SOX6 AU controls myogenesis by cis-modulation of SOX6 in cattle

ABSTRACT

Long non-coding RNAs (lncRNAs) have been shown to be involved in the regulation of skeletal muscle development through multiple mechanisms. The present study revealed that the lncRNA SOX6 AU (SRY-box transcription factor 6 antisense upstream) is reverse transcribed from upstream of the bovine sex-determining region Y (SRY)-related high-mobility-group box 6 (SOX6) gene. SOX6 AU was significantly differentially expressed in muscle tissue among different developmental stages in Xianan cattle. Subsequently, knockdown and overexpression experiments discovered that SOX6 AU promoted primary skeletal muscle cells proliferation, apoptosis, and differentiation in bovine. The overexpression of SOX6 AU in bovine primary skeletal muscle cells resulted in 483 differentially expressed genes (DEGs), including 224 upregulated DEGs and 259 downregulated DEGs. GO functional annotation analysis showed that muscle development-related biological processes such as muscle structure development and muscle cell proliferation were significantly enriched. KEGG pathway analysis revealed that the PI3K/AKT and MAPK signaling pathways were important pathways for DEG enrichment. Notably, we found that SOX6 AU inhibited the mRNA and protein expression levels of the SOX6 gene. Moreover, knockdown of the SOX6 gene promoted the proliferation and apoptosis of bovine primary skeletal muscle cells. Finally, we showed that SOX6 AU promoted the proliferation and apoptosis of bovine primary skeletal muscle cells by cis-modulation of SOX6 in cattle. This work illustrates our discovery of the molecular mechanisms underlying the regulation of SOX6 AU in the development of beef.

KEYWORDS:

• Bovine primary skeletal muscle cells
• SOX6 AU
• proliferation
• apoptosis
• SOX6
• cis-modulation

Introduction

Beef cattle are an important animal husbandry resource in China. The important economic traits of beef cattle mainly include their growth and meat quality traits, which directly affect the yield and quality of beef cattle and are crucial to the physical growth and development of beef cattle [1]. The growth and development of bovine skeletal muscle is a complex and orderly process, that mainly depends on the proliferation and hypertrophy of muscle fibres, including the formation, proliferation, and differentiation of muscle cells, as well as the fusion of myotubes and muscle fibres, ultimately leading to the development of contractile muscle [2–4]. Skeletal muscle growth and development are regulated by a series of key genes. Among these key genes, lncRNAs have been found to be involved in various stages of muscle development, and regulate muscle development through multiple mechanisms.

LncRNAs are defined as transcripts with no or little protein-coding capacity and a length of more than 200 nt [5]. LncRNAs are known to be involved in a variety of cell biological processes, including immune inflammation [6], cell differentiation and development [7], cell proliferation [8] and apoptosis [9]. Notably, an increasing number of studies have reported that lncRNAs play important regulatory roles in skeletal muscle development. For instance, the lncRNA Dum was discovered and characterized in mouse myoblast differentiation, and Dum is transcriptionally induced by myogenic determination (MYOD). Mechanistically, Dum interacts with the developmental pluripotency-associated 2 (Dppa2) promoter and recruits Dnmts, leading to Dppa2 silencing through hypermethylation, thereby promoting myogenesis [10]. Zhou et al. discovered linc-YY1 from the promoter of the transcription factor Yin Yang 1(YY1) gene. Linc-YY1 can interact with YY1 to dissociate and expel the YY1/PRC2 complex from the target promoters of miR-29, miR-1, myosin heavy chain (MyHC) and troponin, resulting in the activation of target genes [11]. lncMGPF is highly expressed in muscle and promotes myogenic differentiation of C2C12 myoblasts by acting as a molecular sponge for miR-135a-5p. Moreover, lncMGPF has a conserved function and mechanism in the process of human myogenesis [12]. IGF2 AS promotes the proliferation and differentiation of bovine myoblasts. Not only can it bind to the precursor of the insulin-like growth factor 2 (IGF2) gene, affecting the stability of IGF2 mRNA, but it can also bind to interleukin enhancer binding factor 3 (ILF3) to regulate myogenesis [13]. Currently, researchers have characterized a large number of lncRNAs related to muscle development in humans and mice, but many lncRNA functions and regulatory mechanisms have yet to be discovered in cattle.

The SOX6 gene is a member of the Sox transcription factor family and is highly expressed in skeletal muscle. Loss of SOX6 leads to the transformation of muscle to a slow-twitch muscle fibre phenotype by directly inhibiting muscle mass and calcium regulatory proteins [14]. This study revealed that the reverse transcribed lncRNA LOC101904740 (GenBank Accession NO: XR_237499.5) is located upstream of the bovine SOX6 gene (GenBank Accession NO: NM_001191418.1), which was named SOX6 AU. Additionally, lncRNAs mainly regulate gene expression in a cis-acting manner to exert their biological effects [15], it is supposed that SOX6 AU may regulate bovine skeletal muscle development. Given to this, the present study explored the effects and molecular mechanisms of SOX6 AU on bovine primary skeletal muscle cell proliferation, apoptosis, and differentiation. This study may provide valuable transcriptional regulatory resources, shedding light on the mechanisms of lncRNA and SOX6 in bovine muscle development. Moreover, this study provides new insights for improving beef production performance and optimizing meat quality.

