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Long non-coding RNAs (lncRNAs) are involved in the process of muscle cell differentiation and play an important role. Previous studies have shown that lncRNA-MEG3 promotes the differentiation of porcine skeletal muscle satellite cells (PSCs), but the regulatory mechanism of MEG3 interaction with target protein has not been well studied. We demonstrated that MEG3 can bind dihydrolipoamide succinyltransferase (DLST) by RNA pull down and RIP-qPCR. Subsequently, knockdown and overexpression experiments showed that DLST promotes PSCs differentiation. Rescue experiments showed that the expression of DLST protein was significantly increased with MEG3 overexpression and decreased with MEG3 knockdown, while its mRNA expression was not changed. Furthermore, we have successfully predicted and validated that the transcription factor myogenic differentiation (MYOD) binds to the MEG3 core promoter though utilizing chromatin immunoprecipitation (CHIP) and luciferase reporter assays. The results indicated that MYOD acts as a transcription factor of MEG3 to promote MEG3 transcription. Knockdown of MEG3 in vivo indicated that MEG3 is involved in skeletal muscle regeneration. It is concluded that MYOD acts as a transcription factor to induce MEG3 expression. MEG3 acts as a molecular scaffold to bind and promote DLST protein expression. This paper provides a new molecular mechanism for MEG3 to promote the differentiation of PSCs.

Myogenic differentiation is a complex biological process that is regulated by multiple factors, among which long noncoding RNAs (lncRNAs) play an essential role. However, in-depth studies on the regulatory mechanisms of long noncoding RNAs (lncRNAs) in myogenic differentiation are limited. In this study, we characterized the role of the novel lncRNA TCONS_00323213 , which is upregulated during porcine skeletal muscle satellite cell (PSC) differentiation in myogenesis. We found that TCONS_00323213 affected the proliferation and differentiation of PSC in vitro. We performed quantitative polymerase chain reaction (qPCR), 5-ethynyl-20-deoxyuridine (EdU), western blotting, immunofluorescence staining, pull-down assays, and cleavage under targets and tagmentation (CUT and Tag) assays to clarify the effects and action mechanisms of TCONS_00323213 . LncRNA TCONS_00323213 inhibited myoblast proliferation based on analyses of cell survival rates during PSC proliferation. Functional analyses revealed that TCONS_00323213 promotes cell differentiation and enhances myogenin ( MyoG ), myosin heavy chain ( MyHC ), and myocyte enhancer factor 2 ( MEF2C ) during myoblast differentiation. As determined by pull-down and RNA immunoprecipitation (RIP) assays, the lncRNA TCONS_00323213 interacted with PBX/Knotted Homeobox 2 (PKNOX2). CUT and Tag assays showed that PKNOX2 was significantly enriched on the MyoG promoter after lncRNA TCONS_00323213 knockdown. Our findings demonstrate that the interaction between lncRNA TCONS_00323213 and PKNOX2 relieves the inhibitory effect of PKNOX2 on the MyoG promoter, increases its expression, and promotes PSC differentiation. This novel role of lncRNA TCONS_00323213 sheds light on the molecular mechanisms by which lncRNAs regulate porcine myogenesis.

