Transcriptome-Based Identification of the Muscle Tissue-Specific Expression Gene CKM and Its Regulation of Proliferation, Apoptosis and Differentiation in Chicken Primary Myoblasts

Simple Summary The study of skeletal muscle growth and development has historically been viewed as an entry point and as a breakthrough event in the improvement of animal breeding in the livestock industry. Muscle is the major constituent of any meat product, and its growth performance and the resulting product quality are the core foci of modern breeding. In this study, we found that creatine kinase M-type-like (CKM) was specifically highly expressed in chicken muscle tissue. The quantitative real-time PCR (qPCR) results were consistent with the sequencing results. By overexpressing CKM and interfering with CKM expression in chicken primary myoblasts, we found that CKM could inhibit the proliferation and promote the apoptosis and differentiation of chicken primary myoblasts. Moreover, the transcriptome sequencing results after interference with CKM expression indicated that CKM is involved in chicken skeletal muscle development. These results suggest that CKM may play a crucial role in chicken muscle growth and development. Abstract Skeletal muscle is an essential tissue in meat-producing animals, and meat-producing traits have been a hot topic in chicken genetic breeding research. Current research shows that creatine kinase M-type-like (CKM) is one of the most abundant proteins in skeletal muscle and plays an important role in the growth and development of skeletal muscle, but its role in the development of chicken skeletal muscle is still unclear. Via RNA sequencing (RNA-seq), we found that CKM was highly expressed in chicken breast muscle tissue. In this study, the expression profile of CKM was examined by quantitative real-time PCR (qPCR), and overexpression and RNA interference techniques were used to explore the functions of CKM in the proliferation, apoptosis and differentiation of chicken primary myoblasts (CPMs). It was shown that CKM was specifically highly expressed in breast muscle and leg muscle and was highly expressed in stage 16 embryonic muscle, while CKM inhibited proliferation, promoted the apoptosis and differentiation of CPMs and was involved in regulating chicken myogenesis. Transcriptome sequencing was used to identify genes that were differentially expressed in CPMs after CKM disruption, and bioinformatics analysis showed that CKM was involved in regulating chicken myogenesis. In summary, CKM plays an important role in skeletal muscle development during chicken growth and development.


Introduction
The proper development of skeletal muscle in meat-producing animals is positively correlated with increased meat production, making it an economically important tissue; in addition, skeletal muscle plays an important role in initiating locomotion, supporting respiration, and maintaining homeostasis in vivo [1]. Skeletal muscle is composed of a series of muscle fibers made up of multinucleated muscle cells and is formed during development by the fusion of several myoblasts into multinucleated myotubes [2]. Therefore, the study of the proliferation and differentiation mechanisms of myoblasts has become a hot topic in scientific research [3]. Muscle development is generally divided into the embryonic and postnatal stages [4]. In the embryonic stage, muscle progenitor cells differentiate and proliferate to form myoblasts that fuse into multinucleated myotubes, which later mature into muscle fibers with contractile properties [5]. Moreover, the deposition of many substances related to the flavor of meat products begins during the embryonic period [6]. The development of muscle after birth depends on the proliferation and differentiation of myoblasts and the function of muscle satellite cells [7]. Among other events, the morphological structure and number of muscle fibers are finalized during the embryonic period, highlighting the need to explore embryonic muscle development in poultry [8].
Recent studies have shown that many genes are involved in the regulation of chicken skeletal muscle growth and development and that understanding these functional genes is important for breeding animals with a high muscle yield and quality [9,10].
Creatine kinase (CK) exhibits isoenzyme-specific subcellular localization at sites of energy production and consumption. Cells and tissues with intermittent high energy needs, such as bone, myocardial, brain, retinal and sperm cells and tissues, require large amounts of immediately available energy, and creatine kinase plays a key role in the cellular energy metabolism in each of these cells [11]. CK is also required for the synthesis of adenosine triphosphate (ATP), which provides energy for muscle contraction. ATP is replenished by the reversible transfer of an N-phosphoryl group from phosphocreatine to adenosine triphosphate (ADP) [12]. CK constitutes a unique family of oligomeric isoforms with tissue-specific expression and isozyme-specific subcellular localization. There are three cytoplasmic isoforms, namely, the ubiquitous brain-type CK isoform (CK-BB), muscle-type CK isoform (CK-MM, encoded by the CKM gene) and CK-MB heterodimer [13], and two mitochondrial isoforms, the ubiquitous CK-MM, encoded by the CKM gene (Mia-CK), and Mib-CK [14].
In fully differentiated skeletal muscle, CK-MM is abundant, along with mitochondrial Mib-CK [15]. In chickens, the creatine kinase M-type-like isozyme (LOC107051134, CK-MM, Gallus gallus; also called CKM) is specifically expressed in skeletal muscle. In humans, CKM is specific to skeletal muscle and is used as a marker for muscle damage [16], muscular dystrophy [17], infectious diseases [18], acute renal failure, myocardial infarction [19], rheumatoid arthritis [20], and some liver diseases [21]. Previous studies have shown that the ATP synthesis capacity is significantly decreased in the muscle tissue of CKMknockout mice [22]. Xiong et al. found that CKM may be related to the differentiation of myoblasts [23].
Many studies have shown that the CKM gene plays an important role in the development of skeletal muscle, but its function in chicken myogenesis is unclear. In this study, the potential regulatory gene CKM, which is specifically highly expressed in muscle tissues, was identified through the integrated analysis of RNA sequencing (RNA-seq) data from 21 tissues, and the effects of CKM on the proliferation, apoptosis and differentiation of chicken myoblasts were investigated using overexpression and interference techniques.