Materials and methods

Animal ethics

The ethics of all aspects of this study were approved by the Animal Care and Use Committee of Henan Agricultural University, China (Permit Number: 11–0085). Nine cattle were purchased from Miyang Xianan Cattle Technology Development Co., Ltd. None of the cattle suffered from any disease. Tissue samples were collected from newborn cattle (0 months old), 12-month-old, and 24-month-old Xianan cattle. Tissues such as heart, liver, spleen, lung, kidney, limb skeletal muscle, rumen and ileum were collected. All the samples were quickly frozen in liquid nitrogen and then stored in a −80°C freezer.

Isolation and culture of bovine primary skeletal muscle cells

Primary Bovine skeletal muscle cells were isolated from the leg muscles of 90-day-old fetal bovines and cultured as previously described [13]. Briefly, the leg muscles were minced and digested with 2 mg/mL type II collagenase. The digested tissue was resuspended in DMEM medium supplemented with 15% FBS, and the tissue was filtered through 200-mesh or 400-mesh filters. The filtrate was collected and centrifuged to remove the supernatant. Then, 10% FBS was added to the DMEM medium to resuspend the cell suspension. Finally, the cell suspension was spread into culture flasks, and the cells were cultured at 37°C in a 5% CO₂ environment. C2C12 myoblasts were obtained from the laboratory. For myogenic differentiation, confluent cells were cultured in DMEM medium supplemented with 2% horse serum.

Total RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from tissues and cells using TRIzol reagent (Takara, Kyoto, Japan) following standard protocols. After the RNA concentration and quality were measured, reverse transcription was performed to synthesize cDNA using a PrimeScript RT reagent kit (Takara, Kyoto, Japan). The qRT-PCR for RNA analysis was performed using SYBR Green PCR Master Mix (Takara, Kyoto, Japan). Relative mRNA expression levels were calculated using the 2^−∆∆Ct method [16]. A list of primers used in this study is provided in Table S1 and Table S2.

Fluorescence in situ hybridization (FISH)

The Cyc-labelled SOX6 AU probe and FISH Kit were purchased from RiboBio (Guangzhou, China). Bovine primary skeletal muscle cells were first fixed with in situ hybridization fixative. The cells were permeabilized in PBS containing 0.5% Triton X-100 at 4°C for 5 min, washed 3 times, and prehybridized at 37°C for 30 min. Then, the cells were incubated overnight with the labelled SOX6 AU probe in hybridization buffer. The next day, the nuclei were stained with DAPI staining solution. The subcellular localization of SOX6 AU in bovine primary skeletal muscle cells was observed using laser confocal microscopy.

Plasmid construction and interfering RNA synthesis

PCR primers were designed according to the sequence information of SOX6 AU in the National Center for Biotechnology Information (NCBI) database. The full-length fragment of SOX6 AU was amplified by touchdown PCR using bovine muscle tissue cDNA as a template. Then, the full-length sequence of SOX6 AU was inserted into the pcDNA3.1 overexpression vector. The promoter region of SOX6 AU was analysed, and its transcription start site was defined as + 1, extending forward by −2016 bp and backward by + 97 bp. Within this region, four deletion fragments (−1767/+97, −1365/+97, −888/+97, and −512/+97) were randomly generated by PCR amplification using bovine genomic DNA as a template. The PCR products were ligated to the pGL3-basic plasmid vector (Promega, Wisconsin, USA), which had been digested with the restriction enzymes NheI and HindIII using a homologous recombination kit (Vazyme, Nanjing, China) following the manufacturer’s instructions. All the constructed plasmids were verified by sequencing.

Small interfering RNAs (siRNAs) targeting SOX6 AU and SOX6 were synthesized by RiboBio (Guangzhou, China). For overexpression, the full-length sequence of SOX6 AU was inserted into the pAdTrack-CMV shuttle vector, and the adenovirus was packaged by Yunzhou (Guangzhou, China). The constructed vector and the sequences of the interfering RNA primers are shown in Table S3.

Cell treatment

According to the manufacturer’s protocol, cells were transfected with siRNA or plasmid using Lipofectamine 3000 reagent (Invitrogen, USA) when they reached 50%–60% confluence. For adenoviral infection of bovine primary skeletal muscle cells, an MOI (multiplicity of infection) of 90 of the infectious virus was added to the cells, and the medium was then replaced with fresh medium after 24 h. The expression efficiency was examined by GFP expression using a fluorescence microscope, and the results were confirmed by qRT-PCR.

Western blotting

Western blotting (WB) analysis was used to detect the protein expression level. Treated bovine primary skeletal muscle cells were lysed using radioimmunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) lysis buffer supplemented with 1% Phenylmethanesulfonyl fluoride (PMSF). After the protein concentration was measured with a BCA protein quantification kit (Epizyme, Shanghai, China), 5 × protein loading buffer (Solarbio, Beijing, China) was added to the sample, which was then heated at 100°C for 5–10 min to denature the protein. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with non-fat dry milk solution at room temperature for 1 h, and incubated with primary antibodies overnight at 4°C. The next day, the PVDF membranes were washed 3 times with 1× TBST solution and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After washing 3 times with 1× TBST solution, the protein signal was detected with ECL plus kit (Beyotime, Shanghai, China). The primary and secondary antibody information is listed in Table S4.

EdU and CCK-8 assays

An EdU (5-ethynyl-2-deoxyuridine) assay kit and Cell Counting Kit-8 (CCK-8) reagent were used to detect cell proliferation. After transfection for 24 hours, the cells were incubated in 50 μm EdU medium. After incubation for 2 h, the proliferating cells were stained according to the manufacturer’s instructions (RiboBio, Guangzhou, China). Images were acquired using a fluorescence microscope. For the CCK-8 assay, cells were seeded into 96-well plates. At 0, 12, 24, and 36 h after transfection, CCK-8 reagent (Vazyme, Nanjing, China) was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance of the CCK-8 solution at 450 nm was measured using an automated microplate reader, and the cell proliferation ability was judged according to the changes in the absorbance.