The difference between the skeletal muscle growth rates of Western and domestic breeds is remarkable, but the potential regulatory mechanism involved is still unclear. Numerous studies have pointed out that long intergenic noncoding RNA (lincRNA) plays a key role in skeletal muscle development. This study used published Yorkshire (LW) and Tibetan pig (TP) transcriptome data to explore the possible role of lincRNA in the difference in skeletal muscle development between the two breeds. 138 differentially expressed lincRNAs (DELs) were obtained between the two breeds, and their potential target genes (PTGs) were predicted. The results of GO and KEGG analysis revealed that PTGs are involved in multiple biological processes and pathways related to muscle development. The quantitative trait loci (QTLs) of DELs were predicted, and the results showed that most QTLs are related to muscle development. Finally, we constructed a co-expression network between muscle development related PTGs (MDRPTGs) and their corresponding DELs on the basis of their expression levels. The expression of DELs was significantly correlated with the corresponding MDRPTGs. Also, multiple MDRPTGs are involved in the key regulatory pathway of muscle fiber hypertrophy, which is the IGF-1-AKT-mTOR pathway. In summary, multiple lincRNAs that may cause differences in skeletal muscle development between the two breeds were identified, and their possible regulatory roles were explored. The findings of this study may provide a valuable reference for further research on the role of lincRNA in skeletal muscle development.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors with the approval of the Editor-in-Chief. The panel SK-N-SH/si-NC from Fig. 2F appears similar to the panel SH-SY5Y/si-NC (vertically flipped) and the panel SK-N-SH/si-DLX6-AS1 appears similar to the panel SH-SY5Y/si-DLX6-AS1 (vertically flipped). In addition, the authors were reportedly not able to retrieve the raw images of these panels., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intramuscular fat (IMF) content is closely related to various meat traits, such as tenderness, juiciness, and flavor. The IMF content varies considerably among pig breeds with different genetic backgrounds. Long intergenic non-coding RNAs (lincRNAs) have been widely identified in many species and found to be an important class of regulators that can participate in multiple biological processes. However, the mechanism behind lincRNAs regulation of pig IMF content remains unknown and requires further study. In our study, we identified a total of 156 lincRNAs in the longissimus dorsi muscle of Wei (fat-type) and Yorkshire (lean-type) pigs using previously published data. These identified lincRNAs have shorter transcript length, longer exon length, lower exon number, and lower expression level as compared with protein-coding transcripts. We predicted potential target genes (PTGs) that are potentially regulated by lincRNAs in cis or trans regulation. Gene ontology and pathway analyses indicated that many potential lincRNAs target genes are involved in IMF-related processes or pathways, such as fatty acid catabolic process and adipocytokine signaling pathway. In addition, we analyzed quantitative trait locus (QTL) sites that differentially expressed lincRNAs (DE lincRNAs) between Wei and Yorkshire pigs co-localized. The QTL sites where DE lincRNAs co-localize are mostly related to IMF content. Furthermore, we constructed a co-expressed network between DE lincRNAs and their differentially expressed PTGs (DEPTGs). On the basis of their expression levels, we suggest that many DE lincRNAs can affect IMF development by positively or negatively regulating their PTGs. This study identified and analyzed some lincRNAs- and PTGs-related IMF development of the two pig breeds and provided new insight into research on the roles of lincRNAs in the two types of breeds.

Long non-coding RNAs (lncRNAs) have been implicated in fundamental and diverse biological processes, including myogenesis. However, the molecular mechanisms involved in this process remain largely unexplored. This study found that H19 affected the differentiation of porcine satellite cells (PSCs) by directly binding to the DNA/RNA-binding protein TDP43. Functional analyses showed that TDP43 knockdown decreased PSC differentiation, whereas TDP43 overexpression exerted opposite effects in vitro. Furthermore, rescue experiments demonstrated that TDP43 can rescue the decrease in PSC differentiation caused by H19 knockdown. Mechanistically, H19 may act as a scaffold to recruit TDP43 to the promoters of MYOD and thereby activate the transcription of MYOD, leading to PSC differentiation. In summary, we elucidate the molecular mechanism by which H19 and TDP43 regulate myogenesis.

Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5'- and 3'-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 ( MEG3 ), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor ( SRF ), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3 . Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3 , miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis.

Muscle growth and fat deposition are the two important biological processes in the development of pigs which are closely related to the pig production performance. Long intergenic noncoding RNAs (lincRNAs), with lack of coding potential and the length of at least 200nt, have been extensively studied to play important roles in many biological processes. However, the importance and molecular regulation mechanism of lincRNAs in the process of muscle growth and fat deposition in pigs are still to be further studied comprehensively. In our study, we used the data, including liver, abdominal fat, and longissimus dorsi muscle of 240 days' age of two F2 full-sib female individuals from the white Duroc and Erhualian crossbreed, to identify 581 putative lincRNAs associated with pig muscle growth and fat deposition. The 581 putative lincRNAs shared many common features with other mammalian lincRNAs, such as fewer exons, lower expression levels, and shorter transcript lengths. Cross-tissue comparisons showed that many transcripts were tissue-specific and were involved in the important biological processes in their corresponding tissues. Gene ontology and pathway analysis revealed that many potential target genes (PTGs) of putative lincRNAs were involved in pig muscle growth and fat deposition-related processes, including muscle cell proliferation, lipid metabolism, and fatty acid degradation. In Quantitative Trait Locus (QTLs) analysis, some PTGs were screened from putative lincRNAs, MRPL12 is associated with muscle growth, GCGR and SLC25A10 were associated with fat deposition, and PPP3CA, DPYD, and FGGY were related not only to muscle growth but also to fat deposition. Therefore, it implied that these lincRNAs might participate in the biological processes related to muscle growth or fat deposition through homeostatic regulation of PTGs, but the detailed molecular regulatory mechanisms still needed to be further explored. This study lays the molecular foundation for the in-depth study of the role of lincRNAs in the pig muscle growth and fat deposition and further provides the new molecular markers for understanding the complex biological mechanisms of pig muscle growth and fat deposition.

