

Isolation, Differentiation, And Culture Of Porcine Intramuscular Adipocyte
Porcine intramuscular adipocyte were isolated from the LD muscle of HN pigs at 2 days of age following methods as previously described [23,24]. Intramuscular adipocytes were cultured with the normal medium: DMEM/F12 supplemented with FBS (10%) and antibiotics (penicillin, 100 IU/mL; streptomycin, 100 µg/mL) at 37 °C and 5% CO 2 in a normal-atmosphere incubator. Two days after 100% con uence, cells were maintained with a differentiation medium (0.5 mM IBMX, 1 mM DEX, 5 µg/mL of insulin add to the "normal medium"). After two days, the medium was changed to a maintenance medium (5 µg/mL of insulin add to the "normal medium") for two days. Then the medium was changed to the "normal medium".

Plasmid Construction
The full lengths of IMFlnc1 and 3' UTR of CAV-1 were cloned into the psiCHECK-2 vector (Promega, USA). The mutant IMFlnc1 and 3' UTR of CAV-1 without the seed region of miR-199a-5p were generated by overlapping extension PCR. An miR-199a-5p sensor was generated by inserting two consecutive miR-199a-5p complementary sequences into psiCHECK-2.

Luciferase Assays
HEK293T cells were seeded in 48-well plates in triplicate, and plasmids were transfected when the cell reached 70% or 80% con uence. Forty-eight hours after transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) on a Fluoroskan Ascent FL instrument (Thermo Fisher Scienti c, USA). For each sample, renilla luciferase activity was normalized to re y luciferase activity.

Statistical analysis
Results were expressed as the mean ± SE, and a p value of < 0.05 was considered statistically signi cant.

lncRNA identi cation 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 signi cant 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 identi ed 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  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, myo bril 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 myo bril 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).
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).
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 veri cation among miR-199a-5p target lncRNAs. IMFlnc1 is located on porcine chromosome 7, and includes two exons.

Im nc1 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 signi cantly reduced its RNA level by 60%, and the expression of CAV-1 and adipogenic markers PPARgama decreased signi cantly (Fig. 4d). Oil O staining indicated that the knockdown of IMFlnc1 inhibited adipogenesis (Fig. 4e).
Im nc1 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, R 2 = 0.590).
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 cotransfected 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.

