Transcriptome analysis of mRNA and miRNA in the development of LeiZhou goat muscles

The progression of muscle development is a pivotal aspect of animal ontogenesis, where miRNA and mRNA exert substantial influence as prominent players. It is important to understand the molecular mechanisms involved in skeletal muscle development to enhance the quality and yield of meat produced by Leizhou goats. We employed RNA sequencing (RNA-SEQ) technology to generate miRNA-mRNA profiles in Leizhou goats, capturing their developmental progression at 0, 3, and 6 months of age. A total of 977 mRNAs and 174 miRNAs were found to be differentially expressed based on our analysis. Metabolic pathways, calcium signaling pathways, and amino acid synthesis and metabolism were found to be significantly enriched among the differentially expressed mRNA in the enrichment analysis. Meanwhile, we found that among these differentially expressed mRNA, some may be related to muscle development, such as MYL10, RYR3, and CSRP3. Additionally,, we identified five muscle-specific miRNAs (miR-127-3p, miR-133a-3p, miR-193b-3p, miR-365-3p, and miR-381) that consistently exhibited high expression levels across all three stages. These miRNAs work with their target genes (FHL3, SESN1, PACSIN3, LMCD1) to regulate muscle development. Taken together, our findings suggest that several miRNAs and mRNAs are involved in regulating muscle development and cell growth in goats. By uncovering the molecular mechanisms involved in muscle growth and development, these findings contribute valuable knowledge that can inform breeding strategies aimed at enhancing meat yield and quality in Leizhou goats.

In recent years, China's goat industry has shown significant growth, particularly in goat meat production.Among low-fat meat options, goat meat is highly favored for its unique flavor, palatability, and relatively lean nature when compared to other red meats 1 .Skeletal muscle makes up approximately 40% of a goat's body weight 2 , highlighting its significance as a crucial component of mutton.Enhancing the growth and development of skeletal muscle is crucial for animal agriculture as it provides a source of meat for human consumption.Consequently, studying the differentiation and development of skeletal muscle in goats is crucial for the advancement of modern agriculture.
Before birth, skeletal muscle growth and development primarily occur through an increase in muscle fibers, resulting in skeletal muscle hypertrophy 3 .After birth, skeletal muscle hypertrophy is achieved through the enlargement of existing muscle fibers and the activation of a population of muscle stem cells.Therefore, postnatal skeletal muscle growth is governed by distinct mechanisms from embryonic muscle development 4 .Research has shown that myostatin is critical for fetal muscle development but is not required for postnatal skeletal muscle growth.Instead, postnatal skeletal muscle growth may be more dependent on Ca2 + signaling and Mef2 proteins, rather than myogenic basic helix-loop-helix (bHLH) factors 5 .Ca2 + signaling can facilitate signal transduction during neonatal muscle development 6 , while Mef2 proteins may serve as a major regulator of skeletal muscle growth, playing a crucial role in postnatal skeletal muscle growth as well 5 .In summary, while embryonic and postnatal skeletal muscle growth share some common features, the underlying mechanisms controlling these processes are distinct.Postnatal skeletal muscle growth is primarily regulated by Ca2 + signaling and Mef2 proteins, with myostatin playing a minor role.
In the past, people who wanted to improve traits such as skeletal muscle in goats to increase meat yield could often only breed strong individuals through individual or group selection, but this approach has the problem of

cDNA library preparation
We used the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and DNase I (Qiagen, Beijing, China) to isolate total RNA.The quality of purified RNA was assessed using 1.5% agarose gel electrophoresis to confirm the absence of genomic DNA, and RNA integrity was estimated using the RNA Nano6000 Assay Kit and a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).Each sample utilized 3 μg of total RNA (with RNA integrity number values larger than 7.0) for cDNA library construction.Ribosomal RNA (rRNA) was removed using the Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA), and the rRNA-free residue was cleaned by ethanol precipitation.The rRNA-deleted RNA was then used to construct sequencing libraries with a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's instructions.After fragmentation, first and second strand cDNA synthesis was performed.Adenylation of the 3' ends of DNA fragments was followed by ligation of NEBNext adaptors containing a hairpin loop structure to prepare them for hybridization.The library fragments were then purified using an AMPure XP system (Beckman Coulter, Beverly, MA, USA), with a preference given to cDNA fragments of 150-200 bp in length.Subsequently, the size selected, adaptor ligated cDNA was incubated with the USER enzyme (NEB) at 37 °C for 15 min, followed by 5 min at 95 °C.PCR amplification was then performed using Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer.Finally, the products were purified using the AMPure XP system, and the library quality was assessed using the Agilent Bioanalyzer 2100 system.

