Effect of LncRNA LOC106505926 on myogenesis and Lipogenesis of porcine primary cells.

BACKGROUND: Skeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving the quality of carcass and meat. In the present study, whole transcriptome sequencing (including RNA-Seq and miRNA-Seq) was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age. RESULTS: The results showed that a total of 245 differentially expressed miRNAs were screened in any two comparisons, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Pearson correlation coefficient analysis showed that long non-coding RNA (lncRNA) LOC106505926 and CXXC5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN. CONCLUSIONS: Collectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.

Construction of LncRNA-Related ceRNA Networks in Longissimus Dorsi Muscle of Jinfen White Pigs at Different Developmental Stages.

The development of skeletal muscle in pigs might determine the quality of pork. In recent years, long non-coding RNAs (lncRNAs) have been found to play an important role in skeletal muscle growth and development. In this study, we investigated the whole transcriptome of the longissimus dorsi muscle (LDM) of Jinfen White pigs at three developmental stages (1, 90, and 180 days) and performed a comprehensive analysis of lncRNAs, mRNAs, and micro-RNAs (miRNAs), aiming to find the key regulators and interaction networks in Jinfen White pigs. A total of 2638 differentially expressed mRNAs (DE mRNAs) and 982 differentially expressed lncRNAs (DE lncRNAs) were identified. Compared with JFW_1d, there were 497 up-regulated and 698 down-regulated DE mRNAs and 212 up-regulated and 286 down-regulated DE lncRNAs in JFW_90d, respectively. In JFW_180d, there were 613 up-regulated and 895 down-regulated DE mRNAs and 184 up-regulated and 131 down-regulated DE lncRNAs compared with JFW_1d. There were 615 up-regulated and 477 down-regulated DE mRNAs and 254 up-regulated and 355 down-regulated DE lncRNAs in JFW_180d compared with JFW_90d. Compared with mRNA, lncRNA has fewer exons, fewer ORFs, and a shorter length. We performed GO and KEGG pathway functional enrichment analysis for DE mRNAs and the potential target genes of DE lncRNAs. As a result, several pathways are involved in muscle growth and development, such as the PI3K-Akt, MAPK, hedgehog, and hippo signaling pathways. These are among the pathways through which mRNA and lncRNAs function. As part of this study, bioinformatic screening was used to identify miRNAs and DE lncRNAs that could act as ceRNAs. Finally, we constructed an lncRNA-miRNA-mRNA regulation network containing 26 mRNAs, 7 miRNAs, and 17 lncRNAs; qRT-PCR was used to verify the key genes in these networks. Among these, XLOC_022984/miR-127/ENAH and XLOC_016847/miR-486/NRF1 may function as key ceRNA networks. In this study, we obtained transcriptomic profiles from the LDM of Jinfen White pigs at three developmental stages and screened out lncRNA-miRNA-mRNA regulatory networks that may provide crucial information for the further exploration of the molecular mechanisms during skeletal muscle development.

CircHOMER1 inhibits porcine adipogenesis via the miR-23b/SIRT1 axis.

Fat deposition is critical to pork quality. However, the mechanism of fat deposition remains to be elucidated. Circular RNAs (circRNAs) are ideal biomarkers and are involved in adipogenesis. Here, we investigated the effect and mechanism of circHOMER1 on porcine adipogenesis in vitro and in vivo. Western blotting, Oil red O staining, and HE staining were used to assess the function of circHOMER1 in adipogenesis. The results showed that circHOMER1 inhibited adipogenic differentiation of porcine preadipocytes and suppressed adipogenesis in mice. Dual-luciferase reporter gene, RIP, and pull-down assays demonstrated that miR-23b directly bound to circHOMER1 and the 3'-UTR of SIRT1. Rescue experiments further illustrated the regulatory relationship among circHOMER1, miR-23b, and SIRT1. Conclusively, we demonstrate that circHOMER1 plays an inhibitory role in porcine adipogenesis through miR-23b and SIRT1. The present study revealed the mechanism of porcine adipogenesis, which may be helpful to improve pork quality.

RNA-seq analysis of small intestine transcriptional changes induced by starvation stress in piglets.