Discussion
Previous studies showed that muscle development during the embryonic stages is different between leanand fat-type pigs. The morphological differences of LD muscle between LT and LR pigs were compared by histological section, and results showed that a few primary bers were present at 35 dpc in LT pigs, but 49 dpc in LR pigs. At 63 dpc, the secondary bers were formed in both breeds, but LR pigs had more bers and larger muscle ber diameter at 91 dpc [12]. Zhao et al., compared the morphological differences of muscle between TC and YK pigs, the number and density of myoblasts in TC was more than that in YK pigs at 30 dpc, primary bers could be found at 40 dpc in both breeds, and the secondary bers appeared at 55 dpc in YK pigs but later in TC pigs [8]. These studies indicated that myoblast differentiation, the formation of primary bers and secondary bers, and muscle ber diameter was different between fatand lean-type pigs. To study the regulation of lncRNA in meat quality between the fat-and lean-type pigs, differentially expressed lncRNAs in LD muscle between HN and LW pigs at 38, 58, and 78 dpc were identi ed. Results indicated that there were more DELs in 58 vs 38 dpc than in 78 vs 58 dpc in HN pigs.
However, there was the completely opposite trend in LW pigs. These coincided with the difference in muscle bers development between fat-and lean-type pigs.
GO analysis of DEL-co-expressed genes was performed to further understand their biological functions (Table 1), and results showed that DELs related to muscle structure/tissue/system development were continuously upregulated in both breeds, with higher expression at 58 dpc, but lower expression at 78 dpc in HN pigs compared to that in LW pigs. Muscle cell differentiation was continuously upregulated in HN pigs, but only upregulated at 78 dpc in LW pigs, and with a higher expressional level at 58 dpc, but lower at 78 dpc in HN than in LW pigs. Similarly, contractile ber and myo bril were continuously upregulated in both breeds, but at 58 dpc their expression was higher in HN than in LW pigs. Genes in the myo bril assembly pathway were continuously upregulated in HN pigs, but upregulated only at 78 dpc in LW pigs, and its expressional level at 78 dpc were higher in LW pigs. These results indicated that compared with LW pigs, HN pigs showed earlier myogenic differentiation. Muscle development-related biological activities were most active at 58 dpc in HN pigs, but most active at 78 in LW pigs.
To identify pathways in muscle development, DEL-co-expressed genes were mapped to the reference canonical pathways in the KEGG database (Table 2). Hypoxia inducible factor-1α (HIF-1α), a key pathway regulating myogenic differentiation, showed higher expression at 58 dpc, but lower expression at 78 dpc in HN than in LW pigs. The pathways associated with energy metabolism showed the same trends, such as biosynthesis of amino acids, adipocytokine pathway, fatty acid metabolism, and fructose and mannose metabolism. These results veri ed that myogenic differentiation in HN pigs was most active at 58 dpc, so more energy is needed at this period. In contrast, LW pigs showed the most active myogenic differentiation at 78 dpc, so energy-related genes had higher expression at 78 dpc.
Previous researchers found that the myoblast proliferation phases of LD in TC pigs were longer than that those in WZS pigs [4], and it was predicted that cell proliferation may contribute to differences in muscle mass and body size between these two breeds [6]. In the current study, GO analysis showed that cell proliferation was at a higher level in LW than in HN pigs at 78 dpc. The insulin [26] and the MAPK [27] pathways both promote muscle cell proliferation. The insulin pathway showed a higher expressional level in LW than that in HN pigs in all three stages. MAPK showed a higher level in LW than in HN pigs at 58 and 78 dpc. These results veri ed that myoblast proliferation was higher in LW than in HN pigs, which coincided with more muscle mass in LW pigs.
It was reported that phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/Akt) participates in the adipogenic differentiation of mesenchymal stem cells [28]. The proliferation and differentiation of porcine intramuscular preadipocytes are activated by G protein-coupled receptor (GPR) 39 through the PI3K/Akt cell signaling pathway [29]. In this research, PI3K/Akt was expressed at a higher level in HN pigs at 78 dpc. The cAMP pathway, which could inhibit adipocyte differentiation and promote lipolysis [30], was continuously downregulated at the prenatal period in both breeds, but its expressional level was lower in HN than in LW pigs in all three stages. These results coincided with the higher IMF content in HN pigs.
The GO and KEGG results of DELs were consistent with previous studies on the differences in embryonic muscle development between fat-and lean-type pigs. To explore how these DELs participated in the regulation of muscle development, interactions between the 37 shared DELs and miRNAs were analyzed by miRanda software. As shown in Fig. 4c, miRNAs enriched in the ceRNA network were associated with meat quality. The expression of miR-125, miR-133, and miR-199 in LD muscle was signi cantly different between German Landrace and Pietrain (Pi) pig breeds at the pre-and post-natal period [31]. Similarly, miR-133 was signi cantly highly abundant in MS (fat-type) than that in LW (lean-type) pigs at all prenatal stages (35,55, and 90 dpc) [32]. The expression of miR-199, miR-423, miR-296, miR-193 and miR-125 was signi cantly different between castrated male pigs (with more fat deposition) and intact male pigs (with less fat deposition) [33]. MiR-423-5p was increased in the plasma of obese children [34]. The expression of miR-342, miR-193a-3p, miR-365 and miR-125 was signi cantly different between obese and lean individuals [35].