Cell cycle and apoptosis assay

After transfection for 24 hours, the cells were harvested, and washed with cold PBS. Then, the cells were fixed with 70% ice-cold ethanol overnight. The next day, after washing with PBS, the staining solution and permeabilization reagent were added for incubation. Finally, changes of in the cell cycle were detected by flow cytometry, and the percentage changes in the number of cells in each stage of the cell cycle were analysed. We also measured cell apoptosis using flow cytometry. Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. All the data were analysed using FlowJo 7.6 software.

Dual luciferase reporter assays

When C2C12 myoblast reached 80% confluence in a 24-well plate, the cells were co-transfected with different promoter dual-luciferase reporter vectors and a pGL-TK plasmid. After 36 hours, the cells were harvested, and the core promoter region of SOX6 AU was analysed using the Dual-Glo® Dual-Luciferase Reporter Gene Detection System Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Renilla luciferase activity served as an internal control to normalize firefly luciferase activity.

Transcriptome sequencing and data analysis

To study the role of SOX6 AU in bovine skeletal muscle development, bovine skeletal primary muscle cells were treated with SOX6 AU adenovirus (4 biological replicates per group), and the extracted RNA was used for transcriptome sequencing analysis. A microvolume spectrophotometer and 1% agarose gel were used to check the quality of the RNA before the RNA-seq libraries were prepared. Three micrograms of RNA from each qualified sample was sent to Paison Biotechnology Co., Ltd (Shanghai, China) for library construction. After PCR amplification and sequencing using an Illumina PE150 instrument, the raw data were stored in FASTQ format. After quality control of the data, the clean reads were mapped to the Bos taurus reference genome using HISAT2 [17]. The software StringTie was used to assemble and splice mapped reads to obtain transcripts without annotation information [18]. Differentially expressed genes analysis was performed using edgeR software [19]. The topGO and clusterProfiler R packages were used to perform differential gene GO and KEGG pathway enrichment analysis [20]. Padj < 0.05 and log2 (fold change ≥ 1.2) were selected as the reliability screening criteria.

Statistical analysis

The data were presented as the mean ± standard deviation (SD), and differences between groups were analysed by Student’s t-test or one-way ANOVA with SPSS24.0 software. p < 0.05 was considered to indicate statistical significance. The results were displayed in a bar graph or a line using GraphPad Prism 7 software.

Results

Expression pattern of SOX6 AU

We found the lncRNA LOC101904740 upstream of the SOX6 gene and named it SOX6 AU according to its coding direction with the SOX6 gene (Figure 1a). To obtain the full-length sequence of SOX6 AU, we used the cDNA of cattle muscle tissue as a template to amplify and obtain a sequence length of 706 bp, which was the same as the reference sequence (GenBank Accession NO: XR_237499.5) (Figure 1b). Analysis of the Coding Potential Calculator 2 (CPC2) website (http://cpc2.gao-lab.org/) revealed that similar to Xist, SOX6 AU had a lower coding ability than the protein-coding genes MyoG and GAPDH, indicating that SOX6 AU is a noncoding RNA (Figure 1c). The expression of SOX6 AU in different stages of Xianan cattle was measured by qRT-PCR. SOX6 AU was expressed in different tissues at each stage, and its expression in muscle was relatively high (Figure S1A). To understand the potential function of SOX6 AU, we analysed the expression characteristics of SOX6 AU in the muscles of Xianan cattle among different developmental stages. The expression level of SOX6 AU in muscle tissue gradually increased with age in Xianan cattle, and the expression level in 24-month-old cattle was significantly higher than that in newborn cattle, which suggested that SOX6 AU plays an important role in the process of bovine skeletal muscle development (Figure 1d). Bovine primary skeletal muscle cells were isolated to detect the expression of SOX6 AU during myogenic differentiation. After 2 days of induction in vitro, bovine primary skeletal muscle cells showed obvious myogenic differentiation characteristics. With the progression of the induced differentiation process, the myotubes continued to fuse and thicken (Figure S1B). After myogenic differentiation, the expression level of SOX6 AU obviously increased (Figure 1e). FISH indicated that SOX6 AU was located mainly in the cytoplasm of bovine primary skeletal muscle cells (Figure 1f).

Figure 1. Expression pattern of SOX6 AU. (a) Location of SOX6 AU in the bovine genome. (b) Full-length SOX6 AU was obtained. (c) The coding ability of SOX6 AU was predicted by the CPC website. (d) Spatiotemporal expression profile of SOX6 AU. (e) Expression profiles of SOX6 AU and MyoG during myogenic differentiation. (f) Detection of the localization of SOX6 AU in bovine primary skeletal muscle cells by FISH. Blue indicates the cell nucleus. Red indicates SOX6 AU. Original magnification, ×200. The data are shown as the mean±SD from three independent experiments. *p < 0.05.

