Screening of lncRNA Proles During Intramuscular Adipogenic Differentiation Inlongissimus Dorsi and Semitendinosus Muscles in Pigs

Background: Intramuscular fat content is an important factor that determines meat quality in pigs. In recent years, epigenetic regulation has increasingly studied the physiological model of intramuscular fat. Although long non-coding RNAs play essential roles in various biological processes, their role in intramuscular fat deposition in pigs remains largely unknown. Results: In this study, intramuscular preadipocytes in the longissimus dorsi and semitendinosus of Large White pigs were isolated and induced into adipogenic differentiation in vitro. High-throughput RNA-seq was then carried out to estimate the expression of lncRNAs at 0, 2, and 8 days post-differentiation. At this stage, 2135 lncRNAs, including 575 novel lncRNAs, were identied. lncRNAs are shorter, less expressed, and less conserved RNAs than protein-coding mRNAs. KEGG analysis showed that the differentially expressed lncRNAs were more common in pathways that are closely involved with adipogenesis and lipid metabolism. Of these, lnc_000368, a previously undescribed lncRNA, was found to gradually increase during the adipogenic process. qRT-PCR and a western blot revealed that the knockdown of lnc_000368 by siRNA signicantly repressed the expression of adipogenic genes (PPARγ, aP2, CEBPβ) and lipolytic genes (ATGL and HSL). As a result, lipid accumulation in porcine intramuscular adipocytes was impaired by the silencing of lnc_000368, as indicated by Oil Red O staining. Conclusions: Overall, our study identied a genome-wide lncRNA prole related to porcine intramuscular fat deposition, and the results suggest that lnc_000368 is a potential target gene that might be targeted in pig breeding in the future.


Background
Pork occupies an important part in the human diet as an important dietary protein source [1]. The intramuscular fat (IMF) content has been shown to be positively correlated with pork quality, as high intramuscular fat content can signi cantly improve the avor and tenderness of the pork [2]. However, in the last decades, extensive breeding selection aimed at increasing lean mass has resulted in low IMF and worsening avor, and thus increasing IMF has become an important concern in the pig breeding industry [3].
Long non-coding RNAs (lncRNAs) are a type of non-coding RNA that are over 200 nt in length, and are involved in various biological processes [4,5], such as adipose deposition [6]. It has been reported that the differentiation of adipocytes is affected by the natural antisense transcript of adiponectin, which forms a duplex with sense mRNA to inhibit the translation of adiponectin [7]. Moreover, PU.1 AS lncRNA has also been found to promote adipogenic differentiation in porcine intramuscular adipocytes [8]. Furthermore, lncRNAs were differentially expressed in pig breeds with differing IMF contents [9]. These studies indicate that lncRNAs may be critical modulators in porcine IMF accumulation, thus potentially providing novel breeding selection markers that select for improved pork quality. Our previous study showed that the intramuscular adipocytes in longissimus dorsi muscles possessed greater lipid-forming ability than those in semitendinosus muscles [10]. In the present study, we isolated intramuscular preadipocytes in longissimus dorsi and semitendinosus muscles, performed high-throughput lncRNA sequencing of these cells, and aimed to identify the common lncRNAs that regulate porcine intramuscular adipogenic differentiation.

Cell culture & transfection
Newborn male Large White piglets (3 days old) were procured from the experimental piglet of Northwest A&F University (Yangling, China). All animals were maintained on a 12:12-h light cycle, and feeding conditions and slaughter methods were in accordance with national animal welfare regulations. In this study, all experiments on animals were approved by the Experimental Animal Management Committee of Northwest A & F University and comply with animal welfare regulations.
Intramuscular preadipocytes were isolated from the longissimus dorsi (LD) and semitendinosus (SD) muscles of these piglets using the method previously described by [10]. To summarize, LD and SD muscles were quickly excised, rinsed twice in sterile pre-cooled phosphate-buffered saline (PBS), and then cut into 1 mm 3 . Muscle fragments were incubated in Dulbecco's Modi ed Eagle's Medium/F12 (DMEM/F12; Hyclone, Logan, UT, USA) containing 0.1% I type collagenase (270 U/mg; Gibco, Carlsbad, CA) for 1.5 hours in a 37 ℃ water bath, with continuous shaking. The products were then sequentially passed through a 70 mesh and then a 200 mesh to obtain single cells. The cells were seeded in a dish with DMEM/F12 medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA). After two hours, we changed the medium to keep only adherent cells.

