MSTN mutant promoted bovine muscle satellite cell proliferation by regulating the binding of SMAD2/SMAD3 with CDKN1C

Background: Myostatin (MSTN), also known as growth/differentiation factor 8, mostly expressed in skeletal muscle and plays negative roles in regulation of muscle development. Previous studies had proved that MSTN have important effect on cell proliferation. Therefore we aimed to investigate the mechanism of MSTN in regulating the proliferation of bovine muscle satellite cells (MSCs). Methods: Bovine MSCs of MSTN mutant (MT) and wild type (WT) were obtained, we detected the cell proliferation and cell cycle by EdU proliferation assay and Flow cytometry. Then we detected the expression of genes associated with cell cycle by Real-time PCR and Western blotting . RNA-seq and Chromatin immunoprecipitation (ChIP)assay were performed to research the mechanism of MSTN in regulating the cell proliferation. Results: In this study, we found that MSTN mutant promoted the proliferation of MSCs. The expression of CyclinA, CyclinD and CyclinE were all increased after MSTN mutant, while the expression of CDKN1C (P57), CDKN2A, CDKN2C and CDKN2D were down-regulated, which were consistent with the promotion of cell proliferation. Among these genes, CDKN1C(P57) down-regulated most signicantly. RNA-seq results showed that MSTN mutant affected the SMAD binding, so we performed ChIP-qPCR and demonstrated that the SMAD2/SMAD3 transcription factor combined with the promoter of CDKN1C thus to increase the expression of CDKN1C, this demonstrating that MSTN regulated the expression of CDKN1C through SMAD2/SMAD3 complex. Finally, overexpression of SMAD3 in wild type cells increased the expression of CDKN1C, further suggested that SMAD3 regulated the expression of CDKN1C. Conclusion: MSTN mutant down-regulated the expression of SMAD2/SMAD3, then reduced the to the expression of CDKN1C, thus to inhibit the expression of CDKN1C, then promoting the cell while the S phase increased on day 1, but decreased after 1 day of myogenesis. These results showed that MT MSCs exited the cell cycle and began to differentiate immediately when induced to myogenic differentiation, but WT MSCs occurred these after 2 days of myogenesis induction. The cell cycles results were consistent with the cell morphology change results observed by microscope.


Background
Myostatin (MSTN), is a member of the transforming growth factor β (TGF-β) family, also known as growth/differentiation factor-8; it is expressed in many tissues but mostly expressed in skeletal muscle, played important roles in the regulation of muscle development [1]. Many studies had proved that MSTN mutant leads to muscle hypertrophy due to both increased myo ber numbers and increased myo ber sizes, and muscle mass is the most important trait in livestock production, so MSTN has received a lot of attention. Up to now, many species of MSTN de ciency animals have been obtained, including cattle [2], humans [3], sheep [4], dogs [5], pigs [6],goats [7] and birds [8] without causing severe adverse consequences.
Previous studies had proved that MSTN have important effect on cell proliferation. Transcriptome analysis of muscle tissue of MSTN knockout chickens found that differentially expressed genes were enriched in the process of cell proliferation and differentiation [9].Knockout of MSTN promote cell proliferation, while overexpression of MSTN inhibit cell proliferation and DNA synthesis [10,11]. McCroskery et al. (2003) showed that overexpression of MSTN up-regulates P21 and downregulates CDK2 expression [12], MSTN negatively regulated the progression of G1/S phase, thus to inhibit the cell cycle and maintain the resting state of satellite cells. Inhibition of MSTN expression by RNAi in C2C12 cells promoted the cell cycle, cells of G0/G1 phase decreased, while S phase cells increased [13,14].
Inhibition and knockout of MSTN expression by RNAi and zinc nger synthase in goat and sheep myoblasts promoted cell proliferation, decreased the expression of P21 while increased CDK2 expression [14,15]. But the further mechanism of MSTN regulating cell cycles were not well known. In this study, we used MSTN mutant bovine muscle satellite cells (MSCs) described in the previous study [16], detected the effect of MSTN mutant to cell cycles and the regulating pathway in the MSTN mutant bovine muscle satellite cells we obtained.

Cell culture
Muscle satellite cells (MSCs) cultured from cattle of MSTN mutant (MT) and wild type (WT) were obtained and identi ed as our previously reported [16]. MSCs of both MT and WT were cultured in DMEM supplemented with 10% horse serum (HS) and 20% fetal bovine serum (FBS) with 5% CO 2 at 38.5°C.
When the con uence of cells reached 100%, subculture was performed.