SOX6 AU promotes bovine primary skeletal muscle cells proliferation and apoptosis

Based on the above results, we preliminarily explored the functional role of SOX6 AU in bovine primary skeletal muscle cells. We synthesized adenoviral and interference fragments of SOX6 AU for SOX6 AU gain-of-function and loss-of-function assays. The overexpression efficiency and interference efficiency of SOX6 AU were carried out in bovine primary skeletal muscle cells. The results showed that Ad-SOX6 AU increased the expression of SOX6 AU by tens of thousands of times, and the interference efficiency of the SOX6 AU interference fragment reached approximately 50% (Figure 2a,b), which could be used in subsequent experiments. The proliferation of bovine primary skeletal muscle cells was detected by EdU reagent, and the results showed that the overexpression or knockdown of SOX6 AU significantly promoted or inhibited cell proliferation respectively (Figure 2c,d), which was consistent with the results obtained by the CCK-8 assay (Figure 2e,f). Cell cycle analysis revealed that overexpression of SOX6 AU significantly increased the percentage of cells in S the phase and decreased the percentage of cells in the G1 phase (Figure 2g); while knockdown of SOX6 AU significantly reduced the percentage of cells in the S phase (Figure 2h), indicating that SOX6 AU can accelerate cell progression from the G1 phase to the S phase, thereby promoting cell proliferation. Next, we detected the expression levels of proliferation marker genes, and found that overexpression of SOX6 AU significantly enhanced the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), while the mRNA levels of the anti-proliferation gene cyclin-dependent kinase (CDK) inhibitor P21 (P21) were significantly decreased (Figure 2i,k). Interference with SOX6 AU had the opposite effect (Figure 2j,l). The protein levels of CDK4 and PCNA are shown in Figure S2A, B. In summary, these results demonstrate that SOX6 AU promotes bovine primary skeletal muscle cell proliferation.

Figure 2. SOX6 AU promotes bovine primary skeletal muscle cells proliferation and apoptosis. (a) Detection of adenovirus-mediated SOX6 AU overexpression efficiency. (b) Detection of the expression levels of SOX6 AU after the transfection of cells with siRNA fragment. (c, d) the number of positive cells in the S phase was detected by the EdU incorporation assay. (e, f) Cell proliferation was measured by CCK-8 assay. (g, h) Flow cytometry was used to detect and analyze the effect of SOX6 AU on different phases of the cell cycle. (i) qRT-PCR was used to detect the relative mRNA expression of the proliferation marker genes CDK4, PCNA, and P21 after overexpression of SOX6 AU. (j) qRT-PCR was used to detect the relative mRNA expression of proliferation marker genes after knockdown of SOX6 AU. (k) Western blotting was used to detect the protein expression of CDK4 and PCNA after overexpression of SOX6 AU. (l) Western blotting was used to detect the protein expression of CDK4 and PCNA after knockdown of SOX6 AU. (m) Flow cytometry was used to detect the apoptosis of cells after overexpression of SOX6 AU. (n) Flow cytometry was used to detect the apoptosis of cells after knockdown of SOX6 AU. (o, p) Relative mRNA expression of apoptosis genes (Bcl2 and Caspase3) was quantified by qRT-PCR in cells overexpressing SOX6 AU and interfering SOX6 AU. (q, r) Relative protein expression levels of apoptosis genes (Bax and Caspase3) were detected by Western blot in cells overexpressing SOX6 AU and interfering SOX6 AU. The data are shown as the mean±SD from three independent experiments. * p < 0.05,** p < 0.01,*** p < 0.001.

We then investigated the effects of SOX6 AU on apoptosis in bovine primary skeletal muscle cells. We first performed flow cytometry and found that infection with the SOX6 AU adenovirus significantly increased the number of apoptotic cells (Figure 2m), while interference with SOX6 AU inhibited apoptosis (Figure 2n). Next, the expression levels of apoptosis marker genes were measured by qRT-PCR. We found that SOX6 AU overexpression significantly promoted the expression of Caspase3 and inhibited the expression of Bcl-2 (Figure 2o); SOX6 AU knockdown dramatically upregulated Bcl-2 mRNA expression and downregulated Caspase3 mRNA expression (Figure 2p). Similarly, Western blot analysis revealed that the protein expression levels of Bax and Caspase3 were significantly upregulated or downregulated upon SOX6 AU overexpression or knockdown, respectively (Figure 2q,r). The protein levels of CDK4 and PCNA are shown in Figure S2C, D. The formation of skeletal muscle fibres mainly depends on the fusion of myoblasts to form myotubes. Therefore, we further explored the effect of SOX6 AU on the differentiation of bovine primary skeletal muscle cells. We infected bovine primary skeletal muscle cells with SOX6 AU adenovirus and detected its expression after 6 days of differentiation. SOX6 AU was successfully overexpressed (Figure S2E), and the mRNA expression levels of MyoD and MyoG (myogenin) were significantly increased (Figure S2F). During myogenic differentiation, approximately 50% of the SOX6 AU was silenced upon siRNA treatment (Figure S2G), which was followed by a significant decrease in the mRNA expression levels of MyoD and MyoG (Figure S2H). Similarly, Western blot analysis showed that MyoG protein expression was significantly upregulated or downregulated after SOX6 AU overexpression or knockdown, respectively (Figure S2I, J). Overall, our data support the idea that SOX6 AU plays vital roles in bovine skeletal muscle development by promoting the proliferation, apoptosis, and differentiation of bovine primary skeletal muscle cells.

