Next Article in Journal
Host Mucosal Niche and Rearing Environment Are Associated with Distinct Gut and Gill Microbiota of L. crocea (Larimichthys crocea)
Next Article in Special Issue
Genome-Wide Association Study of Rib Number and Total Thoracolumbar Vertebrae Number in a Jishen Black Pig Population
Previous Article in Journal
Effects of Dietary Pomegranate Syrup Supplementation on Growth Performance, Carcass Traits, Meat Quality, Oxidative Stress, and GPx7 Gene Expression in Japanese Quail (Coturnix coturnix japonica)
Previous Article in Special Issue
Transcriptomic Analysis Reveals AKT1 Upregulation in Inner Mongolian Cashmere Goats at 12 and 15 Months of Age
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Veterinary Sciences
Volume 13
Issue 7
10.3390/vetsci13070709
vetsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

MAZ Regulates the Proliferation of Skeletal Muscle Satellite Cells via SLPI/Wnt-β-Catenin Signaling in Pigs

by
Mengke Song
1,†,
Rongru Zhu
1,†,
Qian Zhang
1,
Meng Li
1,
Ming Tian
2,
Xinmiao He
2,* and
Xiuqin Yang
1,*
1
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
2
Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Vet. Sci. 2026, 13(7), 709; https://doi.org/10.3390/vetsci13070709
Submission received: 6 June 2026 / Revised: 11 July 2026 / Accepted: 16 July 2026 / Published: 19 July 2026
(This article belongs to the Special Issue Genetic Diversity and Sustainable Breeding of Livestock and Companion Animals)
Browse Figures
Versions Notes

Simple Summary

The growth and development of skeletal muscle is essential for porcine production as it directly determines meat yield and lean meat percentage. Muscle satellite cells (MuSCs) are closely associated with skeletal muscle growth through myogenesis. In the present study, the effects and the underlying mechanisms of MAZ and SLPI on the proliferation of porcine MuSCs (pMuSCs) were analyzed with gain- and loss-of-function methods. It was found that both MAZ and SLPI could increase the proliferation of pMuSCs by promoting cell cycle progression. Then, SLPI was shown to function as a downstream gene of MAZ during the regulation of pMuSCs proliferation. Furthermore, it was revealed that MAZ/SLPI regulated pMuSCs proliferation through canonical Wnt signaling. Additionally, a point mutation, SNP −360C>T, that frequently occurred in the promoter of porcine SLPI was shown to influence gene expression, suggesting potential as a molecular marker for pMuSCs proliferation.

Abstract

Skeletal muscle, accounting for about 40% of the body’s mass, is an important trait for farm animal production. Skeletal muscle satellite cells (MuSCs) are an excellent material for studying the regulatory mechanisms underlying muscle growth and regeneration after birth. Here, we analyzed the role and underlying mechanisms of Myc-associated zinc finger protein (MAZ) and secretory leukocyte protease inhibitor (SLPI) in porcine MuSC (pMuSC) proliferation for the first time. It was revealed that both MAZ and SLPI advanced the proliferation of pMuSCs by promoting cell cycle progression, and that a MAZ/SLPI axis regulated pMuSC proliferation. It was further shown that this MAZ/SLPI axis regulated pMuSC proliferation through the Wnt/β-catenin pathway. The proliferative role obtained above was confirmed in mouse myoblast C2C12 cells. Additionally, a single-nucleotide polymorphism (SNP), −360C>T, which frequently occurred in the promoter of the SLPI gene from Min, Yorkshire, and Longmin black pigs, was found to significantly increase the gene’s expression in vitro and in vivo. The SNP −360C>T in SLPI is a potential molecular marker predicting the proliferative ability of pMuSCs. The data provide a basis for controlling muscle growth and regeneration, which will provide benefits for improving the meat production of pigs and controlling muscle-related diseases in humans.

1. Introduction

Skeletal muscle, accounting for about 40% of the body’s mass [1], is essential for livestock production. Its growth and development directly determine meat yield and lean meat percentage, and thus economic benefits. Additionally, its disorder results in many diseases such as muscular dystrophy/hypertrophy, muscular atrophy, and myosarcoma in humans [2]. Therefore, skeletal muscle is critical for both animal production and human medicine. It is important to reveal the mechanisms behind the growth and development of skeletal muscle, which is the basis for improving the meat production of farm animals and for controlling muscle-related diseases in humans. Researchers have long been working hard to unveil the mystery, and some progress has been made.
It has been revealed that skeletal myogenesis is an orchestrated process controlled by a series of transcription factors such as paired box (Pax) 3 and 5 and myogenic regulatory factors [2,3]. However, the mechanisms underlying muscle growth and development remain far from clarified. Muscle satellite cells (MuSCs), the skeletal muscle-resident stem cells, are an excellent material for studying the regulatory mechanisms underlying muscle growth and regeneration after birth. Their proliferative ability is closely associated with muscle mass and injury repair. Some genetic factors have been demonstrated to be involved in the regulation of MuSCs’ proliferation, including transcription factors, protein-coding genes, and noncoding RNAs [4,5,6].
The transcription factor Myc-associated zinc finger protein (MAZ) is extensively expressed in various tissues [7,8]. The polypeptide contains six C2H2-type zinc fingers responsible for binding to GC-rich sites within the promoter of the target genes at the carboxyl terminus. There is a proline-rich region consisting of 22% proline in the first 150 amino acids at the amino-terminal part. The proline-rich region serves as a transcriptional activation domain [7,9,10]. MAZ has been shown to be involved in every transcriptional process, including initiation, elongation, and termination [7,11], and is thus closely associated with the expression of many genes, including those modulating cell proliferation. For example, MAZ can activate the mitogen-activated protein kinase kinase 2 gene by directly binding to its promoter [12]. Studies also found that MAZ could transactivate muscle-specific genes including muscle creatine kinase, alpha-Actin, Desmin, and alpha-Myosin heavy chain in skeletal myocytes. Additionally, MAZ’s transcripts and DNA-binding activity were upregulated during skeletal myocyte differentiation [13]. However, the specific role of MAZ in MuSC growth and development remains to be identified.
Secretory leukocyte protease inhibitor (SLPI), a ~12 kDa inhibitor of cationic serine protease, is an important regulator of cell proliferation. It has shown a positive role in the proliferation of cells, including CD34+ hematopoietic progenitor cells [14,15], osteoblasts [16], etc. However, no studies on the role of SLPI in the proliferation of MuSCs were found. Although most studies have shown that SLPI promotes cell proliferation, opposite effects were found in a recent study [17], demonstrating the multiplicity and complexity of the SLPI gene’s actions in cell proliferation.
Our previous studies have revealed that MAZ and SLPI are differentially expressed during skeletal muscle development in pigs, suggesting that a MAZ/SLPI regulatory axis exists that regulates muscle growth and development. In the present study, we constructed a MAZ/SLPI/Wnt/β-catenin axis regulating the proliferation of porcine MuSCs (pMuSCs) and confirmed the proliferative role of the axis in mouse myoblast C2C12 cells. The results obtained here provide data further clarifying the mechanisms underlying pMuSCs’ proliferation, which will contribute to improving the meat production of farm animals and controlling muscle-related diseases in humans.