Ethynyl-2'-deoxyuridine (EdU) assays
The proliferation of MT and WT MSCs were detected by using EdU proliferation kit(RuiBo, Guangzhou, China), the operation method is carried out according to the manufacturer's protocol. Both MT and WT bovine muscle satellite cells were incubated with EdU for 12 h and washed twice with PBS for 5min each time; then xed the cells by 4% paraformaldehyde for 30min, permeabilized the cells with 0.5% Triton X-100 for 20min, labeled the cells with Apollo® uorescent dye (RuiBo, Guangzhou, China) for 30min in dark. Finally in ltrated the cells with PBS. Images were captured using a confocal microscope (Nikon, Tokyo, Japan) and calculated the percentage of EdU-positive cells.

Myogenic cells differentiation
Myogenic differentiation was induced when the con uence of the cultured cells reached 80-90% by using low serum medium of which 2% horse serum was supplemented to DMEM. Myogenic differentiation induction lasts for 3d.

Flow cytometry assay
Cell cycles were detected by the Cell Cycle and Apoptosis Analysis Kit (Beyotime, China).MSCs and differentiated MSCs were collected and washed with PBS. Next, cells were xed for over 24h in 4°C with 70% ethanol, then the xed cells were washed with pre-cooled PBS and then labeled with PI solutions avoiding light for 30min in 37°C solid bath, then the cells were detected using a ow cytometer(BD Biosciences). Cell cycles were determined by FlowJo 7.6 software.

Real-time PCR
Expression of genes associated with cell cycle in mRNA level was detected by Real-time PCR. RNA of the myogenic cells and myotubes of MT and WT cattle were extracted by RNAiso Plus kit (9108; Takara), then the RNA was reverse-transcribed into cDNA by reverse transcription reagent (RR047A; Takara). The genes associated with cell cycle were ampli ed using ABI7500 real-time PCR (Applied Biosystems, America). The primers used for PCR are listed in Table S1. DNA ampli cation was performed as the previously described program: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 34 s, and a nal melting curve stage [16]. The cycle threshold (Ct) values of housekeeping gene GAPDH were used to normalized the targeted genes using the 2 -ΔΔCt method [17].

Western blotting
Western blotting was performed according to reference of our previously reported [16]. Brie y, the protein of cell samples were extracted using Radio Immunoprecipitation Assay (RIPA) buffer, then boiled and with secondary antibodies, nally captured the images using Tanon Gel Imaging System (Tanon 4800).

RNA-seq analysis
Total RNA of wild type (WT) and MSTN mutant (MT) bovine muscle tissues were extracted using TRIzol® reagent (Invitrogen). RNA sequencing was operated in Sangon Biotech company. RNA Sequencing data are shown in Table S3. Clean reads were aligned to Bos Taurus genome (NCBI:ID82) as described in reference [18]. KEGG pathway analysis and GO functional enrichment were conducted by Omicshare tools and David online database.

Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using Pierce TM Magnetic ChIP Kit (26157; Thermo Fisher Scienti c) as previously described [19]. anti-SMAD2+SMAD3 (ab202445; Abcam, USA) monoclonal antibodies was used to settle the bound DNA, the settled DNA was analyzed by quantitative PCR. Primers used for ChIP-qPCR were provided in Table S2.

Statistical analysis
T-tests were used to analysis the signi cance difference between MSTN mutant and wild type MSCs. pvalues of less than 0.05 were considered statistically signi cant.

MSTN mutant promoted the proliferation of muscle satellite cells (MSCs)
The muscle satellite cells (MSCs) derived from both MT and WT cattle were shown in Fig 1A, and identi ed in our previously reported reference [16]. We analyzed the proliferation of the MSCs isolated from MT and WT to detect the effect of MSTN mutant to cell proliferation. EdU proliferation assay results showed that MSTN mutant promoted the proliferation of MSCs( Figure 1B, 1C). Then we analyzed the cell cycle of these two cells by Flow cytometry. Results showed that cell numbers of G0/G1 and G2/M phase decreased while the cell numbers of S phase increased compared with WT( Figure 1D, 1E). This results indicated that MSTN mutant promoted the cell cycle mainly by promoting the DNA synthesis of cells.

MSTN mutant promoted the myogenic diffefentiation by exiting the cell cycle earlier
We then induced the MSCs of MT and WT to myogenic differentiation and the cell morphology was observed. Results indicated that myotubes were formed in MT cells after 1 day of myogenic differentiation, while this occurred after 2 days of myogenic differentiation. After 3 day' myogenic differentiation, there were more myotubes formed in MT cells than that in WT cells ( Figure 2A). Then we analyzed the cell cycle of MSCs after 1, 2 and 3 day's myogenic differentiation in MT and WT cells. As shown in Figure 2B,2C and 2D, for MT MSCs, Cell number of G1 phase increased immediately after differentiated, while S phase decreased immediately after 1 day of myogenesis. For WT MSCs, Cell number of G1 phase increased signi cantly after 2 days of myogenesis, while the S phase increased on day 1, but decreased after 1 day of myogenesis. These results showed that MT MSCs exited the cell cycle and began to differentiate immediately when induced to myogenic differentiation, but WT MSCs occurred these after 2 days of myogenesis induction. The cell cycles results were consistent with the cell morphology change results observed by microscope.