Identification and enrichment analysis of DEGs associated with SOX6 AU

To further explore the mechanism by which SOX6 AU affects the development of bovine primary skeletal muscle cells, RNA-seq was performed after the overexpression of SOX6 AU. Firstly, the results of principal component analysis (PCA) between samples showed that the control group and the overexpression group were divided into two independent clusters (Figure 3a), indicating that biological replicates in the group could be used for subsequent analysis. Next, we analysed the DEGs between the two groups and identified 483 DEGs (Padj <0.05 and log2 (fold change ≥ 1.2)), including 224 upregulated DEGs and 259 downregulated DEGs (Figure 3b). In addition, heatmap analysis was performed according to the expression of DEGs (Figure 3c). To verify the accuracy of the sequencing data, we randomly selected 8 DEGs for qRT-PCR, and the results were consistent with the RNA-seq trend, indicating that the sequencing data could be used for further analysis (Figure 3d, e). Next, functional enrichment analysis of the DEGs was performed. GO analysis revealed that the DEGs were mainly enriched in biological processes related to skeletal muscle development, such as muscle structure development, the regulation of ossification, and muscle cell proliferation (Figure 3f). KEGG signaling pathway enrichment analysis revealed that most DEGs were enriched in signaling pathways such as the PI3K/AKT, MAPK, and myocardial contraction pathways (Figure 3g). Further studies revealed that overexpression of SOX6 AU promoted the apoptosis process by affecting the expression of the PI3K/AKT core protein and downstream molecules (Figure S3A). The above results further indicate that SOX6 AU participates in many important processes related to muscle development through its downstream genes and pathways at the transcriptional level, and has clear effects on bovine skeletal muscle development, which is consistent with our conclusion that SOX6 AU promotes the proliferation and apoptosis of bovine primary skeletal muscle cells.

Figure 3. Identification and enrichment analysis of DEGs associated with SOX6 AU. (a) Principal component analysis between samples. (b) Differentially expressed genes between the two groups after the overexpression of SOX6 AU in bovine primary skeletal muscle cells for 36 h. (c) Heatmap of differentially expressed genes. (d) Differential gene qRT-PCR map. (e) Differential gene sequencing expression map. (f) Functional enrichment analysis of differentially expressed genes. (g) KEGG enrichment analysis of differentially expressed genes. The data are shown as the mean±SD from three independent experiments. *p < 0.05,**p < 0.01,***p < 0.001.

Several studies have shown that lncRNAs can be activated by upstream transcription factors to participate in the regulation of biological processes [21,22]. To further explore the regulatory mechanisms of SOX6 AU at the transcriptional level, we successfully constructed SOX6 AU promoter luciferase reporter gene vectors of different lengths (Figure S3B). Next, we transfected C2C12 cells with different promoter dual-luciferase reporter vectors. Dual-luciferase reporter analysis showed that the promoter activity of the −2016/+97 fragment was significantly higher than that of the pGL3-basic vector. The promoter activity significantly decreased when the promoter sequence range was narrowed from −2016 bp to −1767 bp. As the fragment continued to narrow, the fluorescence activity did not change significantly. These results indicate that −2016 bp to −1767 bp is the core promoter region of SOX6 AU (Figure S3C). The transcription factor binding site in the SOX6 AU promoter active region was predicted on the Animal TFDB3.0 website, and we observed that the SOX6 AU core promoter contains a series of putative binding sites for transcription factors related to muscle development. SRF is a key regulator of skeletal muscle development. The knockdown of SRF during embryonic development severely inhibits the formation of muscle tissue, hinders myocyte fusion, and causes severe muscle atrophy [23]. We found that there is a transcription factor SRF binding site in the region from −1826 bp to −1811 bp upstream of the SOX6 AU transcription initiation site, but it is not clear whether the transcription factor SRF regulates its transcription by binding to SOX6 AU and affecting bovine myogenic differentiation (Figure S3D). In summary, we identified the core promoter region of SOX6 AU and predicted that the transcription factor SRF may bind to it. It is speculated that SRF may be involved in the transcription of SOX6 AU and further regulate bovine myogenic differentiation.

Knockdown of SOX6 promotes bovine primary skeletal muscle cells proliferation and apoptosis

Next, we examined the tissue expression profile of SOX6 and found that it was highly expressed in muscles (Figure 4a). Then, we investigated the effect of SOX6 knockdown on the proliferation and apoptosis of bovine primary skeletal muscle cells. In bovine primary skeletal muscle cells, knockdown of SOX6 significantly promoted cell proliferation (Figure 4b–d). This also resulted in a significant decrease in the number of cells in the G0/G1 phase and a significant increase in the number of cells in the S phase (Figure 4e). We observed that knockdown of SOX6 was associated with upregulation of CDK4 and PCNA mRNA levels, compared to their respective levels in the control group (Figure 4f). In addition, we also found that knockdown of SOX6 significantly promoted the apoptosis of bovine primary skeletal muscle cells (Figure 4g,h). Hence, these results suggest that knockdown of SOX6 promotes the proliferation and apoptosis of bovine primary skeletal muscle cells.

Figure 4. Knockdown of SOX6 promotes bovine primary skeletal muscle cells proliferation and apoptosis. (a) Tissue expression profiles of SOX6 in Xianan cattle at different months of age. (b) The mRNA expression levels of SOX6 gene after transfection with si-SOX6 or si-NC. (c) An EdU incorporation assay was used to detect the proliferation of cells after transfection with si-SOX6 or si-NC. (d) CCK-8 assay was used to measure cell growth after transfection with si-SOX6 or si-NC. (e) Cells were collected for cell cycle analysis after transfection with si-SOX6 or si-NC. (f) qRT-PCR was used to analyze the changes in the expression of the proliferation-related genes CDK4, PCNA, and P21 after transfection with si-SOX6 or si-NC. (g) qRT-PCR analyzed the expression changes of apoptosis-related genes Bax, B-cl2, and Caspase3 after transfection with si-SOX6 or si-NC. (h) Cells were collected for cell apoptosis analysis after transfection with si-SOX6 or si-NC. The data are shown as the mean±SD from three independent experiments. *p < 0.05,**p < 0.01,***p < 0.001.