Ethics Approval
In this study, animal experiments and nursing were carried out according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, Beijing, China, 2004), and the experimental scheme was approved by the Animal Care and Use Committee (IACUC) of Henan Agricultural University (approval number: 11-0085). We conducted this study in strict accordance with the ARRIVE guidelines.

Sample Collection
A total of 20 fertilized eggs of Arbor Acres (AA) chickens at 11 embryonic ages (E11, the eleventh day after incubation in an incubator) were provided by Henan Poultry Industry Limited Liability Company (Zhengzhou, China) for the cell culture. The breast muscle and leg muscle tissues of E10, E12, E14, E16, 1-week-old and 3-week-old AA chickens were collected, eight in each period. At the same time, the heart, liver, spleen, lung, kidney, gizzard and ileum tissues of eight AA chickens aged 1-week-old were also collected. Our method of euthanasia for chickens is cervical dislocation. All tissue samples were frozen in liquid nitrogen and placed in a −80 • C freezer for later use.

Rapid Amplification of cDNA Ends (RACE)
Total RNA was first extracted from 1-day-old AA chicken leg muscle, and subsequently, Rapid amplification of cDNA End(RACE PCR) was performed on the CKM gene using the TAKARA SMARTer (TaKaRa, Tokyo, Japan) 5 and 3 RACE kits, according to the manufacturer's instructions. The primer sequences are shown in Table S2.

Plasmid Construction and Small Interfering RNA (siRNA) Synthesis
The coding sequence (CDS) fragment of the CKM gene sequence was obtained as described above, and the pcDNA3.1 vector (Promega, Madison, WI, USA) was used to construct the overexpression plasmid by the insertion of the CDS into the HindIII and EcoRI restriction sites. The siRNA was designed and synthesized by GenePharma (Shanghai, China), and the primer sequences are shown in Table S2.