MSTN mutant promoted cell proliferation mainly by down-regulating CDKN1C
We then detected the expression of cell cycle related genes. Among the Cyclins we detected, the expression of CyclinA, CyclinD and CyclinE were all increased after MSTN mutant, and the most signi cantly increased is CyclinA ( Figure 3A, 3C and 3D). The expression of CDK1, CDK2 and CDK6 were all increased (Fig 3A). The expression of Cyclin dependent kinase inhibitors were not the same after MSTN mutant, CDKN1C (P57), CDKN2A, CDKN2C and CDKN2D were down-regulated, which were consistent with the promotion of cell proliferation. Among these genes, CDKN1C(P57) down-regulated most signi cantly ( Figure 3B, 3C, 3D). These results indicated that MSTN mutant promoted MSCs proliferation mainly by down-regulating CDKN1C, then promoting the CyclinA-CDK2 expression, thus to promote the synthesis of DNA and cell cycle.

RNA-seq analysis
To further analysis the mechanism of MSTN mutant to muscle cell proliferation and cell differentiation, we compared transcripts of MSTN mutant (MT) bovine and the controls. Skeletal muscle of six cattle were performed RNA-seq analysis consist of 3 MT cattle and 3 WT controls. DEGs were identi ed by Pvalue < 0.05 at the same time |fold change (FC)| > 1.5. Notably, expressions of 721 genes were found signi cantly changed has occurred in the MT cattle. Of these, the expressions of 74 genes were upregulated, while 647 genes were down-regulated in the MT cattle ( Figure 4A). We then performed Realtime quantitative PCR to verify the expression of the most signi cant DEGs ( Figure 4B), results were consistent with the RNA-seq results ( Figure 4C), proving that the transcriptome data is usable.
Succeeding Gene Ontology (GO) analysis indicated that the signi cantly differently expressed genes were with regard to the biological processes of muscle cell differentiation regulation, muscle cell proliferation, especially G1/S transition, and TGF-β pathway ( Figure 4D). At the same time, the DEGs were associated with SMAD binding (Figure 4D), this indicated that MSTN mutant may affect the binding of SMAD with downstream genes, thus to regulate cell proliferation.
MSTN regulated the expression of CDKN1C by SMAD2/SMAD3 pathway Since SMAD2/SMAD3 complex were downstream transcription factors of the MSTN type II receptors, MSTN mutant increased the expression of CDKN1C, so we speculated that SMAD2/SMAD3 complex may combine with CDKN1C promoter and regulate the expression of CDKN1C. This was proved by the transcription factor binding prediction analysis using the JASPAR database, results showed that SMAD2/SMAD3 complex bound with the promoter region of CDKN1C (NC_037355.1) from -804 to -792 ( Figure 5B). Furthermore, we also con rmed the binding of SMAD2/SMAD3 complex with promoter of CDKN1C by performing a ChIP-qPCR assessment. We used an anti-SMAD2/SMAD3 monoclonal antibody to settle the DNA bound to SMAD2/SMAD3 complex, then ampli ed the CDKN1C promoter region from the deposited DNA. Region detected was from -926 to -757 on the CDKN1C promoter ( Figure 5C).

Overexpression of SMAD3 promoted the expression of CDKN1C
To further discuss the in uence of SMAD3 to CDKN1C expression, we then constructed the overexpression vector of SMAD3 (Figure6A, 6B), then transfected the WT MSCs with this overexpression vector. Results indicated that the expression of SMAD3 in the transgenic cells increased both in mRNA and protein levels as displayed in Figure 6C, 6D, and 6E. Then we detected the expression of CDKN1C, results showed that the expression of CDKN1C both in mRNA and protein levels increased signi cantly after overexpression of SMAD3( Figure 6F, 6G, 6H). These results indicated that SMAD3 promoted the expression of CDKN1C. So MSTN mutant decreased the expression of SMAD3, then suppressed the expression of CDKN1C, thus to promote the CyclinA-CDK2 complex expression, promoted DNA synthesis and cell proliferation.