Piglets may experience a variety of stress injuries, but the molecular regulatory mechanisms underlying these injuries are not well understood. In this study, we analysed the ileum of Large White (LW) and Mashen (MS) piglets at different times of starvation using chemical staining and transcriptome analysis. The intestinal barrier of piglets was damaged after starvation stress, but the intestinal antistress ability of MS piglets was stronger than LW piglets. A total of 8021 differentially expressed genes (DEGs) were identified in two breeds. Interestingly, the immune capacity (CHUK, TLR3) of MS piglets increased significantly after short-term starvation stress, while energy metabolism (NAGS, PLA2G12B, AGCG8) was predominant in LW piglets. After long-term starvation stress, the level of energy metabolism (PLIN5, PLA2G12B) was significantly increased in MS piglets. The expression of immune (HLA-DQB1, IGHG4, COL3A1, CD28, LAT) and disease (HSPA1B, MINPPI, ADH1C, GAL3ST1) related genes were significantly increased in two breeds of piglets. These results suggest that short-term stress mainly enhances immunity and energy metabolism in piglets, while long-term starvation produces greater stress on piglets, making it difficult for them to compensate for the damage to their bodies through self-regulation. This information can help improve the stress resistance of piglets through molecular breeding.

Genome-wide analysis of circular RNA-mediated ceRNA regulation in porcine skeletal muscle development.

BACKGROUND: As a diverse and abundant class of endogenous RNAs, circular RNAs (circRNAs) participate in various biological processes including cell proliferation and apoptosis. Nevertheless, few researchers have investigated the role of circRNAs in muscle development in cultivated pigs. RESULTS: In this study, we used RNA-seq to construct circRNA expression profiles in skeletal muscle of Jinfen White pigs at the age of 1, 90, and 180 days. Among the 16,990 identified circRNAs, 584 circRNAs were differentially expressed. Moreover, the enrichment analysis of DE circRNA host genes showed that they were mainly involved in muscle contraction, muscle organ development and muscle system processes, as well as AMPK and cAMP-related signal pathways. We also constructed a circRNA-miRNA-mRNA co-expression network to find key circRNAs which many involved in the regulation of porcine skeletal muscle development through the competitive endogenous RNA (ceRNA) mechanism. It is noteworthy that circ_0018595/miR-1343/PGM1 axis may play a regulatory role in the development of porcine skeletal muscle. CONCLUSIONS: This study identified the circRNAs and present the circRNA expression profile in the development of pigs, revealed that DE circRNA host genes participate in different cell fates and enriched the porcine ceRNA network. Thus, this work will become a valuable resource for further in-depth study of the regulatory mechanism of circRNA in the development of porcine skeletal muscle.

Expression patterns and functional analysis of porcine lnc-34015.

Long noncoding RNAs (lncRNAs) play important roles in immune regulation in humans and animals. The lnc-34015 was discovered to be critical for the development of muscles, based on the muscle transcriptome of pigs; however, the underlying molecular mechanism requires better understanding. Here, the sequence characteristics of lnc-34015 were analyzed and a competitive endogenous RNA regulatory network of lncRNA was predicted. The developmental expression trend and tissue expression profiles of lnc-34015 were investigated using quantitative polymerase chain reaction. The lnc-34015 sequence is overlapped with introns 11 and 12 of CWF19L1, while CWF19L1, PKD2L1, and CHUK were identified as cis-regulatory genes of lnc-34015. Bioinformatics analyses revealed that lnc-34015 binds to 15 microRNAs (miRNAs), including miR-3646, miR-377-3p, and miR-190b-3p, to regulate downstream gene expression. GO and KEGG enrichment results show that lnc-34015 was mainly involved in cell proliferation, stress response, transcriptional regulation, and alternative splicing. The expression trend of lnc-34015 in muscle was similar to that of target genes and opposite to that of miRNAs. The expression of lnc-34015 was significantly higher in the porcine small intestine and IPEC-J2 cells. Our findings suggest that lnc-34015 regulates CHUK, ZBTB20, and XIAP gene expression by competing with endogenous RNAs to regulate porcine inflammatory responses.

CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

Transcriptome Analysis Revealed Potential Genes of Skeletal Muscle Thermogenesis in Mashen Pigs and Large White Pigs under Cold Stress.