Isolation, Culture and Transfection of Chicken Primary Myoblasts (CPMs)
CPMs were isolated from the leg muscle of AA chickens at E11, as previously described [24]. CPMs were cultured in high-sugar Dulbecco's modified Eagle medium (DMEM) (Biological Industries, Israel) containing 15% fetal bovine serum (FBS) (Biological Industries, Israel). Then, the cells were placed in an incubator at 37 • C and 5% CO 2 and used for subsequent experiments when the cells were fully grown. For the establishment of the induced differentiation model of CPMs, the cell culture medium was changed to a differentiation medium containing 2% horse serum (Biological Industries, Beit HaEmek, Israel) when the cells reached 95% confluence, and the culture was continued. When CPMs were 95% confluent after differentiation, they were transfected with PC3.1-CKM and PC3.  The total RNA from cells and tissues was extracted using RNA TRIzol (TaKaRa, Japan), according to the manufacturer's instructions. The quality of the total extracted RNA was determined by agarose gel electrophoresis, and the concentration of total RNA was measured using a spectrophotometer (Thermo, Waltham, MA, USA). This was followed by cDNA synthesis using a PrimeScriptTM RT Reagent Kit with a gDNA Eraser (TaKaRa, Japan). Then, qPCR was performed using an SYBR Premix Ex Taq kit (TaKaRa, Japan) on an ABI 7500 instrument (Applied Biosystems, Waltham, MA, USA). The 2 −∆∆Ct method was used for the relative quantification of genes, using GAPDH as the internal reference gene. The primer sequences used for qPCR are shown in Table S2.

Cell Counting Kit-8 (CCK-8) Assay
CPMs were seeded in 96-well cell plates, 10 µL of CCK-8 reagent was added to each well at 10, 22 and 34 h after transfection and incubation was continued in an incubator. After 2 h of incubation, the absorbance values at 450 nm were obtained using a multifunctional microplate reader (BioTek, Winooski, VT, USA) to determine cell viability. Six biological replicates were used for each group.

5-Ethynyl-2 -Deoxyuridine (EdU) Incorporation Assay
CPMs were cultured in 24-well cell plates. Then, 22 h post-transfection, a prepared 50 nM EdU staining solution was added, and the plates were incubated for 2 h. Subsequent processing was performed according to the instructions of the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China), and, finally, a fluorescence inverted microscope (Olympus, Tokyo, Japan) was used to acquire images in three random fields of view. Then, the data were analyzed using ImageJ software (version 6.0) (NIH, Bethesda, MD, USA).

Flow Cytometry Analyses
Cell cycle analysis: CPMs were cultured using six-well cell plates, and after 24 h of transfection, the CPMs were harvested, fixed with precooled 75% ethanol and subsequently stored overnight at 4 • C. The RNase A:PI working solution was prepared as a staining solution and was added to CPMs at a volume ratio of 1:9 and incubated for 30 min.
Apoptosis analysis: CPMs were seeded in six-well cell plates after 48 h of transfection. CPMs were collected and subsequently processed according to the Annexin V-FITC apoptosis assay kit (Beyotime, Shanghai, China) instructions.
Finally, cells from both of these assays were analyzed by a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), with three biological replicates per group.

Immunofluorescence Analysis
CPMs were seeded in 12-well cell plates, and after 48 h of transfection, the cells were fixed for 20 min at 4 • C using a cell fixative (Solarbio, China) and washed twice with PBS (Solarbio, Beijing, China) for 5 min each. Next, 1 mL of 0.1% Triton X-100 was added to each well, the membrane was permeabilized at room temperature for 10 min and the cells were then washed with PBS, as described previously. Goat serum (Bioss, Beijing, China) was then added for 1 h at room temperature, and anti-Desmin or anti-MYHC (DHSB, Iowa City, IA, USA) was then added prior to incubation at 4 • C overnight. After removal, the next day, the cells were washed three times with PBS and then incubated with anti-mouse IgG FITC-conjugated antibodies (Bioss, China) or anti-Cy3 (Proteintech, Wuhan, China) for 2 h at 37 • C in a dark incubator. Finally, three random fields of view were imaged using an inverted fluorescence microscope (Olympus, Japan) and incubated for 3 min. ImageJ software (version 6.0) (NIH, Bethesda, MD, USA) was used for statistical analysis.