Discussion
Myostatin (MSTN), is mainly expressed in skeletal muscle, and has a negative regulatory effect on the development of muscle mass [1]. Studies in many species have shown that MSTN gene mutation can cause abnormal muscle development, including an increase in the number of muscle bers and an increase in the cross-sectional area of muscle bers without causing severe adverse consequences, as previously reported in cattle [2], humans [3], sheep [4], dogs [5], pigs [6],goats [7] and birds [8]. Muscle mass is an important indicator in animal production, so many effective strategies have been made to product animals with increased muscle masses by preventing the expression of MSTN [7,.
Previous studies had proved that MSTN have important effect on cell proliferation. Knockout of MSTN promote cell proliferation, while overexpression of MSTN inhibit cell proliferation and DNA synthesis [10,11,14]. In the present study, we studied the proliferation of MSTN mutant (MT) and wild-type (WT) bovine muscle satellite cells.
The MSTN mutant (MT) satellite cells were found to have a accelerated cell cycle than the control cells.
Then we analyzed the cell cycle of these two cells by Flow cytometry. Results showed that cell number of G0/G1 phase reduced while the cell number of S phase increased compared with WT, these results indicated that MSTN mutant promoted the cell cycle mainly by promoting DNA synthesis. This was consist with the previous studies [10,12]. RNA-seq results also showed that MSTN mutant regulated the cell proliferation and cell cycle, especially G1/S transition. This further demonstrated that MSTN mutant promoted the cell cycle mainly by promoting DNA synthesis.
During the process of myogenic differentiation, myoblasts will withdraw from the cell cycle and obtain the apoptotic phenotype to fuse into myotubes. In the present study, when the MT and WT MSCs were induced to myogenic differentiation, MT cells quit from the cell cycle and began to differentiation earlier than the WT controls. This was consist with the previous study [11,24,25]. Then we detected the cell cycle during myogenic differentiation, results showed that MT MSCs withdraw from the cell cycle and began to differentiate immediately when induced to myogenic differentiation, but WT MSCs occurred these after two days of myogenesis induction. So the promotion of myogenic differentiation due to MSTN mutant had two reasons: on one hand, MSTN mutant promoted the expression of myogenic related genes such as MyoD and MyoG [26,27], then to promote myogenic differentiation; on the other hand, MSTN mutant promoted the cells withdraw from the cell cycle and began to differentiation earlier than the control, thus to promote myogenic differentiation [24].
Cell cycle progression was regulated by a series of related genes, so we detected the expression of cell cycle related genes. The expression of Cyclins and CDKs were all up-regulated, while the Cyclin dependent kinase inhibitors, such as CDKN1C (P57), CDKN2A, CDKN2C and CDKN2D were down-regulated, which were consistent with the promotion of cell proliferation. Among which the expression of CyclinA up-regulated most signi cantly and CDKN1C down-regulated most signi cantly, these results indicated that MSTN mutant promoted MSCs proliferation mainly by down-regulating CDKN1C, then promoting the CyclinA-CDK2 expression, thus to promote the synthesis of DNA and cell cycle.
SMAD were key transcription factors in TGF-β pathway. In this study, we obtained DEGs from MT and WT cattle muscle tissue focused on SMAD binding, this indicated that MSTN may regulate cell cycle by affecting the binding of SMAD transcription factors with the downstream genes. At the same time, previous studies have shown that up-regulation of TGF-β will increase the expression of CDK inhibitor p15, p16, p21 and p27 by increasing the binding of pSMAD3 to the their promoters [28]. So we intend to investigate that if CDKN1C (P57) can also bind with SMAD2/SMAD3. We predicted the binding of SMAD2/SMAD3 complex to the region of CDKN1C promoter by JASPAR database, subsequently a ChIP assay were performed using an anti-SMAD2/SMAD3 monoclonal antibody. Results of ChIP-qPCR con rmed the directly bound of SMAD2/SMAD3 complex with the CDKN1C promoter region. Moreover, overexpression of SMAD3 promoted the expression of CDKN1C. Since MSTN mutations have been found to reduce the activity of SMAD2/SMAD3 complex [29,30], so in the present study, MSTN mutant decreased the promotion of SMAD2/SMAD3 to the expression of CDKN1C, so the expression of CDKN1C was decreased, and then promoted the CyclinA-CDK2 expression, thus to promote the synthesis of DNA and cell cycle.

Conclusion
In summary, the MSTN mutation leads to decreased activity of SMAD2/SMAD3, which bound directly to the promoter region of CDKN1C, SMAD3 overexpression promoted the expression of CDKN1C, so MSTN mutant down-regulated the expression of CDKN1C by SMAD2/SMAD3 pathway. Down-regulation of CDKN1C then promoted the CyclinA-CDK2 complex, then promoted the cell proliferation.   Cyclins and cyclin dependent kinase inhibitors in protein level detected by western blot and quanti ed in D. T-test was used to generate p-value.*p<0.05,**p<0.01,***p<0.001.

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