2. Materials and Methods

2.1. Animals and Nucleic Acids

Min pig, an indigenous pig breed unique to Northeast China, was used in the present study. Animals at the age of 210 d old were obtained from the Heilongjiang Academy of Agricultural Sciences, Harbin, China. The animals were reared in the same environment and had access to their diets and water ad libitum. The individuals were not related by blood, and gender was not taken into account in the present study. The pigs were slaughtered using electric stunning methods, and muscle tissues were collected. After being snap-frozen in liquid nitrogen, the tissues were stored at −80 °C. Animals were treated strictly based on the Chinese national standard for pig slaughtering (GB/T 17236–2019) [18], as approved by the Laboratory Animal Welfare and Ethics Committee of Northeast Agricultural University. RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA).

2.2. Cell Culture and Transfection

pMuSCs were purchased from Ming Zhou Biotechnology Co. (MZ-8377, MINGZHOUBIO, Ningbo, Zhejiang, China). pMuSCs were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS; Gibco BRL, Rockville, MD, USA) and 1% penicillin–streptomycin. C2C12 (CRL-1772) and PK-15 (CCL-33) cells were cultured in DMEM/High Glucose (SEVEN, Beijing, China) containing 10% FBS and 1% penicillin–streptomycin. Transient transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNA was isolated with the normal phenol–chloroform method.

2.3. Reverse Transcription and PCRs

Reverse transcription was carried out with the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China) to obtain cDNA. Regular PCR was carried out with 2× Taq PCR Master Mix (Vazyme). Real-time quantitative PCR (qPCR) was performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme) based on the manufacturer’s instructions, in triplicate. The 2−ΔΔCt method was used to analyze the relative expression level of each gene, with β-actin as a reference. All primers were designed with primer premier 5.0 and synthesized by Genesoul Technology (Harbin, China). The sequences of all the primers used here are given in Table S1.

2.4. Plasmids and siRNA Sequences

An improved plasmid pCAGGS with modified enzyme sites at a polylinker provided by Dr. Honglin Jia, affiliated with the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, was used to construct an overexpression vector. The complete coding sequences (CDSs) were first amplified from cDNA obtained from the muscle tissue of Min pigs, and then, sequences encoding three continuous hemagglutinin tags were spliced into the CDS with overlapping-extension PCR. The fragments were ligated into the improved pCAGGS at the SamI site with a ClonExpress II One Step Cloning Kit (Vazyme). Wild-type (WT) reporter genes containing the SLPI promoter were obtained previously [19]. The promoter was mutated with overlapping-extension PCR and inserted into pGL3-basic as described previously [20] to construct mutant type (MT) reporter genes. siRNAs against MAZ or SLPI were synthesized by General Biol (Hefei, China). The sequences of the primers and siRNAs are listed in Table S1.

2.5. Cell Counting Kit 8 and 5-Ethynyl-2′-Deoxyuridine Incorporation Assay

Cell counting kit 8 (CCK-8) was used, and 5-ethynyl-2′-deoxyuridine (EdU) staining was performed as described previously [21]. Briefly, overexpressing plasmids or siRNAs were transfected into the cells. The cells were collected at 0, 24, 48, 72, 96, and 120 h post-transfection and subjected to a CCK-8 assay according to the manufacturer’s instructions. The optical density (OD) was measured with a Tecan Microplate Reader Infinite F50 (Tean GENios, Mannendorf, Switzerland) at 450 nm. For the EdU assay, the cells were stained with the BeyoClick™ EdU-555 kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol at 24 h post-transfection, and then visualized with the Olympus inverted fluorescence microscope IX71 (Olympus, Tokyo, Japan) at 346 nm (excitation)/460 nm (emission).

2.6. Western Blotting

Western blotting was carried out as described previously [20]. Briefly, when they reached 50% confluence, the cells were transfected and cultured for 48 h. Total protein was isolated with RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor (Invitrogen) and quantified with an enhanced BCA protein assay kit (Beyotime, Shanghai, China). A total of 25–30 µg of protein was separated by SDS–polyacrylamide gel electrophoresis and incubated with anti-PCNA (Proliferating cell nuclear antigen; 1:5000 dilution; Proteintech, Rosemont, IL, USA), β-tubulin (1:20,000 dilution; Proteintech), β-actin (1:20,000 dilution; Proteintech), or -HA tag (1:5000 dilution; Abmart, Shanghai, China). β-tubulin and -actin were used as references. Proteins were visualized with the Odyssey® DLx near-infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The intensities of the bands were measured with ImageStudio software (v5.2.2), and the relative values were calculated.

2.7. Flow Cytometry Analysis

The cells were cultured in six-well plates until 60% confluence and transfected for 24 h. After washing with phosphate-buffered solution (PBS), the cells were digested with trypsin and resuspended. The cells were stained with a cell cycle staining Kit (MultiSciences, Hangzhou, China) according to the manufacturer’s protocol. An Agilent Novo Cyte Flow Cytometer (Palo Alto, CA, USA) was used to analyze the cell cycle.

2.8. Dual-Luciferase Reporter Gene Analysis

Each of the reporter genes was co-transfected into PK-15 with the Renilla luciferase reporter, pRL-TK. At 48 h post-transfection, the luciferase activities were measured with the dual-luciferase reporter gene assay kit (Beyotime). The relative activity was calculated as the ratio of firefly to Renilla luciferase activity.