SOX6 AU cis-acting SOX6 regulates bovine primary skeletal muscle cells proliferation and apoptosis

LncRNAs can regulate the expression of adjacent genes, and SOX6 can participate in the development and maintenance of skeletal muscle. Therefore, we speculate that SOX6 AU may be involved in the regulation of myogenesis by affecting its adjacent gene SOX6. Interestingly, we found that the mRNA and protein levels of SOX6 were significantly decreased after the overexpression of SOX6 AU, while the inhibition of SOX6 AU upregulated the mRNA and protein expression of SOX6 (Figure 5a–c). These results suggest that SOX6 may be a key factor required for the cis-acting activity of SOX6 AU. To further investigate whether SOX6 is involved in the cis-modulation of SOX6 AU, we co-transfected si-SOX6 AU and si-SOX6 into bovine primary skeletal muscle cells. We found that si-SOX6 AU decreased the mRNA expression of proliferation and apoptosis marker genes, while si-SOX6 significantly attenuated the effect of si-SOX6 AU on the mRNA expression of proliferation and apoptosis marker genes (Figure 5d, e). The CCK-8 assay results revealed that cotreatment with si-SOX6 AU and si-SOX6 attenuated proliferative effect of si-SOX6 AU on bovine primary skeletal muscle cells (Figure 5f). In addition, the flow cytometry results revealed that interference with SOX6 AU significantly inhibited the number of bovine primary skeletal muscle cells in the S phase, but combined interference with SOX6 AU and SOX6 significantly attenuated the inhibitory effect on the proliferation of bovine primary skeletal muscle cells (Figure 5g). At the same time, flow cytometry was used to analyse the effect on cell apoptosis. The results suggested that si-SOX6 weakened the inhibitory effect of si-SOX6 AU on the apoptosis of bovine primary skeletal muscle cells (Figure 5h). Collectively, these results support the view that SOX6 AU promotes the proliferation and apoptosis of bovine primary skeletal muscle cells by cis-acting on SOX6.

Figure 5. SOX6 AU cis-acting SOX6 regulates bovine primary skeletal muscle cells proliferation and apoptosis. (a) The mRNA expression levels of SOX6 were detected after overexpressing or interference of SOX6 AU in bovine primary skeletal muscle cells. (b) The protein expression levels of SOX6 were measured after overexpressing or interference of SOX6 AU bovine primary skeletal muscle cells. (c) Quantification of SOX6 protein levels in Figure 5b. (d, e) the mRNA expression levels of CDK4/PCNA/P21/Bax/Caspase3/Bcl2 after cotransfection with si-SOX6 AU and si-SOX6. (f) CCK-8 assay was used to detect the effect of both SOX6 AU and SOX6 on cell proliferation. (g) The cell cycle distribution was analyzed by flow cytometry after cotransfection with si-SOX6 AU and si-SOX6. (h) Cell apoptosis was detected by flow cytometry after cotransfection with si-SOX6 AU and si-SOX6. The data are shown as the mean±SD from three independent experiments. *p < 0.05,**p < 0.01,***p < 0.001.

Conversation analysis of SOX6 AU

Compared with protein-coding RNAs, lncRNAs are not well conserved in sequence, which prompted us to experimentally determine whether SOX6 AU also plays a role in C2C12 myoblasts. Interestingly, we also found the lncRNA Sox6 os (GenBank Accession NO:NR_152104.1) upstream of the mouse SOX6 gene in the NCBI database. Next, we analysed the interspecies conservation of bovine SOX6 AU through the UCSC database (http://genome.ucsc.edu/) and found that bovine SOX6 AU is conserved with of mice (Figure S4A). We also used the AnnoLnc database (http://annolnc.cbi.pku.edu.cn/) to analyse the expression pattern of the lncRNA Sox6 os in mouse tissues and found that it was expressed in various tissues (Figure S4B). In addition, subcellular localization prediction found that lncRNA Sox6 os was located mainly in the cytoplasm, which was similar to the localization of bovine SOX6 AU (Figure S4C). C2C12 myoblasts are the most widely studied skeletal muscle development culture system. To study whether the myogenic function of SOX6 AU is the same in mice, we overexpressed SOX6 AU in C2C12 myoblasts and detected its effect on the proliferation, differentiation, and apoptosis of C2C12 myoblasts. We transfected C2C12 myoblasts with SOX6 AU overexpression plasmid and found that SOX6 AU was successfully overexpressed in C2C12 myoblasts (Figure 6a). The effect of SOX6 AU on cell proliferation was detected by an EdU assay, and the percentage of EdU-positive cells decreased significantly after SOX6 AU was overexpressed (Figure 6b). The flow cytometry results showed that the number of cells in the S phase decreased significantly after overexpression of SOX6 AU, and the number of cells in the G1 phase increased (Figure 6c). Next, we found that the increase in SOX6 AU expression significantly decreased the mRNA and protein expression levels of CDK4 and PCNA (Figure 6d–f). We also examined the effect of SOX6 AU on the expression of apoptosis genes. After overexpression of SOX6 AU, the mRNA and protein levels of the Bcl-2 gene were significantly decreased, the mRNA and protein levels of the Bax gene were significantly increased, and the mRNA level of the Caspase3 gene was also significantly increased (Figure 6g–i). Next, the effects of SOX6 AU on myogenic genes MyoD, MyoG, and Myf5 (family myogenic regulatory factor 5) were examined. After overexpression of SOX6 AU, the mRNA expression of MyoD and MyoG was significantly increased (Figure 6j,k), and the protein expression of MyoG and Myf5 was significantly increased (Figure 6l, m). The above results indicate that the function of SOX6 AU in myoblast apoptosis and differentiation is conserved between mice and bovines.