There were also muscle-development-related miRNAs in the ceRNA network, such as miR-133, which was signi cantly more highly expressed in MS pigs skeletal muscle compared with that in LW pigs at all prenatal stages [32]. SNPs in miR-133 were signi cantly associated with total muscle ber number, loin eye area, and muscle pH [36]. Meanwhile, it was reported that miR-133 could promote myoblast differentiation and inhibit proliferation through the extracellular signal-regulated kinase (ERK) signaling pathway [37]. It also participates in the regulation of the metabolic difference between glycolytic and oxidative myo bers [38]. MiR-365 could inhibit myoblast proliferation by targeting insulin-like growth factor I (IGF-I) or cyclinD1 [39][40][41]. MiR-125 regulates muscle differentiation by target myocyte-speci c enhancer factor 2D (Mef2d) [42,43].
In the ceRNA network there are the miRNAs associated not only with muscle development, but also with adipocyte development, such as miR-199a-5p, which has the most targets in the current ceRNA network.
As the key regulator in the Wnt signaling pathway, miR-199a-5p induced muscle proliferation by targeting HIF-1, which is the key regulator in smooth muscle hypertrophy [49][50][51]. It also regulates cardiomyocyte apoptosis by targeting JunB (JunB proto-oncogene) [52]. In diabetic individuals, miR-199a-5p may be involved in skeletal muscle insulin resistance by inhibiting glucose transporter 4 (GLUT4) and hexokinase 2 (HK2) [53]. MiR-199a-5p also participates in lipid metabolism. In Italian Large White pigs, miR-199a-5p showed a lower expressional level in the backfat of the lean groups than that in the fat group [54]. Its expressional level was also higher in undifferentiated 3T3-L1 adipocytes, and rapidly reduced during differentiation [55]. Overexpression of miR-199a-5p in human bone-marrow-derived mesenchymal stem cells, the marker gene of adipocytes-FABP4 (ap2) was inhibited [56]. In mice, groin fat pads weight was reduced when the Dnm3os (DNM3 opposite strand/antisense RNA) was knocked down, which served as a precursor of miR-199a-5p and miR-199a-3p [57]. In porcine preadipocytes, miR-199a-5p signi cantly promotes proliferation, and reduced adipogenesis by targeting CAV-1 [58]. MiR-199a-5p could be regulated by PPARgama in a transcription-independent manner, and it regulates adipogenic differentiation by targeting the expression of transforming growth factor beta induced (TGFBI) [59]. In general, miR-199a-5p was not only associated with the proliferation and differentiation of muscle cells, but also with the proliferation and adipogenesis of pre-adipocytes. Similarly, miR-370 was not only related to lipid metabolic homeostasis and lipid deposition, [44] but it also regulates muscle cell apoptosis [60]. MiR-149 promoted porcine satellite cell proliferation through the Notch signaling pathway [61], while miR-149-3p induced a subcutaneous-to-visceral fat switch by suppressing PR domain-containing 16 (PRDM16) [62].
There are several coding genes associated with meat quality, such as adipocyte determination and differentiation dependent factor 1 (ADD1), which stimulates PPARgama expression and plays an important role in adipogenic differentiation [63], whose expressional level is positively correlated with IMF content (P < 0.05) [64], The stearoyl-CoA desaturase (SCD) gene plays a key role in the desaturation of fatty acids and synthesis of oleic fatty acid, and SCD overexpression in muscle is correlated with a higher IMF content [65]. SCD showed a signi cantly different expressional level in LD muscle between LT and LR pigs [66]. IGF-I regulates myo ber hypertrophy postnatally through the PI3K-Akt-mTOR pathway, which increased skeletal muscle mass in economic livestock, while IGF-I promotes free fatty acid oxidation and improves insulin sensitivity in muscle [67]. As a major fatty acid binding protein in adipocytes, CAV-1 moves from the plasma membrane to lipid droplets under the action of free fatty acids [68,69]. Compared with Large YK pigs, CAV-1 showed a signi cantly higher expressional level in the backfat(P < 0.01)and longissimus dorsi muscle P < 0.05 in LT [70]. CAV-1-de cient mice showed a lean body phenotype, insulin resistance, and hypertriglyceridemia with adipocyte abnormalities [71]. Activated by TNF-α, CAV-1 reduces 3T3-L1 cell differentiation and blocks insulin-mediated glucose uptake [72]. CAV-1 was associated with lipoprotein metabolism [73], lipolysis [73], cholesterol homeostasis [74], fatty acid intake [75], and atherosclerosis [76], so CAV-1 is an important regulator of adipogenic differentiation and glycolipid metabolism.
These muscle development and lipid metabolism related coding genes and miRNAs in the ceRNA network indicated that these DELs may participate in the regulation of meat quality via the ceRNA mechanism. So, the miR-199a-5p-IMFlnc1-CAV-1 pathway was selected to be veri ed in porcine intramuscular adipocytes (Figs. 4 and 5). The results indicated that IMFlnc1 had broad-spectrum expression in different tissue types, and its expressional level was upregulated during the differentiation of intramuscular preadipocytes. Oil red O staining suggested that the reduction of IMFlnc1 inhibits adipogenesis. Luciferase assay revealed that IMFlnc1 could function as a ceRNA that sequesters miR-199a-5p, thereby protecting CAV-1 transcripts from miR-199a-5p-mediated suppression. At the same time, miR-199a-5p also participated in the regulation of myoblast differentiation. The role of IMFlnc1 in muscle cell proliferation and differentiation could be veri ed in further research.

Conclusion
In summary, a genome-wide view of the expression pro ling of lncRNAs in pocine LD muscle at 38, 58, and 78 dpc between HN and LW pigs was investigated. These ndings were of great signi cance for understanding the molecular regulation mechanisms of meat quality trait differences between fat-and lean-type pigs.