2.9. Polymorphism Analysis

The promoter of SLPI (Gene ID: 396886) was amplified, and single-nucleotide polymorphisms (SNPs) were identified through the direct sequencing of PCR products. SnapGene (v6.0.2) was used to scan the sequencing diagram to profile the genotypes of individuals. The diagram was manually inspected to confirm the SNPs and genotypes identified.

2.10. Statistical Analysis

All experiments were performed three times independently, each with triplicates. GraphPad Prism (version 9.5.1; GraphPad, San Diego, CA, USA) was used to analyze and visualize the data. Differences between two groups were compared with an unpaired t-test, and those between multiple groups were compared with one-way ANOVA. *, p < 0.05; **, p < 0.01.

3. Results

3.1. MAZ Promotes Skeletal Muscle Satellite Cell Proliferation

Plasmids overexpressing MAZ and siRNA against MAZ functioned efficiently in pMuSCs, as revealed by Western blotting and qPCR assays (Figure S1). Compared to the negative control (NC), the overexpression of MAZ increased the cell viability of pMuSCs, while the knockdown of MAZ inhibited it, as revealed by CCK-8 assays (Figure 1A). EdU assays showed that overexpressing MAZ promoted DNA synthesis in pMuSCs, while knocking down MAZ weakened it (Figure 1B). Additionally, the expression of MKI67 and PCNA, marker genes of cell proliferation, was increased by ectopic MAZ and decreased by knocking down MAZ in pMuSCs at the mRNA level (Figure 1C). The protein levels of MKI67 and PCNA changed similarly in response to MAZ treatment, as revealed by ELISA and Western blotting, respectively (Figure 1D,E). At the same time, C2C12 cells overexpressing MAZ were used to confirm the role of MAZ in cell viability and DNA synthesis, and consistent results were obtained (Figure 1).

3.2. MAZ Promotes Skeletal Muscle Satellite Cell Proliferation by Facilitating Cell Cycle Progression

To analyze how MAZ affects the proliferation process of pMuSCs, the changes in cell cycle distribution were analyzed. Cells overexpressing MAZ showed reduced cell numbers in G0/G1-phase and increased numbers in S-phase compared to the NC groups in pMuSCs, while the knockdown of MAZ had opposite effects (Figure 2A). Similar results were obtained in C2C12 cells overexpressing MAZ, confirming the role of MAZ in cell proliferation (Figure 2A). To explore the mechanisms underlying the regulation of the cell cycle by MAZ, the expression changes in two representative cell cycle regulatory proteins—CCND1 and Cyclin B1 (CCNB1), which are G1/S- and G2/M-phase regulators, respectively—were measured in cells overexpressing or knocked down for MAZ. We found that MAZ promoted CCND1 levels’ stability in both pMuSCs and C2C12 cells. The changes in CCNB1 expression were not consistent across the two cell lines: MAZ had no effect on its expression in pMuSCs but enhanced it in C2C12 cells (Figure 2B), showing differences in the mechanisms behind the proliferative effects of MAZ between species. Nevertheless, the results indicated that MAZ promoted cell proliferation.

3.3. SLPI Functions as a Gene Downstream of MAZ to Regulate Skeletal Muscle Satellite Cell Proliferation

To analyze the role of SLPI in pMuSC proliferation, we first successfully constructed overexpression plasmids (Figure S2); the optimum siRNAs against SLPI in pMuSCs (si-173) or C2C12 (si-145) cells were also obtained (Figure S2). Studies in pMuSCs showed that ectopic expression of SLPI significantly increased cell viability and DNA synthesis, while knockdown of SLPI inhibited them (Figure 3A,B). Consistent results were obtained in C2C12 cells (Figure 3A,B). Furthermore, the expression changes in PCNA or MKI67 in response to SLPI treatment further showed a positive role of SLPI in pMuSC proliferation because the overexpression of SLPI increased their expression at both the mRNA and protein levels, and knocking down SLPI decreased it in both cell lines (Figure 3C–E).
Cell cycle distribution analysis showed that overexpressing SLPI reduced the numbers of cells in G0/G1-phase and increased those in S- and G2/M-phase in both pMuSCs and C2C12 cells, and that opposite results were obtained when the expression of SLPI was knocked down (Figure 4A). The expression of CCND1 and CCNB1 was then analyzed in cells overexpressing or knocked down for SLPI, and the results showed that SLPI significantly enhanced the expression of CCND1 in both cells (Figure 4B).
Rescue experiments showed that knocking down SLPI decreased the promoting effects of MAZ on the cell viability and DNA synthesis of pMuSCs according to CCK-8 and EdU assays, and the results were confirmed in C2C12 cells (Figure 5A,B), indicating that MAZ promotes MuSC proliferation via regulating SLPI.

3.4. MAZ/SLPI/Wnt/β-Catenin Axis Exists in Regulating Skeletal Muscle Satellite Cell Proliferation

The canonical Wnt/β-catenin pathway plays a pivotal role in regulating MuSC proliferation. We next investigated whether there was an association between the MAZ/SLPI axis and the Wnt/β-catenin pathway in regulating MuSC proliferation. As shown in Figure 6A, SLPI positively regulates the expression of β-catenin because overexpressing SLPI increased the expression of β-catenin, while knocking down SLPI decreased it in both pMuSCs and C2C12 cells. Further experiments revealed that IWR-1, an inhibitor of the canonical Wnt/β-catenin pathway, reversed the promoting effects of SLPI on the proliferation of pMuSCs and C2C12 cells, as revealed by CCK-8 and EdU assays (Figure 6B,C), confirming that Wnt/β-catenin is involved in the regulation of MuSC proliferation through the MAZ/SLPI axis.