Aim: We aimed to identify previously unreported long intergenic noncoding RNAs (lincRNAs) in the porcine liver, an important metabolic tissue, and further illustrate the epigenomic landscapes and the evolution of lincRNAs., Materials & Methods: We used porcine omics data and comprehensively analyzed and identified lincRNAs and their methylation, expression and evolutionary patterns during pig domestication., Results: LincRNAs exhibit highly methylated promoter and downstream regions, as well as lower expression levels and higher tissue specificity than protein-coding genes. We identified a batch of lincRNAs with selection signals that are associated with pig domestication, which are more highly expressed in the liver than in other tissues (19:10/8/6/3/2/1/1). Interestingly, the lincRNA linc-sscg1779 and its target gene C6, which is crucial in liver metabolism, are differentially expressed during pig domestication., Conclusion: Although they may originate from noisy transcripts, lincRNAs may be subjected to artificial selection. This phenomenon implies the functional importance of lincRNAs in pig domestication.

Long intergenic non-coding RNAs (lincRNAs) play essential roles in numerous biological processes and are widely studied. The skeletal muscle is an important tissue that plays an essential role in individual movement ability. However, lincRNAs in pig skeletal muscles are largely undiscovered and their biological functions remain elusive. In this study, we assembled transcriptomes using RNA-seq data published in previous studies of our laboratory group and identified 323 lincRNAs in porcine leg muscle. We found that these lincRNAs have shorter transcript length, fewer exons and lower expression level than protein-coding genes. Gene ontology and pathway analyses indicated that many potential target genes (PTGs) of lincRNAs were involved in skeletal-muscle-related processes, such as muscle contraction and muscle system process. Combined our previous studies, we found a potential regulatory mechanism in which the promoter methylation of lincRNAs can negatively regulate lincRNA expression and then positively regulate PTG expression, which can finally result in abnormal phenotypes of cloned piglets through a certain unknown pathway. This work detailed a number of lincRNAs and their target genes involved in skeletal muscle growth and development and can facilitate future studies on their roles in skeletal muscle growth and development.

Background: The domestic pig Sus scrofa domesticus originated from the wild boar S. scrofa about 10,000 years ago. During domestication, drastic morphological, physiological, and behavioral changes developed between domestic pigs and wild boars through artificial and natural selection. The long non-coding RNA (lncRNA) H19, which is located within the imprinting gene cluster H19-IGF2, plays an important role in regulating muscle development in humans and mice. This study systematically analyzed the molecular evolution of H19 and its possible epigenetic changes during pig domestication and breeding to explore the genetic and epigenetic contributions of H19 to pig domestication., Results: The molecular evolution of H19 was initially analyzed on a large phylogenetic scale. Results showed that the gene was highly conserved within a broad range, especially in the 5' terminal sequence. The molecular evolution of the gene was then analyzed using published re-sequencing data of 30 wild boars from Tibet, 3 wild boars from Sichuan, and 15 native pigs from other regions in China. Eight polymorphic sites were identified, and the nucleotide diversity (π) value within the H19 gene body was significantly higher (Z-test, P < 0.05) in domesticated pigs than in wild pigs. However, no significant divergence occurred between domesticated and wild pigs. Single nucleotide polymorphisms in the 3' terminal sequence were surveyed in other Chinese local breeds and foreign pig breeds. We observed a consistently higher diversity in domesticated pigs than in wild pigs. The methylation pattern of the H19 gene in pigs was subsequently analyzed using published methylated DNA immunoprecipitation data and an unpublished single-base resolution liver methylome. Analysis results showed distinct methylation levels in some tissues. Among the samples surveyed, Landrace showed the lowest methylation level, followed by the Guizhou wild boar, whereas the Enshi pig exhibited the highest methylation level in the 2 kb upstream region of the H19 gene. Liver transcriptome data suggested that Landrace harbored the highest expression of the H19 gene, followed by the Guizhou wild boar, whereas the Enshi pig harbored the lowest expression of the gene. Differential methylation sites (DMSs) among the three breeds were mainly identified in the 2 kb upstream region of the H19 gene. In the Enshi pig, we detected allele-specific methylation (ASM) regions in the 2 kb upstream region of the H19 gene. Most of the DMSs in the upstream 2 kb region of the gene were also located in the ASM region in this breed., Conclusions: Molecular analyses suggest that the H19 gene was highly conserved during large-scale evolution and exhibited genotype differentiation during domestication and breed differentiation. The drastic diversity pattern between domestic and wild pigs in the H19 gene body, which was highly conserved during large-scale evolution, suggests that this gene might have played roles in the breed differentiation of domestic pigs. Methylation analysis indicates an opposite epigenetic regulation direction between Chinese and European pig (EU) domestication, which resulted in opposite expression changes in this gene between the two domesticated groups. Our preliminary analyses on DMSs among different pig breeds and ASM imply that imprinting was associated with methylation differences. This study systematically demonstrates the genetic and epigenetic patterns of H19 during pig domestication and provide valuable cues and basis for further research on the function of H19 in pig domestication.