RNA extraction and transcriptome sequencing
Trizol (Takara Bio, Otsu, Japan) was used to extract total RNA as per the manufacturer's instructions. The concentration of RNA was measured using NanoDrop 2000 (Thermo Fisher, Waltham, MA, USA), and the RNA was then stored in a -80℃ refrigerator for storage.
The mRNA fragment was then reverse transcribed into rst-strand cDNA using reverse transcriptase (Takara Bio, Otsu, Japan) and random primers. The RNA template was then removed, and doublestranded cDNA was produced. End repair, poly-A tail processing, adapto r ligation and cDNA puri cation and enrichment were then performed. Sequencing was carried out using an Illumina HiSeq 2500 sequencing system, with 100 bp paired-end sequencing.
Next, raw data were measured by each internal script in the fastq format. Moreover, with the strict screening criteria, the clean data from raw data were collected. Synchronously, the related indicators of clean data were calculated, including Q20, Q30, and GC content. The high quality clean data were used for further downstream analyses.
Scripture (beta2) [11] and Cu inks (v2.1.1) [12] were used to combine the mapping metrics of each sample. Scripture was run with default parameters, while Cu inks was run with 'min-frags-per-transfrag = 0' and '--library-type', while other parameters set to default. Coding-Non-Coding Index (CNCI), Coding Potential Calculator (CPC) and Pfam-scan were used to predict the encoding ability of novel lncRNA.

Quanti cation of gene expression level
Cuffdiff (V2.1.1) was used to calculate the abundance of lncRNA. The FPKM of lncRNA was calculated by summing the FPKM of the transcripts in each sample genome. The Cuffdiff program was used to perform statistical modelling based on a negative binomial distribution to determine differential expression in digital transcripts of gene expression data. Statistical results were considered differential expressed when P * <0.05.

Bioinformatic analysis
PhyloFit was used to compute phylogenetic models for conserved and non-conserved regions between species, and HMM transition parameters were set to phyloP to compute a set of conservation scores for lncRNA and coding genes [13].
For cis-acting lncRNA, coding genes 10 kb/100 kb upstream and downstrea m of each lncRNA were searched for functional analysis of lncRNA. Meanwhile, for trans-acting lncRNA, the correlation between lncRNA and the coding gene was calculated using a custom script; these genes are considered as target genes of lncRNA predicted by position. KOBAS software (http://kobas.cbi.pku.edu.cn) was used to perform KEGG pathway enrichment analysis for each target gene predicted by lncRNA.

Real-time quantitative PCR
Approximately 500 ng of RNA was reverse transcripted using the PrimeScript RT Enzyme Mix (Takara Bio, Otsu, Japan) system to produce cDNA. Real-time quantitative PCR was performed on ABI StepOne Plus using the SYBR Premix Ex Taq™ system (Vazyme Biotech, Nanjing, China). The relative expression level of target genes was evaluated by the 2-ΔΔ Ct method, with β-actin acting as an internal reference gene. Sequences for all primers are shown in Table 1.

Statistical analysis
Each experiment was repeated at least three times independently, and representative results are shown. Differences between groups were assessed for signi cance with the t test, and the results are presented as mean ± SEM. P * < 0.05 was considered statistically signi cant. P * <0.01 is considered to have a very signi cant relationship.

Characteristics of lncRNA expressed in porcine intramuscular adipocytes
To pro le the lncRNA expressed during adipogenic differentiation in porcine intramuscular adipocytes, high-throughput RNA-seq was used to screen the transcriptome at 0, 2, and 8 days after differentiation.
From this, 707,853,440 raw reads were identi ed by RNA-seq, and 664,015,984 clean reads (66.41 GB, 0.01% error rate) remained after excluding low-quality data. The cleaned data were then compared to the pig genome (tax ID: 9823) to search for potential lncRNAs (Fig. 1A). In total, 2135 lncRNAs, including 575 novel lncRNAs, were identi ed using 3 different coding potential prediction software (CNCI, CPC and Pfam-scan) (Fig. 1B). Compared with protein-coding mRNAs obtained in the RNA-seq, lncRNAs were generally shorter (Fig. 1C), contained fewer exons (Fig. 1D), and had signi cantly lower ORF numbers (Fig. 1E). The novel lncRNAs identi ed in our study consisted of 500 lincRNAs and 75 antisense lncRNAs (Fig. 1F). The expression of lncRNAs was also much lower than that of mRNAs (Fig. 1G). Lastly, a phastCons analysis revealed that the lncRNAs were less conserved than mRNAs (Fig. 1H).