Western Blotting (WB)
CPMs were seeded in six-well cell plates, and after 48 h of transfection, proteins were extracted from the CPMs using a cell extraction kit (Bioss, China) and quantified with a bicinchoninic acid (BCA) protein assay kit (Epizyme, Tianjin, China). The proteins were denatured by adding loading buffer at 100 • C for 10 min and were then stored at −20 • C. Electrophoresis was performed on a 10% polyacrylamide gel (Epizyme, China), and the proteins were transferred to a PVDF membrane (Beyotime, China) prior to blocking with 5% skim milk powder (Yili, Hohhot, China) for 1 h. Anti-MYHC (DHSB, USA) and antiβ-actin (Proteintech, China) antibodies were added, and the membrane was incubated overnight at 4 • C. The next day, the membrane was incubated with an HRP-conjugated secondary antibody (Proteintech, China) for 1 h 30 min at room temperature. Finally, an ECL chromogenic kit was used for color development, images were acquired using an Odyssey Fc Imager (version 5.2.5) (LI-COR, Lincoln, NE, USA) and ImageJ software was used for analysis (NIH, Bethesda, MD, USA). β-actin was used as an internal control.

RNA-seq and Differential Expression Genes (DEGs) Analysis
CPMs were isolated, cultured in vitro and transfected with si-CKM and si-NC for 48 h; the total RNA was then extracted using TRIzol (TaKaRa, Japan) prior to transcriptome library construction. mRNA was purified from the total RNA by oligo magnetic beads and the mRNA was randomly fragmented by adding fragmentation buffer. First-strand cDNA was synthesized by using six-base random primers and reverse transcriptase; second-strand cDNA was then synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. The purified double-stranded cDNA was then end-repaired, A-tailed and ligated to sequencing junctions, and then the library fragments were purified. Finally, the cDNA library was enriched by PCR. The library was evaluated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (effective concentration >2 nM) and sequenced using the Illumina NovaSeq 6000 platform.
Subsequently, quality control was performed on raw reads, using FASTQ to filter lowquality data in order to obtain clean reads. The reference genome Gallus gallus 6.0 and pairedend clean reads were mapped to the reference genome using HISAT2 (version 2.1.0). For each transcribed region, the fragments per kilobase million (FPKM) values were calculated using StringTie software (version 2.2.0) to quantify their expression abundance. Differential expression analysis of the samples was performed using DESeq2 (version 1.40.2) and edgeR software (version 2.4), and we identified genes with a false discovery rate (FDR) <0.05 and |log2 fold change| >1 as DEGs. Our analysis of the DEGs associated with 169 transcriptome data downloaded from the NCBI database is the same as above.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Analyses
GO analysis was used to screen for differentially expressed genes to predict potentially affected biological processes and functions. KEGG pathway analysis was used to determine the functions of molecular pathways potentially involving the differentially expressed genes. The statistical significance was set at p < 0.05.

Statistical Analysis
All data are expressed as the mean ± standard error of the mean (SEM) values. Student's t test was performed on the data using IBSS SPSS (SPSS for Windows, standard version 24.0; SPSS, Chicago, IL, USA). Differences were considered significant when 0.01 ≤ p ≤ 0.05 and highly significant when p < 0.01 (* p < 0.05; ** p < 0.01, *** p < 0.001). The calculated data were visualized and analyzed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). All data are presented as the results of three technical and biological replicates.

Identification and Analysis of Chicken Tissue-Specific Genes
A total of 24,403 genes were detected by integrating and analyzing the collected datasets for tissue-specific high-expression gene screening, with an FPKM value greater than 1000. Statistical analysis revealed the number of genes specifically highly expressed in tissues including the breast muscle, heart muscle, gizzard fat, liver, pancreas, proventriculus and skin ( Figure 1A, Table S3). These tissue-specific highly expressed genes are important for identifying gene function and expression characteristics in various tissues [25,26]. As meat production traits are the most economically important traits in broiler production, we focused on genes that are specifically expressed in muscle tissue. We identified a gene called CKM (LOC107051134) that was specifically highly expressed in chicken breast muscle tissue ( Figure 1B).