3.5. Polymorphisms Affecting SLPI Expression

SNP −360C>T was detected with high frequency in the promoter of SLPI by the direct sequencing of PCR products (Figure 7A). Genotyping analysis showed that the SNP frequently occurred in all three breeds/lines detected, including Min, a Chinese indigenous pig breed, and Yorkshire, Yorkshire pig and LMB pigs, Landrace × Min pig crossbred pigs. Allele T was dominant in Min and Yorkshire pigs, while in LMB pigs, allele C was dominant. Compared to Yorkshire pigs, allele C occurred more frequently in Min pigs (Table 1).
To reveal the role of SNP −360C>T in the expression of the SLPI gene, MT reporters containing the nucleotide T at position −360 were obtained based on WT reporters (Figure 7B). A C-to-T mutation significantly increased the promoter activity (Figure 7C). To further reveal the SNP’s impact on the expression of the SLPI gene, individuals with different genotypes were screened to measure the expression of SLPI in vivo. It was shown that genotype TT had the highest mRNA level of SLPI among the three genotypes (Figure 7D), which is consistent with the stronger promoter activity of reporter genes containing nucleotide T at that site.

4. Discussion

MuSCs, located between the plasma membrane and the basal lamina of myofibers, are a population of postnatal stem cells essential for skeletal muscle growth and regeneration through myogenesis [22,23,24,25], and they are therefore important for farm animal production and human muscle health. It is vital to reveal the mechanisms underlying the proliferation and differentiation of MuSCs. In the present study, it was revealed that MAZ promoted the proliferation of pMuSCs by facilitating cell cycle progression, and that SLPI functioned as a gene downstream of MAZ to regulate pMuSC proliferation. It was further revealed that the MAZ/SLPI axis regulated cell proliferation via canonical Wnt signaling. Additionally, an SNP influencing the expression of SLPI was identified in the promoter. The results enrich our knowledge of the regulatory mechanisms behind pMuSC proliferation and will contribute to controlling muscle growth and regeneration.
MAZ plays important roles during growth and development in mammals. As a transcriptional regulator, it has been demonstrated to be involved in various biological processes, including aerobic glycolysis [26], genitourinary development [27], ocular development [28], etc. Additionally, MAZ is involved in chromatin domain insulation and genome organization [29,30,31]. In the present study, it was first revealed that MAZ upregulated the proliferation of pMuSCs. Further analysis showed that MAZ could reduce cell numbers in G0/G1-phase, and thereafter facilitate cell cycle progression. The results extend the effects of MAZ to MuSCs, highlighting a role of MAZ in muscle growth and development.
A number of genes have been found to mediate the regulatory role of MAZ in cell proliferation, such as LOC547, BRAF, and KRAS [32,33]. In particular, MAZ could directly bind to the promoters of BRAF or KRAS to increase their transcription and expression levels during the proliferation of papillary thyroid carcinoma cells [33]. MAZ drives the tumor-specific expression of peroxisome proliferator-activated receptor gamma I, a regulator of cell cycle arrest [34,35,36]. However, the mechanism by which MAZ regulates pMuSCs’ proliferation remains to be further clarified. We found that MAZ might be a transcription factor for SLPI through the Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing [19]. As a protease inhibitor, SLPI was first highlighted as a molecule that benefits the cell via its anti-proteolytic and immunomodulatory activities [37]. As research proceeds, increasing data have emerged to support this protein as a regulator of cell proliferation in various cells in humans [14,15,16,38]. However, no relationship between SLPI and the proliferation of MuSCs was found.
Thereafter, SLPI was selected as a candidate for mediating MAZ’s regulation of pMuSC proliferation. As expected, a promoting effect of SLPI on pMuSC proliferation was characterized, and SLPI was also shown to promote cell cycle progression. The similar effects of MAZ and SLPI indicate that there might be a relationship between them in regulating pMuSC proliferation. Through rescue experiments, it was confirmed that SLPI functions as a downstream gene regulating pMuSC proliferation because the promoting role of MAZ in cell proliferation was reversed when SLPI expression was inhibited. However, we did not make it clear whether MAZ regulated SLPI directly or indirectly here.
Additionally, it was found that both MAZ and SLPI could increase the expression of the CCND1 gene. CCND1, an essential regulator of cell cycle progression, was tightly regulated by MAZ/SLPI during pMuSC proliferation, showing a positive correlation between MAZ/SLPI and CCND1 expression. Studies have revealed that there was a high G/C content, characteristic of the MAZ binding motif, in the promoter of the human CCND1 gene, especially in the region adjacent to the transcription initiation site, and that MAZ bound to the CCND1 gene promoter by binding to the region [39]. There is also a G/C-rich region in the promoter of the porcine or mouse CCND1 gene promoter; therefore, MAZ could be expected to directly transcriptionally regulate CCND1 expression in the two species. Studies also indicated a link between SLPI and CCND1 transactivation in that SLPI dose-dependently modulated the activity of the CCND1 gene promoter in human cells [40]. Our results confirmed the regulatory effects of SLPI on CCND1 in pigs and mice. However, it was not revealed whether SLPI functioned as a direct transcription factor to regulate CCND1 gene expression. Further efforts should be made to help answer this question.
Wnt/β-catenin signaling is a key pathway regulating MuSC proliferation. Activation of the canonical Wnt signaling pathway robustly increases MuSC proliferation, and in activated MuSCs, the expression of β-catenin was strongly increased [41,42,43]. Additionally, β-catenin could bind the promoters of proliferative genes such as CCND1 and c-myc via TCF/Lef transcription factors to activate their expression [42]. Thereafter, it was speculated that canonical Wnt signaling might be involved in MAZ/SLPI’s regulation of pMuSCs’ proliferation. As expected, β-catenin expression was significantly increased by ectopic SLPI, while it was decreased by silencing SLPI, indicating a link between the SLPI level and Wnt/β-catenin activation. Further rescue experiments showed that inhibiting Wnt/β-catenin signaling reversed the promoting effects of SLPI on satellite cell proliferation, validating that SLPI regulates pMuSC proliferation through the canonical Wnt signaling pathway.
Additionally, we identified a functional point mutation, SNP −360C>T, in the promoter of the SLPI gene, which frequently occurs among Min, Yorkshire, and LMB pigs. Through dual-luciferase reporter and qPCR assays, we found that a C-to-T mutation increased the promoter activity of SLPI, and pigs with the TT genotype had high levels of SLPI mRNA. Combining these findings with the result that SLPI promotes pMuSC proliferation, the SNP should be considered a molecular marker for predicting the proliferative ability of pMuSCs, contributing to controlling muscle growth and regeneration.