Figure 6. Conversation analysis of SOX6 AU. (a) Detection of the overexpression efficiency of SOX6 AU in C2C12 myoblasts. (b) Effect of SOX6 AU overexpression on the DNA replication activity of C2C12 myoblasts. (c) After overexpression of SOX6 AU, the percentage of cells in each stage of the cell cycle was detected by flow cytometry. (d) After overexpression of SOX6 AU in C2C12 myoblasts, the effects of SOX6 AU on CDK4 and PCNA gene expression were measured by qRT-PCR. (e) After overexpression of SOX6 AU in C2C12 myoblasts, the effects of SOX6 AU on CDK4 and PCNA gene protein expression were detected by western blotting. (f) Protein quantification of CDK4 and PCNA. (g) qRT-PCR was used to detect the expression levels of the apoptosis genes Bax, Caspase3, and Bcl-2. (h) Western blot analysis of the expression levels of the apoptosis genes Bax and Bcl-2. (i) Protein quantification of Bax and Bcl-2. (j) The expression efficiency of SOX6 AU during myogenic differentiation of C2C12 myoblasts. (k) The mRNA expression levels of MyoD and MyoG were detected by qRT-PCR. (l) The expression of Myf5 and MyoG were measured by Western blotting. (m) Protein quantification of Myf5 and MyoG. The data are shown as the mean±SD from three independent experiments. *p < 0.05,**p < 0.01,***p < 0.001.

Discussion

Here, we identified a bovine skeletal muscle-related lncRNA located upstream of the SOX6 gene, named SOX6 AU. In general, the expression of lncRNAs has tissue and spatiotemporal specificity. In this study, SOX6 AU was widely expressed in various tissues, especially in the lung and heart. However, we also found that its expression level in skeletal muscle tissue was relatively high, the expression level of SOX6 AU in muscle tissue gradually increased with age in cattle, and the expression level in 24-month-old cattle was significantly greater than that in newborn cattle. MyoD upstream noncoding RNA (MUNC) is specifically expressed in skeletal muscle and can affect myoblast differentiation during myogenesis by stimulating the adjacent MyoD gene [24]. Thus, we support that SOX6 AU might be a skeletal muscle development-related lncRNA. To elucidate the functional role of SOX6 AU in myogenesis, gain-of-function or loss-of-function assays were performed. PCNA is a crucial protein involved in the DNA synthesis process of eukaryotic cells and plays a pivotal role in regulating the cell cycle and DNA replication [25]. The CDK4 protein also plays a crucial role in regulating the cell cycle [26]. The P21 protein can induce G1 arrest and prevent cells from progressing into the S phase by inactivating CDKs or inhibiting PCNA activity [27]. Hence, in this study, we selected the CDK4, PCNA, and P21 genes as molecular indicators to assess cell proliferation. Our findings showed that after overexpression of SOX6 AU in bovine primary skeletal muscle cells, the expression of PCNA and CDK4 was upregulated, while the expression of P21 was downregulated, indicating that cell proliferation was promoted. Consistent with many studies, this study selected Caspase3, Bcl-2, and Bax as indicators to evaluate skeletal muscle cell apoptosis [28]. In this study, Caspase3 and Bax were significantly upregulated, while the expression of Bcl-2 was significantly downregulated. In addition, the expression of genes related to myogenic differentiation, such as MyoG and MyoD, significantly increased after SOX6 AU overexpression. The expression of MyoD marks the beginning of cells entering the differentiation stage and forming myotube [29]. MyoG is essential for cell fusion and terminal differentiation stages [30,31]. According to the changes in cell phenotype and marker genes, we demonstrated that overexpression of SOX6 AU promoted the proliferation, apoptosis and differentiation of bovine primary skeletal muscle cells. The results of the SOX6 AU knockdown experiments supported this conclusion.

SOX6 is a regulator of the proliferation, apoptosis, and differentiation of multiple cell types and organ development [32]. SOX6 indirectly regulates muscle terminal differentiation by interacting with transcription factors related to muscle development [33]. SOX6 can also upregulate the expression of chicken muscle growth-related genes and promote the proliferation and differentiation of chicken skeletal muscle cells [34]. As cis-regulatory elements, lncRNAs are involved in the regulation of various biological processes. LncKdm2b regulates cortical neuronal differentiation by cis-activating the transcription of Kdm2b and binding to hnRNPAB [35]. MK5-AS1 was confirmed to accelerate colorectal cancer progression by cis-regulating the nearby gene MK5 (MAPK activated protein kinase 5) and acting as a let-7f-1-3p sponge [36]. Here, we demonstrated that SOX6 is highly expressed in bovine muscle tissue and inhibits the proliferation and apoptosis of bovine primary skeletal muscle cells. SOX6 AU affects the proliferation and apoptosis of bovine primary skeletal muscle cells by negatively regulating the expression of the adjacent gene SOX6. We demonstrated that SOX6 attenuates the effect of SOX6 AU on the proliferation and apoptosis of bovine primary skeletal muscle cells through SOX6 AU and SOX6 functional compensation experiments. In summary, SOX6 AU affects bovine muscle growth by reducing the expression of the neighboring gene SOX6 through a cis-regulatory module.