Expression Pattern of CKM in Chickens
Based on the known gene sequence information, amplification was performed using the RACE technique, and a product band of approximately 1.0 kbp in length was found to be amplified by 3 PCR (Figure 2A), yielding 674 bp of an unknown sequence; the 5 PCR amplification product had a length of 0.6 kbp ( Figure 2B), yielding 395 bp of an unknown sequence. The RACE results confirmed that the length of the PCR amplification product was consistent with the expected length (approximately 1.3 kbp) of size for the target fragment ( Figure 2C). The full-length sequence of 1321 bp was obtained by sequencing, and the sequence obtained by 3 RACE and 5 RACE was verified to have an A-to-G synonymous mutation at position 381 in the NCBI sequence of CKM ( Figure S1). NCBI BLAST analysis showed that CKM was indeed present in the chicken genome. We further explored the expression pattern of CKM in AA chickens by qPCR and semi-quantitative assays, showing that the CKM showed specific high expression in the breast and leg muscles of 1-week-old AA chickens ( Figure 2D,E). However, it was not expressed or not highly expressed in the heart, liver and spleen (they may exhibit other muscle subtypes of CK), which is consistent with the sequencing data results, further suggesting that the CKM may be associated with the growth and development of chicken skeletal muscle. In addition, we found that the expression of CKM was gradually upregulated during the development of breast and leg muscles in chicken embryos until E16 reached the peak, while the expression level after hatching showed a downward trend ( Figure 2F,G). Therefore, we speculated that CKM may be involved in embryonic muscle development.

CKM Inhibits the Proliferation of CPMs
We first validated the purity of the CPMs isolated from the leg muscles of 11-day-old AA chickens by an Desmin immunofluorescence assay ( Figure S2). To further explore the role played by the CKM in the proliferation of CPMs, we transfected the CKM overexpression vector and control vector, as well as si-CKM and si-NC, into CPMs and examined the relative expression of mRNA using qPCR. The results in Figure 3A show that the overexpression of CKM resulted in a highly significant difference in mRNA expression levels compared to the control (p < 0.01). In addition, the results in Figure 3B show that our designed CKM interference fragment resulted in a highly significant reduction in CKM gene expression compared to the control group (p < 0.01); thus, both the CKM overexpression vector and the interference fragment were used for further experiments. Subsequently, we transfected the CKM overexpression vector and measured the expression of genes capable of regulating cell cycle-related factors, such as cyclin B2 (CCNB2), cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA) and cyclin kinase inhibitor (P21). The results in Figure 3C show that CCNB2, CCND1, and PCNA expression was significantly downregulated (p < 0.05), while P21 expression showed highly significant upregulation (p < 0.01) after the overexpression of CKM compared to the control group. In addition, the expression of proliferation-related factors (CCNB2, PCNA, CCND1) was significantly upregulated (p < 0.05), and the expression of the cell proliferation factor P21 was significantly downregulated (p < 0.05) after transfection with si-CKM compared to that after transfection with si-NC ( Figure 3D). In addition, as shown in Figure 3E, the results of the CCK-8 assay showed that the overexpression of CKM decreased the viability of myoblasts; the inhibition rate increased with an increasing treatment time, and CKM overexpression extremely significantly inhibited the proliferation of myoblasts at 24 h and 36 h (p < 0.01) compared to that in the control group. Moreover, the results of the EdU incorporation assay ( Figure 3G) indicated that overexpression of the CKM gene was able to inhibit the proliferation of myoblasts. In contrast, the CCK-8 and EdU incorporation assay results showed that interference with CKM expression significantly promoted the proliferation of myoblasts ( Figure 3F,H). In addition, as shown in Figure 3I,J, the cell cycle analysis results showed that, after CKM overexpression, the proportion of cells in the G1 phase was increased compared to that in the control group, and the proportion of cells in the S phase was lower than that in the control group, indicating that cell division was inhibited after CKM overexpression. The proportion of cells in the G1 phase was significantly higher (p < 0.05) than that in the control group. Taken together, these results suggest that CKM can inhibit the proliferation and promote the apoptosis of CPMs.

CKM Promotes Apoptosis in CPMs
We further examined the effect of CKM on apoptosis in CPMs. First, we examined the changes in the relative mRNA expression of the apoptosis-related factor cystatin protease 3 (Caspase3) and the apoptosis-related factor cystatin protease 9 (Caspase9) by qPCR. The results in Figure 4A show that Caspase3 expression was very significantly upregulated (p < 0.01) and Caspase9 expression was significantly upregulated (p < 0.05) after the overexpression of CKM compared to the control group. Moreover, the transfection of si-CKM significantly downregulated (p < 0.05) the expression of the apoptosis-related factors Caspase3 and Caspase9 compared with that in the si-NC group ( Figure 3B). In addition, as shown in Figure 4C,D, the results of the apoptosis analysis by flow cytometry showed that the number of apoptotic cells was significantly increased after the overexpression of CKM compared to that in the control group (p < 0.01) and was significantly lower after interference with CKM (p < 0.01) than that in the control group. In summary, these results suggest that CKM can promote apoptosis in CPMs.