Sequencing and transcriptome assembly
We used the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and DNase I (Qiagen, Beijing, China) to isolate total RNA.The quality of purified RNA was assessed using 1.5% agarose gel electrophoresis to confirm the absence of genomic DNA.RNA integrity was estimated using the RNA Nano6000 Assay Kit and a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).Each sample used 3 μg of total RNA (with RNA integrity number values greater than 7.0) for cDNA library construction.Ribosomal RNA (rRNA) was removed using the Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA), and the rRNA-free residue was purified through ethanol precipitation.The rRNA-depleted RNA was then used to construct sequencing libraries using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's instructions.After fragmentation, first-and second-strand cDNA synthesis was performed.Adenylation of the 3' ends of DNA fragments was followed by ligation of NEBNext adaptors containing a hairpin loop structure to prepare them for hybridization.The library fragments were purified using an AMPure XP system (Beckman Coulter, Beverly, MA, USA), with a preference for cDNA fragments ranging from 150 to 200 bp in length.Subsequently, the size-selected cDNA with ligated adaptors was incubated with the USER enzyme (NEB) at 37 °C for 15 min, followed by 5 min at 95 °C.PCR amplification was then performed using Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer.Finally, the products were purified using the AMPure XP system, and the library quality was assessed using the Agilent Bioanalyzer 2100 system.

Prediction of the differentially expressed mRNAs and miRNAs
To identify known miRNAs, we first mapped small RNA tags and used miRBase 20.0 as a reference with customized software, miRDeep2 20 , and sRNA-tools-cli.We calculated miRNA counts and base bias using custom scripts for identified miRNA positions of a specific length.To eliminate tags originating from non-miRNA sources such as protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, we aligned small RNA tags to RepeatMasker, Rfam database, or data specific to the selected species.
To predict new miRNAs, we utilized the properties of miRNA precursor hairpin structures.We utilized miREvo 20 and miRDeep2 21 software to analyze the secondary structure, Dicer cleavage site, and minimal free energy of short RNA tags that had not been annotated in previous phases.We used custom scripts to obtain the detected miRNA counts and base bias for identified miRNA positions of a specific length at each position of all identified miRNA.

Target gene prediction
MiRanda was used to predict the target genes of miRNA in animals.The miRanda algorithm is based on the complementarity of nucleotides (A = U or G≡C).The scoring matrix used for this analysis also accommodates G = U 'wobble' pairs.Complementarity parameters at individual alignment positions are: + 5 for G≡C, + 5 for A = U, + 2 for G = U, and − 3 for all other nucleotide pairs.The algorithm uses affine penalties, which are linear in the length of a gap, for gap opening (− 8) and gap extension (− 2).In addition, after observing known target sites, complementarity scores (both positive and negative values) at the first eleven positions are multiplied by a scaling factor (set at 2.0 in this case) to account for the observed 5'-3' asymmetry.Finally, the following four empirical rules are applied, with positions counted starting at the 5' end of the miRNA: no mismatches at positions 2 to 4; fewer than five mismatches between positions 3-12; at least one mismatch between positions 9 and L-5 (where L is the total alignment length); and fewer than two mismatches in the last five positions of the alignment.With these parameters, the dynamic programming algorithm optimizes the complementarity score between a miRNA sequence and an mRNA sequence (usually a 3' UTR), summed over all aligned positions.It identifies all non-overlapping hybridization alignments in decreasing order of complementarity score down to a specified cutoff value (default value 80) 22 .

GO and KEGG enrichment analysis
We conducted Gene Ontology (GO) enrichment analysis on the target gene candidates of differentially expressed miRNAs.For Gene Ontology (GO) enrichment analysis, we employed a Wallenius non-central hypergeometric distribution based on GOseq 23 , which can adjust for gene length bias.KEGG 24 is a database resource for inferring high-level functions and utilities of biological systems such as the cell, organism, and ecosystem from molecularlevel data, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http:// www.genome.jp/ kegg/).The statistical enrichment of target gene candidates in KEGG pathways was tested using the KOBAS 25 program.