To further identify the regulatory factors of SOX6 AU in the myogenesis of bovine primary skeletal muscle cells, RNA-seq was performed to analyse the regulatory mechanism of SOX6 AU on cell proliferation and apoptosis. GO enrichment and KEGG pathway analyses revealed that SOX6 AU affects muscle structure development, DNA replication, the cell cycle and other related pathways. The cell cycle is a highly regulated process that enables cell growth, replication of genetic material, and cell division [37]. The analysis of DEGs showed that regulatory factors related to muscle growth and development, such as EGR1 (early growth response factor 1) [38], GDF15 (growth differentiation factor 15) [39], IGF2 (insulin-like growth factor 2) [40], and IGFBP5 (insulin-like growth factor-binding protein 5) [41], were significantly enriched. This finding also confirms the important role of SOX6 AU in the regulation of bovine primary skeletal muscle cell development at the transcriptional level, but the specific key regulatory pathways involved need to be further explored. Increasing evidence shows that lncRNAs can interact with transcription factors to activate or inhibit transcription [42]. The lncRNA PANDA transcribed from the CDKN1A (cyclin-dependent kinase inhibitor 1A) promoter interacts with the transcription factor NF-YA to inhibit the expression of proapoptotic genes [43]. In this study, a dual-luciferase reporter system was used to identify the core promoter of SOX6 AU. We found that promoter activity suddenly decreased from P1 to P2 (−2016 bp to −1767 bp), suggesting that this region may play an important role in SOX6 AU promoter transcription. Transcription factors are DNA-binding proteins that activate or inhibit gene transcription. We predicted that the transcription factor binding site was in the core promoter region of SOX6 AU and found that there is a binding site for the transcription factor SRF. SRF is an important regulator of skeletal muscle development, and skeletal muscle-specific deletion of SRF can lead to severe muscle hypoplasia, resulting in severe muscle atrophy [44]. In this study, we predicted that SRF binds to the SOX6 AU promoter region, but whether SRF can regulate its transcription by binding to SOX6 AU and affecting the proliferation, apoptosis, and differentiation of bovine primary skeletal muscle primary cells requires further experimental verification.

Compared with protein-coding genes, lncRNAs exhibit lower expression levels and poorer conservation [45]. Gong et al. overexpressed mouse lncMyoD or human hlncMyoD in lncMyoD-knockdown mouse myoblasts, and had little effect on MHC, suggesting that lncMyoD is necessary but not sufficient to drive differentiation. After endogenous mouse lncMyoD was downregulated, both mouse lncMyoD and human hlncMyoD could restore MHC expression, demonstrating that lncMyoD function is conserved in mouse and human myogenic differentiation [46]. In the present study, C2C12 myoblasts were used to overexpress SOX6 AU to verify its effect on the proliferation, apoptosis, and differentiation of C2C12 myoblasts. SOX6 AU promoted the apoptosis and differentiation of C2C12 myoblasts, which is consistent with its function in bovine primary skeletal muscle cells. Interestingly, SOX6 AU promoted the proliferation of bovine primary skeletal muscle cells but inhibited the proliferation of C2C12 myoblasts. This indicates that the functions of SOX6 AU in apoptosis and differentiation processes are conserved between cattle and mice, but there are also cases in which the functions are not similar. Considering the important role of SOX6 AU in muscle development, further elucidation of the function and mechanism of action of SOX6 AU in muscle development in cattle and mice is warranted.

In conclusion, we identified SOX6 AU as a differentially expressed lncRNA in skeletal muscle among different developmental stages of Xianan cattle. Gain-of-function and loss-of-function experiments showed that SOX6 AU promoted the apoptosis and differentiation of bovine primary skeletal muscle cells, which was consistent with the results in C2C12 myoblasts. Interestingly, SOX6 AU promoted the proliferation of bovine primary skeletal muscle cells but inhibited the proliferation of C2C12 myoblasts. Knockdown of SOX6 promoted bovine primary skeletal muscle cells proliferation and apoptosis. Furthermore, SOX6 AU regulated the proliferation and apoptosis of bovine primary skeletal muscle cells by cis-acting on the SOX6 gene (Figure 7). Taken together, our results indicated that SOX6 AU could be a novel molecular marker for the breeding of beef cattle.

Figure 7. Summary figure of this study. SOX6 AU regulates the proliferation and differentiation of bovine primary skeletal muscle cells by cis-acting on the SOX6 gene.

Author Contributions

XL, SX, performed the experimental process; XL wrote the manuscript; SM, ZH, and LY were involved in validation of experimental data; MC and DY participated in data curation and visualization. HX, HC, and ML supervised the study. HC and ML conceived the study and provided overall supervision and manuscript revision. All authors have read and agreed the final manuscript.

Acknowledgment

Thanks to all participants for their suggestions and support for this study. Thanks for the funding of related projects for this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw data used in this study are publicly available. If necessary, please contact the corresponding authors. For the RNA-seq data involved in this manuscript, we promise to upload all data to the specified database as required by this journal when accepted.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2024.2341578

Additional information

Funding

This research was funded by the National Natural Science Foundation of China (No. 32002168), the Key Scientific and Technological Project of Henan Province Department of China (222102110152), and Special Fund for the Henan Agriculture Research System (No. HARS-22-13-G1).