Screening and identi cation of differentially expressed genes (DEGs) during intramuscular adipogenic differentiation
In intramuscular adipocytes isolated from longissimus dorsal, 199 lncRNAs were found to be differentially expressed between 0 and 2 days after adipogenic induction (77 up-regulated, 122 downregulated). There were 98 up-regulated and 45 down-regulated lncRNAs in well-differentiated intramuscular adipocytes (8 days after differentiation) when compared with preadipocytes (0 days after differentiation). From the 2nd to the 8th day after adipogenic differentiation, there were 144 up-regulated and 43 down-regulated lncRNAs ( Fig. 2A).
With regards to the intramuscular adipocytes derived from semitendinosus, 350 differentially expressed lncRNAs were identi ed in total. There were 78 up-regulated and 89 down-regulated lncRNA in adipocytes of 2 days after adipogenic differentiation compared with preadipocytes (day 0), and 115 ascending and 51 descending lncRNAs in lipid-laden adipocytes (8 days after induction) in comparison with preadipocytes. Moreover, 132 lncRNAs increased and 64 lncRNAs decreased between 2 and 8 days after differentiation (Fig. 2B).
Heat maps were made of differentially expressed lncRNAs of interest (Fig. 2C).
To further validate the expression of some differentially expressed lncRNAs in RNA-seq, 2 annotated (ALDBSSCT0000011643, ALDBSSCT0000004851) and 5 novel lncRNAs of interest (lnc_000368, lnc _000359, lnc _000170, lnc _000076, lnc _000108) were selected, and their expression patterns were further con rmed by RT-qPCR (Fig. 2D). Overall, the expression patterns of these lncRNAs are mostly consistent with the results of RNA-seq.

KEGG analysis of differentially expressed lncRNAs during intramuscular adipogenic differentiation
KEGG analysis of target genes predicted by lncRNAs. KEGG analysis results show, on day 0 and day 2 of LD differentiation, KEGG analysis enriched signal pathways such as Ras signaling pathway, NF-kappa B signaling pathway (Fig. 3A), and on day 0 and day 8 enriched signal pathways such as Fatty acid metabolism, Fatty acid biosynthesis (Fig. 3B). The KEGG analysis on day 0 and day 2 of SD differentiation enriched signal pathways such as NF-kappa B signaling pathway (Fig. 3C), and on day 0 and day 8 enriched signal pathways such as PPAR signaling pathway and Insulin signaling pathway (Fig. 3D). Among the signi cantly enriched pathways, fat deposition related pathways such as PI3K-AKT, adipocytokine signaling, PPAR signaling pathway, Toll-like receptor signaling pathway, and Jak-STAT signaling pathway, were up-regulated, indicating that lncRNAs likely participated in intramuscular fat deposition through these pathways.

Characterization of lnc_000368
Lnc_000368 is an intergenic lncRNA located on pig Chr 2, 143856229-143907415, and is composed of two exons (Fig. 4A). The concentration of lnc_000368 gradually increased during intramuscular adipogenic differentiation in both longissimus dorsal and semitendinosus, as shown in the RNA-seq and RT-qPCR results (Fig. 2D). In order to explore the expression pattern of lnc_000368 in pig growth and development, we tested the tissues of Large White pigs at different ages. Further RT-qPCR analysis showed that lnc_000368 was highly enriched in back fat pads in 180-day-old Large White pigs (Fig. 4B), indicating that lnc_000368 is potentially involved in adipogenesis. The expression of lnc_000368 in longissimus dorsal and semitendinosus muscles reached a peak at 30 and 90 days, respectively (Fig. 4C,  D). By predicting target genes based on their location in the genome, interaction protein analysis was performed using Cytoscape software (Fig. 4E). Among these potential targets, TGFB1 (Transforming Growth Factor Beta 1) and GNAS (Guanine Nucleotide Binding Protein, Alpha) have been demonstrated to be involved in adipogenesis [14,15]. RNA-FISH showed that lnc_000368 was distributed in both the nucleus and the cytoplasm in the back subcutaneous fat pads of 90-day-old pigs (Fig. 4F).

Lnc_000368 promotes intramuscular adipocyte differentiation
To explore the role lnc_00368 potentially plays during intramuscular adipogenesis, siRNAs targeting lnc_00368 were designed and transfected into intramuscular preadipocytes isolated from longissimus dorsal. The well-differentiated cells were harvested and subjected to further analysis at day 8 after adipogenic differentiation. Results showed that lnc_00368 siRNAs effectively reduced the expression of lnc_000368 (Fig. 5A). Adipogenic transcription factors such as PPARγ, CEBPβ, and aP2, were signi cantly repressed by lnc_000368 siRNA at the mRNA level, while the key enzymes of lipolysis, ATGL and HSL, were down-regulated (Fig. 5B). The expression of these marker genes at the protein level was reduced (Fig. 5C, D). Oil Red O staining revealed that lipid accumulation was signi cantly reduced upon siRNA transfection (Fig. 5F). Collectively, our data showed that lnc_000368 is a potential enhancer for intramuscular adipogenesis.