Quantitative real-time polymerase chain reaction validation
For the qRT-PCR study, 1 µg of total RNA was reverse transcribed using RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instructions.The qRT-PCR was conducted using Fast Start Universal SYBR Green Master (ROX) based on routine procedures (Roche, Mannheim, Germany) with a StepOnePlus Real-Time PCR System (Life Technologies, Gaithersburg, MD, USA).

Enrichment analysis of the differentially expressed mRNAs
For the identification of differentially expressed genes, we used a significance threshold of p < 0.05, which resulted in the detection of 977 mRNAs.Among these, 77 mRNAs were found to be upregulated, and 75 mRNAs were downregulated in the M3 group compared to the M0 group.The M6 group exhibited 515 upregulated and 402 downregulated mRNAs in comparison to the M0 group.In the comparison between the M3 and M6 groups, we observed 21 upregulated mRNAs and 32 downregulated mRNAs (Fig. 1).To better understand the relationships and similarities between the different libraries, we conducted systematic cluster analyses to analyze the expression patterns of the DEmRNAs.In this article, we cite the research results of Zhao 1 , mainly focusing on mRNA research data.We would like to express our gratitude to Zhao for sharing mRNA-related information with us, as it has laid an important foundation for our research.
In order to understand the functional implications of these mRNAs in regulating litter size, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.We identified a total of 519 significant Gene Ontology (GO) terms with a p-value < 0.05.Among these terms, the M0 vs. M3 group showed significant enrichment in processes related to glucose and glycogen metabolism, generation of precursor metabolites and energy, as well as oxidation-reduction processes (Fig. 2A) (Supplementary Information 1).The differential expression analysis revealed that a significant portion of genes between the M0 and M6 groups were associated with cellular protein metabolic processes, glycolysis, and protein metabolic processes (Fig. 2B) (Supplementary Information 2).The M6 vs. M3 group was mainly enriched in nucleoside and nucleoside phosphate biosynthetic processes, calcineurin-NFAT signaling cascade, and organonitrogen compound biosynthetic processes (Fig. 2C) (Supplementary Information 3).Moreover, within the M0 vs. M3 group, we discovered five statistically significant KEGG pathways (P < 0.05) related to muscle growth and development.These pathways include metabolic pathways, glycine, arachidonic acid metabolism, serine, and threonine metabolism, biosynthesis of amino acids, and focal adhesion (Fig. 2D) (Supplementary Information 4).The M0 vs. M6 group is also enriched in the ribosome, biosynthesis of amino acids, calcium signaling pathway, focal adhesion, and AMPK signaling pathway (Fig. 2E) (Supplementary Information 5).Interestingly, we identified differentially expressed genes (DEGs).One of these genes, OR2AP1 (gene ID: 100861186), was consistently found in all three groups (Supplementary Information 6).In the investigation comparing the M0 and M3 groups, MYL10 emerged as one of the top 50 differentially expressed genes (DEGs) (Supplementary Information 7).Intriguingly, MYL10 demonstrated active involvement in the focal adhesion signaling pathway, indicating its plausible influence on the regulation of skeletal muscle growth and development.In the first 50 DEGs of the M0 vs. M6 group, RYR3 exhibited significant involvement in the calcium signaling pathway (Supplementary Information 7), emphasizing its relevance to cell differentiation and muscle development.Meanwhile, CSRP3 was found to be highly expressed in the first 50 degrees of both M0 vs. M6 and M0 vs. M3 groups.This gene is associated with muscle development, muscle cell maintenance, transcription-related regulation, signal transduction, cytoskeletal organization, and tissue-specific cell growth and development.www.nature.com/scientificreports/

Prediction and functional analysis of differentially expressed miRNA target genes
Through miRNA target gene prediction using miRanda, we constructed a prediction of DEmiRNA-mRNA interactions.We identified a total of 162 mRNAs targeting 15 differentially expressed miRNAs in the three groups.Out of these 162 mRNAs, 13 were differentially expressed, and there was minimal overlap in the target genes that were differentially expressed.Among them, there were 15 miRNA-mRNA interaction pairs involving 5 miRNAs and 13 mRNAs (Fig. 5).Functional analyses of these differentially expressed target genes revealed that they were mainly enriched in the binding of various ions, including cations, metal ions, and transition metal ions, as well as in primary metabolic processes, protein binding, and oxidoreductase activity (Fig. 6).www.nature.com/scientificreports/