CKM Promotes the Differentiation of CPMs
To further determine the role of CKM in CPMs differentiation, we first used qPCR to measure the mRNA expression levels of CKM and myosin heavy chain (MYHC) in the CPMs proliferation and differentiation model established earlier. As shown in Figure 5A,B, the overall trends in CKM and MYHC expression were consistent, first decreasing, then increasing and then decreasing, with the lowest expression on day 1 of differentiation. Both expression levels showed an increasing trend upon cell entry into the redifferentiation stage, indicating that CKM is involved in the differentiation of myoblasts. Then, we used qPCR to measure the changes in the mRNA expression of marker genes related to cell differentiation, including a myoblast factor (MYOD), myogenin (MYOG) and MYHC, after transfection with the CKM overexpression vector ( Figure 5C). The results in Figure 5D show that interference with CKM significantly decreased the mRNA expression levels of MYOD and MYHC (p < 0.05) and very significantly decreased the mRNA expression level of MYOG (p < 0.01). In addition, after the transfection of the CKM overexpression vector, we evaluated the differentiation degree of CPMs using MYHC immunofluorescence staining ( Figure 5E). The results showed that the overexpression of CKM significantly promoted the expression of the protein encoded by MYHC, thus promoting the differentiation of CPMs. Moreover, the opposite effect was observed by MYHC immunofluorescence staining after interference with CKM ( Figure 5F). Furthermore, we examined the protein expression levels of CKM and MYHC by WB. As shown in Figure 5G, the expression of the proteins encoded by both CKM and MYHC was highly significantly upregulated after the overexpression of CKM (p < 0.01, Figure 5H,I). In contrast, the levels of the proteins encoded by both CKM and MYHC were significantly decreased after disrupting the CKM expression (p < 0.05, Figure 5J,K). At the translational level, the changes in CKM expression were consistent with the changes in the expression of the differentiation marker MYHC, which more fully shows that the overexpression of CKM could promote myoblast differentiation, while interference with CKM expression showed the inverse effect. In summary, these results suggest that CKM can promote the differentiation of CPMs.

RNA-seq Analysis of Chicken Primary Myoblasts after CKM Interference
To further explore the molecular mechanism of the CKM regulation of chicken growth and development, we used transcriptome sequencing technology to compare the differentially expressed genes in primary CPMs treated with si-CKM and si-NC by bioinformatic methods. First, the results of the principal component analysis (PCA) are shown in Figure 6A. The two treatment groups can be clearly distinguished, and the si-CKM interference group (Group 1) and the si-NC interference control group (Group 2) are clustered together; therefore, the trends in the measured samples remain consistent. This pattern further indicates the reliability of the samples and ensures the accuracy of subsequent tests. There were 439 differentially expressed genes in the si-CKM group compared to those of the si-NC group, with 81 upregulated genes and 357 downregulated genes, as shown in the volcano plot ( Figure 6B). The results of the GO term analysis of the differentially expressed genes after CKM interference showed that the molecular functions of the genes included skeletal muscle fiber adaptation, the positive regulation of sarcomere tissue and myoblast differentiation ( Figure S3A). The cellular components related to these genes included the Z-line (a disc-like structure at the junction of two sarcomeres adjacent to myofibrils, which is the location of α-actinin and is connected with fine myofilaments); cardiac myofibrils; myofibrils and other cellular components ( Figure S3B). The biological processes included creatine kinase activity, actin binding and calcium-dependent protein kinase activity ( Figure S3C). In addition, MAPK signaling pathways, the regulation of the actin cytoskeleton and amino acid metabolism and biosynthesis, which are related to the regulation of skeletal muscle development, were the main KEGG pathways enriched in the differentially expressed genes ( Figure S4).
In addition, we analyzed and screened differentially expressed genes associated with important GO terms and KEGG pathways based on our sequencing results and references to other studies on skeletal muscle development. Based on the available literature, we identified highly significantly differentially expressed genes that regulate myoblast development and participate in the muscle growth process, including CAPN3 [27], PDK4, RBM24 [28], SMYD1 [29], KLHL40 [30], KLHL41 [31], STAC3 [32], FAM65B [33], LMOD3 [34], ANKRD1 [35], EGF [36] and CAMK2A [37] (p < 0.01, Figure 6C). Subsequently, we screened the differentially expressed genes with |log2 fold change| ≥1.5 and randomly selected four genes that were upregulated (FST, NOX3, PDK4 and S100A2) ( Figure 6D-G) and four that were downregulated (CAPN3, MYL10, MYL1 and MYOD1) ( Figure 6H-K) for qPCR validation. The results were consistent with the RNA-seq results, demonstrating the accuracy of the sequencing data and showing that the expression of these highly significantly downregulated differentiation-related genes was reduced after interference with CKM expression. In conclusion, these results indicate that CKM plays an important role in the development of CPMs.