Pigs are susceptible to cold stress due to the absence of brown fat caused by the partial deletion of uncoupling protein 1 during their evolution. Some local pig breeds in China exhibit potential cold adaptability, but research has primarily focused on fat and intestinal tissues. Skeletal muscle plays a key role in adaptive thermogenesis in mammals, yet the molecular mechanism of cold adaptation in porcine skeletal muscle remains poorly understood. This study investigated the cold adaptability of two pig breeds, Mashen pigs (MS) and Large White pigs (LW), in a four-day cold (4 °C) or normal temperature (25 °C) environment. We recorded phenotypic changes and collected blood and longissimus dorsi muscle for transcriptome sequencing. Finally, the PRSS8 gene was randomly selected for functional exploration in porcine skeletal muscle satellite cells. A decrease in body temperature and body weight in both LW and MS pigs under cold stress, accompanied by increased shivering frequency and respiratory frequency, were observed. However, the MS pigs demonstrated stable physiological homeostasis, indicating a certain level of cold adaptability. The LW pigs primarily responded to cold stress by regulating their heat production and glycolipid energy metabolism. The MS pigs exhibited a distinct response to cold stress, involving the regulation of heat production, energy metabolism pathways, and robust mitochondrial activity, as well as a stronger immune response. Furthermore, the functional exploration of PRSS8 in porcine skeletal muscle satellite cells revealed that it affected cellular energy metabolism and thermogenesis by regulating ERK phosphorylation. These findings shed light on the diverse transcriptional responses of skeletal muscle in LW and MS pigs under cold stress, offering valuable insights into the molecular mechanisms underlying cold adaptation in pigs.

The long non-coding RNA lncMYOZ2 mediates an AHCY/MYOZ2 axis to promote adipogenic differentiation in porcine preadipocytes.

Long non-coding RNAs (lncRNAs) play a vital role in regulating adipogenesis. However, the associated regulatory mechanisms have yet to be described in detail in pig. In this study, we demonstrate a critical role for lncMYOZ2 in adipogenesis from porcine preadipocytes. Specifically, lncMYOZ2 was more abundant in the adipose tissue of Mashen (fat-type) pigs than for Large White (lean-type) pigs, and knockdown of this lncRNA significantly inhibited the differentiation of porcine preadipocytes into adipocytes. Mechanistically, we used RNA pull-down and RIP assays to establish that lncMYOZ2 interacts with adenosylhomocysteinase (AHCY). Moreover, lncMYOZ2 knockdown increased promoter methylation of the target gene MYOZ2 and lowered its expression. Finally, we describe a positive regulatory role for MYOZ2 in adipogenesis. Collectively, these findings establish lncMYOZ2 as an important epigenetic regulator of adipogenesis via the aforementioned AHCY/MYOZ2 pathway, and provide insights into the role of lncRNAs in porcine adipose development.

CircCSDE1 Regulates Proliferation and Differentiation of C2C12 Myoblasts by Sponging miR-21-3p.

The growth and development of skeletal muscle is regulated by many factors, and recent studies have shown that circular RNAs (circRNAs) can participate in this process. The model of porcine skeletal muscle injury was constructed to search for circRNAs that can regulate the growth and development of skeletal muscle in pigs. Using whole-transcriptome sequencing and bioinformatics analysis, a novel circRNA (circCSDE1) was screened out, which is highly expressed in skeletal muscle. Functional studies in C2C12 cells demonstrated that circCSDE1 could promote proliferation and inhibit myoblast differentiation, while opposing changes were observed by circCSDE1 knockdown. A dual-luciferase reporter assay revealed that circCSDE1 directly targeted miR-21-3p to regulate the expression of the downstream target gene (Cyclin-dependent kinase 16, CDK16). Moreover, miR-21-3p could inhibit proliferation and promote myoblast differentiation in C2C12 cells, opposite with the effects of circCSDE1. Additionally, the rescue experiments offered further evidence that circCSDE1 and its target, miR-21-3p, work together to regulate myoblast proliferation and differentiation. This study provides a theoretical basis for further understanding the regulatory mechanisms of circRNAs.