Gene network validation by quantitative real-time polymerase chain reaction (qRT-PCR)
We randomly selected three differentially expressed miRNAs and three differentially expressed mRNAs for qRT-PCR analysis.Results demonstrated that the gene expression trend in the two experimental groups was consistent between RNA-Seq and qRT-PCR methods, although the fold change of the expression level, to some extent, differed between the two sets of data (Fig. 7).It is proven that the transcriptome sequencing results are reliable and can be used for follow-up studies.

Discussion
In the past decade, high-throughput sequencing technology has become increasingly widespread in animal science, enabling the discovery of functional genes.This technique has been instrumental in advancing our understanding of miRNAs in studies on muscle growth and development in animals.It has also influenced studies conducted specifically in sheep and goats.MiRNAs, short for microRNAs, are small RNA molecules that www.nature.com/scientificreports/function as post-transcriptional regulators by selectively binding to complementary sequences within specific messenger RNAs (mRNAs).This binding often leads to mRNA degradation or translational repression 26 .Studies have shown that miRNAs play a crucial role in the development and differentiation of skeletal muscle, which is a highly coordinated biological process regulated by a network of myogenic transcription factors 27 .
Several studies have identified specific miRNAs that play a role in muscle growth and development in goats.For example, Lyu et al. 28 discovered that chi-miR-487b-3p regulates goat myoblast growth by targeting IRS1 and  affecting the PI3K/Akt signaling pathway, thereby inhibiting skeletal myogenesis.Liu et al. 29 established that miR-124a functions as a regulatory factor for VMP1 in muscle tissue.Modulating the expression of miR-124a, either through overexpression or inhibition, can exert effects on myoblast proliferation and autophagy.Shen et al. 30 conducted a comparison of transcriptome profiles in the dorsal longus muscle of Liaoning cashmere goats and Ziwuling black goats.They identified several genes that exhibited differences in skeletal muscle growth and development, such as PEBP1, SPARC, COL3A1, SOCS2, BTG2, and MB.Tripathi AK et al. 31 found that miR-1, miR-133b, miR-222, and miR-30a are involved in the specificity of muscle development and differentiation.Furthermore, Ling et al. 32 performed a comprehensive analysis of miRNA in goat fetuses and newborns at seven different stages.They conducted miRNA profiling and identified a total of 421 known miRNAs and 228 novel miRNAs, which provided valuable insights into the temporal expression patterns during skeletal muscle development in goats.These studies provide compelling evidence of the importance of miRNAs in coordinating muscle growth and development.
In order to further investigate the role of miRNAs in muscle growth and development in goats, we conducted transcriptome sequencing and carefully examined the different expression patterns of mRNAs and miRNAs in three distinct groups of Leizhou goats at 0, 3, and 6 months of age.In our study, we identified a number of valuable mRNAs and miRNAs that may play important roles in promoting and influencing muscle growth and development, thus shedding light on the complex mechanisms involved in this process.This is also important for improving the meat production of livestock in production practice.Meanwhile, this will provide a theoretical basis for the future elucidation of the molecular mechanisms of skeletal muscle development in Leizhou goats, and provide new ideas for the molecular breeding and development of Leizhou goats (Supplementary table 1).
Among the three groups, we observed significantly high expression levels of five muscle-specific miRNAs (miR-127-3p, miR-133a-3p, miR-193b-3p, miR-365-3p, miR-381).Situated within the Dlk1-Dio3 region's miRNA cluster, miR-127 has been reported to participate in the development of normal cell lineages, including embryonic stem cells 33 and fibroblasts 34 .However, it is worth noting that the miR-127 precursor can transcribe two distinct types of mature miRNAs, namely miR-127-3p and miR-127-5p 35 .In earlier investigations, miR-127-3p was found to be abundantly expressed in skeletal muscle, myocardial muscle of adult mice, and proliferating C2C12 cell lines.Moreover, it was shown to exert regulatory effects on muscle cell proliferation through its interaction with