Discussion
Recently, several studies have been conducted at the transcriptional level in poultry to investigate and analyze the genes and regulatory pathways that influence skeletal muscle growth and development; these studies have helped to reveal genes and pathways associated with muscle development by comparing gene expression profiles for specific traits in various animal populations, thereby facilitating the selection of candidate genes associated with important traits [38]. To date, RNA-seq has been used to discover and study specific genes and pathways for muscle development under different conditions in animals such as chickens [39] and ducks [40]. Wang et al. found that CKM may be a key gene involved in the regulation of bovine skeletal muscle development through multi-omics analysis [41]. Bouwman F.G. et al. have shown that a subtype of CKM is a new type of fatigue biomarker in horses, which indicates that the change in energy distribution in muscle cells is part of the etiology of the disease [42]. However, there have been fewer studies on skeletal muscle development in chickens. Therefore, in this study, based on the integrated analysis of expression data from different tissues of chickens, we screened for a high expression of CKM specifically in muscle tissues and performed qPCR in various tissues of 1-week-old AA chickens, and we found that CKM showed specifically high expression in muscle tissues. However, muscle development occurs in two main stages: embryonic and postnatal. Muscle fibers are formed during the embryonic stage, and their number is maintained after birth [43]. We found that CKM expression was the highest at E16 during the development of breast muscle and leg muscle. At the same time, studies have shown that CKM is involved in the gene regulation of skeletal muscle development and has an important impact on muscle differentiation [41,44]. In addition, CKM levels are altered in response to muscle damage and are therefore used as a marker to identify muscle integrity [16]. This finding led to the hypothesis that CKM is involved in skeletal muscle development and may play a major role in muscle development during the embryonic p stage in chickens.
Numerous studies have shown that RNA overexpression interference can be used as a means to manipulate gene expression in experiments and investigate gene function on a genome-wide scale [45]. Recently, it has been found that the expression of genes associated with muscle development can influence the development of muscle and that the degree of active gene expression reflects the progress of muscle development [46]. Examples of such genes include CCNB2 [47], CCND1 [48], PCNA [49], P21 [50], Caspase3 [51,52] and Caspase9 [53], which instruct myoblasts to exit the cell cycle, express muscle-specific genes and block the expression of other cell-or tissue-specific genes. Therefore, in this study, by overexpressing and interfering with CKM gene expression, we found that CKM could significantly promote the downregulation of proliferation-related genes (CCNB2, CCND1 and PCNA), promote the upregulation of the antiproliferative gene P21, promote the regulation of apoptosis-related genes (Caspase3 and Caspase9), reduce cell viability, decrease the proportion of cells entering the S phase and decrease the cell proliferation rate. In summary, we found that CKM can inhibit myoblast proliferation and promote myoblast apoptosis.
Skeletal muscle differentiation is a multistep process in which myoblasts fuse to give rise to muscle fibers, which are tubular multinucleated fibers containing the contractile machinery necessary for muscle contraction [8]. One study revealed that porcine MYHC IIA/X-AS stimulates skeletal satellite cells to exit the cell cycle to promote fusion with myotubes [54]. Therefore, to further investigate the role of CKM in the differentiation of chicken myoblasts, we first analyzed the expression pattern of MYHC and CKM during myoblast differentiation. We showed that MYHC expression in myoblasts tended to increase and then decrease with time after the initiation of differentiation and that the changes in CKM expression during the differentiation period were consistent with those in the expression of MYHC, the differentiation marker gene. In addition, the expression levels of differentiation-related marker genes (MYOD, MYOG