5. Conclusions

This study focused on revealing the role of MAZ and SLPI and exploring the relationship between them in the regulation of pMuSCs. Gain- and loss-of-function analysis showed that both MAZ and SLPI could advance pMuSC proliferation by promoting cell cycle progression, and a MAZ/SLPI/Wnt-β-catenin axis regulated pMuSC proliferation. Additionally, an SNP, −360C>T, was found to occur frequently in the promoter of the SLPI gene among pigs from Min, Yorkshire, and LMB breeds. It was further revealed that the SNP −360C>T could influence the expression of SLPI, indicating its potential as a molecular marker for predicting the proliferative ability of pMuSCs. These findings will contribute to controlling muscle growth and regeneration, which will benefit pig production and human muscle injury.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vetsci13070709/s1; Figure S1: Detection of MAZ overexpression and interference efficiency; Figure S2: Detection of SLPI overexpression and interference efficiency; Figure S3: Effects of MAZ on cell cycle progression; Figure S4: Effects of SLPI on cell cycle progression; Figure S5: Original Western blot images for detecting the overexpression efficiency of MAZ and SLPI; Figure S6: Original Western blot images showing the effects of MAZ on the protein expression levels of PCNA and β-actin in porcine muscle satellite cells (pMuSCs) and C2C12 cells; Figure S7: Original Western blot images showing the effects of SLPI on the expression levels of PCNA, β-actin and β-tubulin in porcine muscle satellite cells (pMuSCs) and C2C12 cells; Table S1: Oligonucleotide sequences used in this study.

Author Contributions

Conceptualization, X.Y. and X.H.; validation, M.S., R.Z., and Q.Z.; investigation, M.S., R.Z., and Q.Z.; data curation, M.L. and M.T.; writing—original draft preparation, M.L. and M.S.; writing—review and editing, X.Y., M.T., and X.H.; visualization, M.T. and Q.Z.; supervision, X.Y. and X.H.; funding acquisition, X.Y. and X.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Heilongjiang Province for Distinguished Young Scholars (JQ2024C004), the National Natural Science Foundation of China (32172696), and the China Agriculture Research System of MOF and MARA (CARS-35).

Institutional Review Board Statement

The animal study protocol was approved by the tab of the Laboratory Animal Welfare and Ethics Committee of Northeast Agricultural University No. NEAUEC202402116, approval date: 20 June 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
MuSCMuscle satellite cell
MAZMyc-associated zinc finger protein
SLPISecretory leukocyte protease inhibitor
CCNB1Cyclin B1
CCK-8Cell counting kit 8
EdU5-ethynyl-2′-deoxyuridine
PMuSCsPorcine MuSCs
qPCRReal-time quantitative PCR
CDSCoding sequence
WTWild type
MTMutant type
SNPsSingle-nucleotide polymorphisms
PaxPaired box