KMT5a 36 .In another study, it was observed that miR-127-3p induces myogenesis through its interaction with Vamp2.By transfecting miR-127-3p into C2C12 cells, researchers demonstrated that miR-127-3p directly targets Vamp2, thereby regulating myoblast proliferation and differentiation 35 .Additionally, it was discovered that miR-127-3p also affects myoblast proliferation by targeting Sept7 37 .In the investigation, we observed a differential expression of FHL3, a target of miR-127-3p, across the 0-6 months age range.Its expression consistently increases from 0 to 6 months.In C2C12 myoblasts, FHL3 was primarily localized in the nucleus.When integrin engagement occurs, it undergoes translocation to actin stress fibers and focal adhesions.Moreover, FHL3's ability to inhibit α-actinin-mediated actin bundling emphasizes its crucial role as a regulator of actin cytoskeletal dynamics in skeletal myoblasts 38 .Additionally, the interaction between FHL3 and MyoD may play a role in regulating MyoD-dependent transcription of muscle genes, thereby contributing to myogenesis 39 .Therefore, www.nature.com/scientificreports/we predict that miR-127-3p regulates myogenesis by targeting FHL3.We observed a significant increase in the expression of miR-133a-3p from month 0 to month 6 in DE-miRNAs (p < 0.05).By targeting PRRX1 expression, miR-133a-3p exerts a significant inhibitory effect on myogenic cell proliferation and promotes myogenic cell differentiation 40 .The Akt/mTOR/S6K pathway plays a crucial role in muscle growth and hypertrophy.The reduction of miR-133a-3p expression can stimulate muscle growth through the activation of the Akt/mTOR/S6K signaling 41 .According to our research, SESN1, which is a differentially expressed target gene of miR-133a-3p, is a stress-responsive protein involved in regulating inflammatory responses, muscle atrophy, reactive oxygen species, and oxidative stress 42 .Simultaneously, overexpression of SESN1 significantly increased the proliferation of myogenic cells, inhibited apoptosis, and promoted differentiation 43 .The expression of SESN1 from 0 to 6 months of age showed a tendency to increase and then decrease, which may be related to its involvement in muscle growth and differentiation.Therefore, we hypothesized that the overexpression of SESN1 in Leizhou goats after birth resulted in rapid proliferation of myoblasts and muscle growth, but this trend slowed down with increasing age.In chickens, PPARGC1A promotes intramuscular fatty acid oxidation, drives the conversion of fast-twitch to slow-twitch myofibers, and enhances skeletal muscle mass.Notably, miR-193b-3p is the key regulator that governs PPARGC1A expression 44 .Recent investigations have demonstrated that miR-193b-3p in goats enhances myoblast proliferation by activating insulin-like growth factor-2 mRNA-binding protein through binding to its 3' untranslated region (UTR).In our study, PACSIN3, the gene targeted by miR-193b-3p, is involved in vesicle formation and transport and is predominantly expressed in muscle tissue 45 .Recent studies have shown that PACSIN3 affects the differentiation of primary skeletal muscle cells by interacting with IL-6 in skeletal muscle cells 46 .Meanwhile, we found that another differentially expressed target gene of miR-193b-3p, LMCD1, also appears to be associated with muscle growth and differentiation.It has been found that LMCD1 can act as a gene closely related to skeletal muscle function.It inhibits the expression of muscle regulatory proteins while also increasing muscle protein synthesis and fiber size.LMCD1 is a positive regulator of muscle mass 2 .It has also been found that LMCD1 can enhance cell proliferation, glycolytic capacity, epithelial-mesenchymal transition (EMT), and lower the epigenetic barrier by up-regulating epigenetic factors (EZH2, WDR5, BMI1, and KDM2B) in the early stages of reprogramming, making cells more likely to acquire pluripotency 3 .Hence, we hypothesized that miR-193b-3p influences the differentiation and proliferation of skeletal muscle cells by targeting PACSIN3 and LMCD1.The expression level of miR-365-3p among differentially expressed miRNAs significantly increased from 0 to 6 months (p < 0.05).MiR-365-3p regulates skeletal muscle development by targeting SelT through muscle development-related factors, mitochondrial biosynthesis, and the calcium ion pathway 47 .In a study on primary bovine myoblast cell proliferation and differentiation, the upregulation of miR-365-3p significantly reduced the