and MYHC) in CPMs showed a highly significant increase after the overexpression of CKM, with the opposite result observed after the interference of CKM. This result was also confirmed by the detection of MYHC protein expression levels. Myogenesis is the differentiation process that drives the formation of multinucleated myotubes. However, cell proliferation and phenotypic differentiation are mutually exclusive events [55]. Initially, myoblasts fuse to form small nascent myotubes, which subsequently fuse with other myoblasts to form large, mature myotubes [56]. Therefore, the detection of the differentiation status of myoblasts by MYHC immunofluorescence showed a significant increase in the number of myotubes formed after the overexpression of CKM, with the inverse result after interference with CKM. It has been shown that the CKM promoter has transcriptional initiation activity only during the induced differentiation of myoblasts, and it is speculated that CKM is associated with myoblast differentiation [23], which is consistent with our findings. In summary, CKM can promote the differentiation of myoblasts, providing new evidence for further revealing the regulatory mechanism of muscle development and mass increase.
To elucidate the molecular regulatory mechanism of CKM function in myoblasts, we performed transcriptome sequencing on chicken myoblasts with disrupted CKM expression, endeavoring to provide useful references for studies on muscle growth and development in livestock. The results showed that 438 genes were differentially expressed, of which 81 were upregulated and 357 were downregulated. Interestingly, as we expected, CKM was one of the top five downregulated genes. Moreover, the sequencing results were examined using qPCR and revealed that the expression levels of NOX3, PDK4, FST and S100A12 were significantly higher in the interference CKM group compared to those in the control group, while the expression levels of CAPN3, MYL10, MYL1 and MYOD1 were reduced, consistent with and indicating the reliability of the sequencing results. In addition, we performed GO enrichment analysis of these differentially expressed genes and found that they were enriched mainly in skeletal muscle fiber development, the regulation of myoblast differentiation, creatine kinase activity and other processes. In addition, KEGG pathway analysis showed that the differentially expressed genes were enriched mainly in the MAPK signaling pathway [57,58], the calcium signaling pathway [59], the regulation of actin cytoskeleton, the amino acid metabolism, biosynthesis and other related signaling pathways. In conclusion, these differentially expressed genes were enriched mainly in pathways related to skeletal muscle development, thus suggesting that CKM may play an important role in skeletal muscle development.

Conclusions
In summary, we identified a gene, CKM, that was specifically highly expressed in chicken muscle tissue. CKM can inhibit the proliferation of CPMs and promote their apoptosis and differentiation. The transcriptome sequencing results showed that interference with the CKM gene identified 12 genes that may regulate the development of CPMs and participate in the muscle growth process with significant differential expression; these differentially expressed genes were enriched mainly in pathways related to skeletal muscle development. These results suggest that CKM plays an important role in chicken muscle growth and development.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13142316/s1, Figure S1: CKM gene sequence alignment; Figure S2: Identification of CPMs; Figure S3: GO-annotated categorical statistics of differentially expressed genes after the transfection of si-CKM and si-NC; Figure S4: Scatter plot of the KEGG pathway enrichment of differentially expressed genes after transfection with si-CKM and si-NC; Table S1: Information on 169 RNA sequencing datasets from 21 chicken tissues; Table S2: Primers list;  Table S3: Chicken tissue-specific highly expressed genes.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data in this study are included in the article content as well as in the Supplementary Materials. Transcriptome data have been uploaded to the NCBI database sequence read archive, accession number PRJNA993231.

Conflicts of Interest:
The authors declare no conflict of interest.