References

  1. Proctor, D.N.; O’Brien, P.C.; Atkinson, E.J.; Nair, K.S. Comparison of techniques to estimate total body skeletal muscle mass in people of different age groups. Am. J. Physiol. 1999, 277, E489–E495. [Google Scholar] [CrossRef] [PubMed]
  2. Morgan, J.; Partridge, T. Skeletal muscle in health and disease. Dis. Model. Mech. 2020, 13, dmm042192. [Google Scholar] [CrossRef] [PubMed]
  3. Tajbakhsh, S.; Rocancourt, D.; Cossu, G.; Buckingham, M. Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 1997, 89, 127–138. [Google Scholar] [CrossRef] [PubMed]
  4. Sato, T.; Yamamoto, T.; Sehara-Fujisawa, A. miR-195/497 induce postnatal quiescence of skeletal muscle stem cells. Nat. Commun. 2014, 5, 4597. [Google Scholar] [CrossRef] [PubMed]
  5. Zhu, H.; Xiao, F.; Wang, G.; Wei, X.; Jiang, L.; Chen, Y.; Zhu, L.; Wang, H.; Diao, Y.; Wang, H.; et al. STAT3 Regulates Self-Renewal of Adult Muscle Satellite Cells during Injury-Induced Muscle Regeneration. Cell Rep. 2016, 16, 2102–2115. [Google Scholar] [CrossRef] [PubMed]
  6. Aloysius, A.; DasGupta, R.; Dhawan, J. The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence. Sci. Signal. 2018, 11, eaan3000. [Google Scholar] [CrossRef] [PubMed]
  7. Bossone, S.A.; Asselin, C.; Patel, A.J.; Marcu, K.B. MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination. Proc. Natl. Acad. Sci. USA 1992, 89, 7452–7456. [Google Scholar] [CrossRef] [PubMed]
  8. Song, J.; Murakami, H.; Tsutsui, H.; Tang, X.; Matsumura, M.; Itakura, K.; Kanazawa, I.; Sun, K.; Yokoyama, K.K. Genomic organization and expression of a human gene for Myc-associated zinc finger protein (MAZ). J. Biol. Chem. 1998, 273, 20603–20614. [Google Scholar] [CrossRef] [PubMed]
  9. Parks, C.L.; Shenk, T. Activation of the adenovirus major late promoter by transcription factors MAZ and Sp1. J. Virol. 1997, 71, 9600–9607. [Google Scholar] [CrossRef] [PubMed]
  10. Song, J.; Ugai, H.; Kanazawa, I.; Sun, K.; Yokoyama, K.K. Independent repression of a GC-rich housekeeping gene by Sp1 and MAZ involves the same cis-elements. J. Biol. Chem. 2001, 276, 19897–19904. [Google Scholar] [CrossRef] [PubMed]
  11. Gromak, N.; West, S.; Proudfoot, N.J. Pause sites promote transcriptional termination of mammalian RNA polymerase II. Mol. Cell. Biol. 2006, 26, 3986–3996. [Google Scholar] [CrossRef] [PubMed]
  12. Ren, L.X.; Qi, J.C.; Zhao, A.N.; Shi, B.; Zhang, H.; Wang, D.D.; Yang, Z. Myc-associated zinc-finger protein promotes clear cell renal cell carcinoma progression through transcriptional activation of the MAP2K2-dependent ERK pathway. Cancer Cell Int. 2021, 21, 323. [Google Scholar] [CrossRef] [PubMed]
  13. Himeda, C.L.; Ranish, J.A.; Hauschka, S.D. Quantitative proteomic identification of MAZ as a transcriptional regulator of muscle-specific genes in skeletal and cardiac myocytes. Mol. Cell. Biol. 2008, 28, 6521–6535. [Google Scholar] [CrossRef] [PubMed]
  14. Goselink, H.M.; Hiemstra, P.S.; van Noort, P.; Barge, R.M.; Willemze, R.; Falkenburg, J.H. Cytokine-dependent proliferation of human CD34+ progenitor cells in the absence of serum is suppressed by their progeny’s production of serine proteinases. Stem Cells 2006, 24, 299–306. [Google Scholar] [PubMed]
  15. Klimenkova, O.; Ellerbeck, W.; Klimiankou, M.; Ünalan, M.; Kandabarau, S.; Gigina, A.; Hussein, K.; Zeidler, C.; Welte, K.; Skokowa, J. A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation. Blood 2014, 123, 1239–1249. [Google Scholar] [CrossRef] [PubMed]
  16. Leelasukseree, R.; Chouyratchakarn, W.; Phutiyothin, C.; Pikwong, F.; Srisopar, O.; Baipaywad, P.; Udomsom, S.; Mongkolpathumrat, P.; Supanchart, C.; Kumphune, S. Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation. Sci. Rep. 2023, 13, 23013. [Google Scholar] [CrossRef] [PubMed]
  17. Sun, J.; Li, J.; Wu, Z.; Liang, Y.; Duan, R.; Zheng, M.; Wang, J.; Kong, D. SLPI suppresses hepatocellular carcinoma progression via endoplasmic reticulum stress induced apoptosis. Int. J. Biol. Sci. 2022, 18, 140–153. [Google Scholar] [CrossRef] [PubMed]
  18. GB/T 17236-2019; Operating Procedures of Livestock and Poultry Slaughtering. State Administration for Market Regulation and Standardization Administration of the People’s Republic of China: Beijing, China, 2019.
  19. Zhang, D.; Zhang, Q.; Wu, X.; Wang, L.; Zhang, X.; Liu, D.; Yang, X. Integrative Analysis of Chromatin Accessibility and Transcriptional Landscape Identifies Key Genes During Muscle Development in Pigs. Cells 2024, 13, 2118. [Google Scholar] [CrossRef] [PubMed]
  20. Yang, X.; Zhang, X.; Yang, Z.; Zhang, Q.; Hao, W.; Pang, Y.; Zhang, D.; Liu, D. Transcriptional Regulation Associated with Subcutaneous Adipogenesis in Porcine ACSL1 Gene. Biomolecules 2023, 13, 1057. [Google Scholar] [CrossRef] [PubMed]
  21. Zhang, X.H.; Li, J.X.; Wu, X.X.; Zhang, Q.; Tian, M.; Yang, S.Q.; Liu, D.; Yang, X.Q. PABPN1 functions as a downstream gene of CREB to inhibit the proliferation of preadipocytes. Anim. Biosci. 2025, 38, 41–53. [Google Scholar] [CrossRef] [PubMed]
  22. Yin, H.; Price, F.; Rudnicki, M.A. Satellite cells and the muscle stem cell niche. Physiol. Rev. 2013, 93, 23–67. [Google Scholar] [CrossRef] [PubMed]
  23. Relaix, F.; Zammit, P.S. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development 2012, 139, 2845–2856. [Google Scholar] [CrossRef] [PubMed]
  24. Persson, P.B. Skeletal muscle satellite cells as myogenic progenitors for muscle homoeostasis, growth, regeneration and repair. Acta Physiol. 2015, 213, 537–538. [Google Scholar] [CrossRef]
  25. Guilhot, C.; Catenacci, M.; Lofaro, S.; Rudnicki, M.A. The satellite cell in skeletal muscle: A story of heterogeneity. Curr. Top. Dev. Biol. 2024, 158, 15–51. [Google Scholar] [CrossRef] [PubMed]
  26. Zhang, F.; Wang, B.; Zhang, W.; Xu, Y.; Zhang, C.; Xue, X. Transcription Factor MAZ Potentiates the Upregulated NEIL3-mediated Aerobic Glycolysis, thereby Promoting Angiogenesis in Hepatocellular Carcinoma. Curr. Cancer Drug Targets 2024, 24, 1235–1249. [Google Scholar] [CrossRef] [PubMed]