expression of cell cycle proteins D1 (CCND1), cyclin-dependent kinase 2 (CDK2), and proliferating cell nuclear antigen (PCNA).This reduction in expression led to the inhibition of cell proliferation but also facilitated myotube formation.It can be hypothesized that skeletal muscle development and muscle tube formation occur in Leizhou goats from 0 to 6 months.Among the miRNAs analyzed, miR-381 exhibited the most significant down-regulation among all the differentially expressed miRNAs in the three groups.In a study focused on the skeletal muscle of Liaoning cashmere goats, researchers observed that miR-381 exhibited the most significant down-regulation among the examined miRNAs.Moreover, miR-381 was found to significantly increase the expression levels of myosin heavy chain (MyHC), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C) in primary goat skeletal muscle satellite cells (SMSCs).This effect resulted in the promotion of myogenic differentiation in goat skeletal muscle stem cells (SMSCs).Recent studies on miRNAs in sheep skeletal muscle have shown that miR-381 targets several muscle-associated mRNAs, and the genes it targets are implicated in skeletal muscle development 48 .Therefore, miR-381 has the potential to regulate skeletal muscle development by targeting multiple myogenic mRNAs in Leizhou goats.Nevertheless, more research is required to elucidate the underlying mechanism.Within the group of identified differentially expressed genes (DEGs), MYL10 is a gene that encodes a protein.In livestock and poultry research focused on the molecular mechanisms of meat quality in crossbred sheep, researchers observed the involvement of MYL10 in regulating muscle growth and development.In a study utilizing whole-genome bisulfite sequencing and mRNA sequencing on cloned piglets, the examination of the tongue and biceps femoris muscles revealed that MYL10 plays a critical role in the development of embryos, as well as tissues and organs 49 .We observed a decrease in MYL10 expression in the M0 vs. M3 group.Through bioinformatics analysis, we found that MYL10 is primarily involved in calcium ion binding pathways.The expression of the RyR3 gene was found to be reduced in the M0 vs. M3 group and M0 vs. M6 group.In the adult diaphragm muscle, RyR3 accounts for 1-4% of the total ryanodine binding sites 50 , while in most adult mammalian fast-twitch muscles, it constitutes only 0-0.2% of the total 22,51 .Neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane.A study has revealed that RyR3 is strengthened through calcium-induced calcium release (CICR), which leads to strong, uniform, and simultaneous activation of Ca (2 +) release throughout the cell body.This mechanism plays a critical role in Ca (2 +) signaling for cell differentiation and muscle contraction 52 .In mice, during the neonatal stage of diaphragm muscle development (2-15 days after birth), RyR3 was found to be more highly expressed compared to the same muscle in adult mice.This observation indicates a preference for RyR3 expression in differentiated skeletal muscle cells 53 .Functional analysis indicated that RyR3 is involved in the KEGG pathway "Calcium signaling pathway."Based on our findings, we hypothesize that RyR3 plays a role in the skeletal muscle development and differentiation of Leizhou goats.CSRP3, a member of the cysteine-rich protein family, belongs to the group of muscle-specific LIM-only factors and shows specific expression in skeletal muscle.In chicken primary muscle cells, siRNA-mediated CSRP3 knockout demonstrated that silencing CSRP3 resulted in the downregulation of myogenic genes and the upregulation of atrophy-related genes 54 .In livestock and poultry research, the inhibition of CSRP3 through knockdown has been found to suppress chicken satellite cell differentiation by regulating Smad3 phosphorylation in the TGF-β signaling pathway 55 .The expression of

Figure 5 .
Figure 5. Interaction diagram of differentially expressed miRNA targeted differentially expressed mRNA.

Figure 6 .
Figure 6.GO enrichment analysis of differentially expressed target genes.
The animal study received approval from the Institutional Animal Care and Use Committees of South China Agricultural University (Permit number: 2018-P002).All experimental procedures and sample collection methods adhered to the Regulation on the Administration of Laboratory Animals (CLI.2.293192, 2017 Revision, State Council, China).

Table 1 .
Summary of clean reads mapped to the Capra hircus reference genome.