Discussion
Although lncRNAs have gained increasing attention in recent years, understanding of how lncRNA affects intramuscular fat deposition in pigs is still limited due to the interspecies speci city of lncRNA, even though intramuscular fat is an economically important feature in determining meat quality [2]. Past studies have shown that lncRNAs are associated with fat deposition [16], and thus we proposed that lncRNAs may also be involved in the regulation of intramuscular fat deposition in pigs. An interesting characteristic some studies have found is that even under the same differentiation conditions, intramuscular adipocytes in longissimus dorsi are bigger and contain more lipid droplets than in semitendinosus [10]. Resultantly, the intramuscular fat content in longissimus dorsi is higher than in semitendinosus in adult pigs [17,18]. For this reason, in this study we selected intramuscular adipocytes from longissimus dorsi and semitendinosus muscles of Large White pigs for lncRNA-sequencing, aiming to identify the key lncRNAs that may be involved in the regulation of intramuscular adipocyte differentiation.
In this study, a total of 2135 lncRNAs, including 575 novel lncRNAs, were identi ed. Compared with protein-coding mRNAs, the number of exons and ORFs of lncRNAs were signi cantly lower in lncRNAs. In terms of conservation between species, lncRNAs were also far less conserved than mRNAs. The lncRNAs obtained through RNA-seq were roughly the same in terms of length, number of exons, and conservation, as those found in previous studies on porcine adipose tissues [19] and intramuscular fat [20]. KEGG analysis showed that the target genes of these lncRNAs were enriched in some classical signaling pathways related to fat formation, in ammation, and growth. This suggests that these differentially expressed lncRNAs may be involved in regulating the adipogenesis of intramuscular adipocytes.
Of the lncRNAs, lnc_000368 was gradually upregulated in intramuscular adipogenic differentiation in both longissimus dorsi and semitendinosus muscles; thus, we speculate that it may promote intramuscular adipocyte differentiation. Lnc_000368 was highly expressed in the adipose tissue of pigs, and additional analysis indicated that the knockdown of lnc_000368 might signi cantly repress lipid accumulation and reduce the expression of adipogenesis-related genes, such as PPARγ, CEBPβ, and aP2.
Among them, PPARγ is a very important regulator of adipogenesis. In previous studies, PPARγ was considered to be almost the most critical factor that determines adipocytes differentiation [21,22]. This suggests that lnc_000368 may be a novel enhancer of intramuscular adipogenesis.
In order to explore the underlying mechanism, cellular localization of lnc_000368 was detected using a FISH probe, and results showed that lnc_000368 was distributed in both the nucleus and cytoplasm. It illustrates that nuclear lncRNAs may participate in cellular activity through transcriptional regulation, dose compensation effect, enhancer regulation [23]. Cytoplasmic lncRNA may be involved in regulating mRNA stability, regulating mRNA translation, and acting as a competitive endogenous RNA or a precursor of microRNA [24]. Considering the cellular distribution, lnc_000368 may be a part of any of the mechanisms discussed above. Furthermore, among the target genes predicted by genomic position of lnc_000368, TGFB1 (Transforming Growth Factor Beta 1) [25] and GNAS (Guanine Nucleotide Binding Protein, Alpha) [26] have the potential to regulate adipocyte differentiation. Thus lnc_000368 likely regulates the differentiation of intramuscular adipocytes by regulating these two genes. However, the speci c mechanism of action requires further research.
In summary, our study screened out regulatory lncRNAs during the differentiation of porcine intramuscular adipocytes using RNA-sEq. With regards to intramuscular fat accumulation, adipocyte proliferation and differentiation are two important factors that contribute to lipid deposition [27]. In previous studies, lncRNAs were found to exert a regulatory role in the proliferation of porcine intramuscular adipocytes [9]. Our study showed that lncRNA may also modulate the adipogenic process of intramuscular adipocytes. Both of these regulatory lncRNAs could potentially be used as genetic markers in pig breeding.

Conclusions
In this study, we screened a group of lncRNAs that may be involved in the regulation of porcine intramuscular adipocyte differentiation, and performed qRT-PCR veri cation and KEGG analysis on them. From this we carried out a detailed analysis of lnc_000368, and then silenced in porcine intramuscular adipocytes, and found that the differentiation ability of the cells was reduced. Therefore, we concluded that lnc_000368 is a lncRNA that promotes the differentiation of porcine intramuscular adipocytes.