  27. Haller, M.; Au, J.; O’Neill, M.; Lamb, D.J. 16p11.2 transcription factor MAZ is a dosage-sensitive regulator of genitourinary development. Proc. Natl. Acad. Sci. USA 2018, 115, E1849–E1858. [Google Scholar] [CrossRef] [PubMed]
  28. Medina-Martinez, O.; Haller, M.; Rosenfeld, J.A.; O’Neill, M.A.; Lamb, D.J.; Jamrich, M. The transcription factor Maz is essential for normal eye development. Dis. Model. Mech. 2020, 13, dmm044412. [Google Scholar] [CrossRef] [PubMed]
  29. Xiao, T.; Li, X.; Felsenfeld, G. The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization. Proc. Natl. Acad. Sci. USA 2021, 118, e2023127118. [Google Scholar] [CrossRef] [PubMed]
  30. Ortabozkoyun, H.; Huang, P.Y.; Cho, H.; Narendra, V.; LeRoy, G.; Gonzalez-Buendia, E.; Skok, J.A.; Tsirigos, A.; Mazzoni, E.O.; Reinberg, D. CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters. Nat. Genet. 2022, 54, 202–212. [Google Scholar] [CrossRef] [PubMed]
  31. Darbellay, F.; Rouco, R.; Andrey, G. MAZ regulates chromatin domain insulation and genome architecture. Nat. Genet. 2022, 54, 96–97. [Google Scholar] [CrossRef] [PubMed]
  32. Guetta-Terrier, C.; Karambizi, D.; Akosman, B.; Zepecki, J.P.; Chen, J.S.; Kamle, S.; Fajardo, J.E.; Fiser, A.; Singh, R.; Toms, S.A.; et al. Chi3l1 Is a Modulator of Glioma Stem Cell States and a Therapeutic Target in Glioblastoma. Cancer Res. 2023, 83, 1984–1999. [Google Scholar] [CrossRef] [PubMed]
  33. Zeng, J.; Zhang, L.; Huang, L.; Yu, X.; Han, L.; Zheng, Y.; Wang, T.; Zhang, N.; Yang, M. MAZ promotes thyroid cancer progression by driving transcriptional reprogram and enhancing ERK1/2 activation. Cancer Lett. 2024, 602, 217201. [Google Scholar] [CrossRef] [PubMed]
  34. Mueller, E.; Smith, M.; Sarraf, P.; Kroll, T.; Aiyer, A.; Kaufman, D.S.; Oh, W.; Demetri, G.; Figg, W.D.; Zhou, X.P.; et al. Effects of ligand activation of peroxisome proliferator-activated receptor gamma in human prostate cancer. Proc. Natl. Acad. Sci. USA 2000, 97, 10990–10995. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, X.; Southard, R.C.; Allred, C.D.; Talbert, D.R.; Wilson, M.E.; Kilgore, M.W. MAZ drives tumor-specific expression of PPAR gamma 1 in breast cancer cells. Breast Cancer Res. Treat. 2008, 111, 103–111. [Google Scholar] [PubMed][Green Version]
  36. Zaytseva, Y.Y.; Wang, X.; Southard, R.C.; Wallis, N.K.; Kilgore, M.W. Down-regulation of PPARgamma1 suppresses cell growth and induces apoptosis in MCF-7 breast cancer cells. Mol. Cancer 2008, 7, 90. [Google Scholar] [CrossRef] [PubMed]
  37. Zhu, J.; Nathan, C.; Jin, W.; Sim, D.; Ashcroft, G.S.; Wahl, S.M.; Lacomis, L.; Erdjument-Bromage, H.; Tempst, P.; Wright, C.D.; et al. Conversion of proepithelin to epithelins: Roles of SLPI and elastase in host defense and wound repair. Cell 2002, 111, 867–878. [Google Scholar] [CrossRef] [PubMed]
  38. Bouchard, D.; Morisset, D.; Bourbonnais, Y.; Tremblay, G.M. Proteins with whey-acidic-protein motifs and cancer. Lancet Oncol. 2006, 7, 167–174. [Google Scholar] [CrossRef] [PubMed]
  39. Wang, W.; Li, D.; Xu, Q.; Cheng, J.; Yu, Z.; Li, G.; Qiao, S.; Pan, J.; Wang, H.; Shi, J.; et al. G-quadruplexes promote the motility in MAZ phase-separated condensates to activate CCND1 expression and contribute to hepatocarcinogenesis. Nat. Commun. 2024, 15, 1045. [Google Scholar] [CrossRef] [PubMed]
  40. Zhang, D.; Simmen, R.C.; Michel, F.J.; Zhao, G.; Vale-Cruz, D.; Simmen, F.A. Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes. J. Biol. Chem. 2002, 277, 29999–30009. [Google Scholar] [CrossRef] [PubMed]
  41. Otto, A.; Schmidt, C.; Luke, G.; Allen, S.; Valasek, P.; Muntoni, F.; Lawrence-Watt, D.; Patel, K. Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J. Cell Sci. 2008, 121, 2939–2950. [Google Scholar] [CrossRef] [PubMed]
  42. Tanaka, S.; Terada, K.; Nohno, T. Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells. J. Mol. Signal. 2011, 6, 12. [Google Scholar] [CrossRef] [PubMed]
  43. Jones, A.E.; Price, F.D.; Le Grand, F.; Soleimani, V.D.; Dick, S.A.; Megeney, L.A.; Rudnicki, M.A. Wnt/β-catenin controls follistatin signalling to regulate satellite cell myogenic potential. Skelet. Muscle 2015, 5, 14. [Google Scholar] [CrossRef] [PubMed]
Vetsci 13 00709 g001
Figure 1. MAZ promotes muscle cell proliferation. (A,B) Effects of MAZ on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. (C) Effects of MAZ on expression of cell proliferation marker genes as revealed with qPCR. (D) Effects of MAZ on expression of MKI67 as revealed with ELISA. (E) Effects of MAZ on expression of PCNA as revealed with Western blotting. OE, overexpression; KD, knockdown; si, RNAi; NC, negative control. *, p < 0.05; **, p < 0.01. The same as below. Scale bar: 50 μm.
Figure 1. MAZ promotes muscle cell proliferation. (A,B) Effects of MAZ on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. (C) Effects of MAZ on expression of cell proliferation marker genes as revealed with qPCR. (D) Effects of MAZ on expression of MKI67 as revealed with ELISA. (E) Effects of MAZ on expression of PCNA as revealed with Western blotting. OE, overexpression; KD, knockdown; si, RNAi; NC, negative control. *, p < 0.05; **, p < 0.01. The same as below. Scale bar: 50 μm.
Vetsci 13 00709 g001
Vetsci 13 00709 g002
Figure 2. MAZ facilitates cell cycle progression. (A) Effects of MAZ on cell cycle progression. (B) Effects of MAZ on the expression of cell cycle regulators CCND1 and CCNB1. OE, overexpression; si, RNAi; NC, negative control. CCND1, Cyclin D1; CCNB1, Cyclin B1; G1, Gap 1 phase; S, Synthesis phase; G2/M, Gap 2/Mitosis phase. *, p < 0.05; **, p < 0.01.
Figure 2. MAZ facilitates cell cycle progression. (A) Effects of MAZ on cell cycle progression. (B) Effects of MAZ on the expression of cell cycle regulators CCND1 and CCNB1. OE, overexpression; si, RNAi; NC, negative control. CCND1, Cyclin D1; CCNB1, Cyclin B1; G1, Gap 1 phase; S, Synthesis phase; G2/M, Gap 2/Mitosis phase. *, p < 0.05; **, p < 0.01.
Vetsci 13 00709 g002
Vetsci 13 00709 g003
Figure 3. SLPI promotes muscle cell proliferation. (A,B) Effects of SLPI on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. (C) Effects of SLPI on expression of cell proliferation marker genes as revealed with qPCR. (D) Effects of SLPI on expression of MKI67 as revealed with ELISA. (E) Effects of SLPI on expression of PCNA as revealed with Western blotting. OE, overexpression; si, RNAi; KD, knockdown; NC, negative control. *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Figure 3. SLPI promotes muscle cell proliferation. (A,B) Effects of SLPI on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. (C) Effects of SLPI on expression of cell proliferation marker genes as revealed with qPCR. (D) Effects of SLPI on expression of MKI67 as revealed with ELISA. (E) Effects of SLPI on expression of PCNA as revealed with Western blotting. OE, overexpression; si, RNAi; KD, knockdown; NC, negative control. *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Vetsci 13 00709 g003
Vetsci 13 00709 g004
Figure 4. SLPI facilitates cell cycle progression. (A) Effects of SLPI on cell cycle progression. (B) Effects of SLPI on the expression of cell cycle regulators CCND1 and CCNB1. OE, overexpression; si, RNAi; NC, negative control. CCND1, Cyclin D1; CCNB1, Cyclin B1; G1, Gap 1 phase; S, Synthesis phase; G2/M, Gap 2/Mitosis phase. *, p < 0.05; **, p < 0.01.
Figure 4. SLPI facilitates cell cycle progression. (A) Effects of SLPI on cell cycle progression. (B) Effects of SLPI on the expression of cell cycle regulators CCND1 and CCNB1. OE, overexpression; si, RNAi; NC, negative control. CCND1, Cyclin D1; CCNB1, Cyclin B1; G1, Gap 1 phase; S, Synthesis phase; G2/M, Gap 2/Mitosis phase. *, p < 0.05; **, p < 0.01.
Vetsci 13 00709 g004
Vetsci 13 00709 g005
Figure 5. SLPI functions as a gene downstream of MAZ to promote cell proliferation. (A,B) Effects of co-transfection of MAZ and SLPI on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. OE, overexpression; si, RNAi; NC, negative control; EV, empty vector. *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Figure 5. SLPI functions as a gene downstream of MAZ to promote cell proliferation. (A,B) Effects of co-transfection of MAZ and SLPI on muscle cell proliferation as revealed with CCK-8 (A) and EdU (B) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. OE, overexpression; si, RNAi; NC, negative control; EV, empty vector. *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Vetsci 13 00709 g005
Vetsci 13 00709 g006
Figure 6. MAZ/SLPI axis regulates satellite cell proliferation through the Wnt-β-catenin pathway. (A) SLPI regulates the expression of β-catenin in muscle cells. (B,C) SLPI regulates muscle cell proliferation through the canonical Wnt pathway as revealed with CCK-8 (B) and EdU (C) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. In the panel: OE, overexpression; KD, knockdown; EV, empty vector; *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Figure 6. MAZ/SLPI axis regulates satellite cell proliferation through the Wnt-β-catenin pathway. (A) SLPI regulates the expression of β-catenin in muscle cells. (B,C) SLPI regulates muscle cell proliferation through the canonical Wnt pathway as revealed with CCK-8 (B) and EdU (C) assays. Red fluorescence indicates EdU-positive proliferating cells; blue fluorescence (Hoechst) labels all cell nuclei. Merge represents the overlay of EdU and Hoechst signals. In the panel: OE, overexpression; KD, knockdown; EV, empty vector; *, p < 0.05; **, p < 0.01. Scale bar: 50 μm.
Vetsci 13 00709 g006
Vetsci 13 00709 g007
Figure 7. Single-nucleotide polymorphism (SNP) is associated with the expression of SLPI. (A) Sequencing diagram of three genotypes at the SNP −360C>T site. Blue corresponds to C, green to A, red to T, and black to G. (B) Schematic structure of reporter genes. (C) Effects of SNP −360C>T on the expression of reporter genes. (D) Effects of SNP −360C>T on endogenous expression of SLPI. **, p < 0.01. Bars with different letters indicate significant differences (p < 0.01).
Figure 7. Single-nucleotide polymorphism (SNP) is associated with the expression of SLPI. (A) Sequencing diagram of three genotypes at the SNP −360C>T site. Blue corresponds to C, green to A, red to T, and black to G. (B) Schematic structure of reporter genes. (C) Effects of SNP −360C>T on the expression of reporter genes. (D) Effects of SNP −360C>T on endogenous expression of SLPI. **, p < 0.01. Bars with different letters indicate significant differences (p < 0.01).
Vetsci 13 00709 g007
Table 1. Distribution of SNP −360C>T in the promoter of SLPI among porcine populations.
Table 1. Distribution of SNP −360C>T in the promoter of SLPI among porcine populations.
BreedNumbersGenotype Frequency (Numbers)Allele Frequency
CCCTTTCT
Min1370.25 (34)0.29 (40)0.46 (63)0.390.61
Yorkshire300.1 (3)0.07 (2)0.83 (25) 0.130.87
LMB920.46 (42)0.41 (38)0.13 (12)0.660.34
Notes: Min, Min pig (a Chinese indigenous pig breed); Yorkshire, Yorkshire pig; LMB, Landrace × Min pig crossbred pigs. CC, CT and TT represent three genotypes at the target locus. C and T refer to two alleles at the detected locus.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Song, M.; Zhu, R.; Zhang, Q.; Li, M.; Tian, M.; He, X.; Yang, X. MAZ Regulates the Proliferation of Skeletal Muscle Satellite Cells via SLPI/Wnt-β-Catenin Signaling in Pigs. Vet. Sci. 2026, 13, 709. https://doi.org/10.3390/vetsci13070709

AMA Style

Song M, Zhu R, Zhang Q, Li M, Tian M, He X, Yang X. MAZ Regulates the Proliferation of Skeletal Muscle Satellite Cells via SLPI/Wnt-β-Catenin Signaling in Pigs. Veterinary Sciences. 2026; 13(7):709. https://doi.org/10.3390/vetsci13070709

Chicago/Turabian Style

Song, Mengke, Rongru Zhu, Qian Zhang, Meng Li, Ming Tian, Xinmiao He, and Xiuqin Yang. 2026. "MAZ Regulates the Proliferation of Skeletal Muscle Satellite Cells via SLPI/Wnt-β-Catenin Signaling in Pigs" Veterinary Sciences 13, no. 7: 709. https://doi.org/10.3390/vetsci13070709

APA Style

Song, M., Zhu, R., Zhang, Q., Li, M., Tian, M., He, X., & Yang, X. (2026). MAZ Regulates the Proliferation of Skeletal Muscle Satellite Cells via SLPI/Wnt-β-Catenin Signaling in Pigs. Veterinary Sciences, 13(7), 709. https://doi.org/10.3390/vetsci13070709

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop