A Novel miRNA Y-56 Mediates The Proliferation of Porcine Skeletal Muscle Satellite Cells By Targeting IGF-1R

The skeletal muscle phenotype of the Bama Xiang pig (BM) is signicantly different from the Landrace pig (LP). Uncovering the mechanism of porcine skeletal muscle growth will be of great signicance to elucidate the mechanism of the different formation. As the key post-transcriptional regulators, miRNAs play an indispensable role in skeletal muscle development. The proliferation of skeletal muscle satellite cells not only maintain the muscle stem cell population, but also provide a large number of muscle derived cells. Thus, the goal of this study is to explore the effects of a novel miRNA Y-56 on the porcine skeletal muscle satellite cells (PSCs). IGF-1R G2/M inhibits proliferation and induces apoptosis of prostate cancer cells by blocking the Raf/MEK/ERK and PI3K/Akt/mTOR signal pathways[J]. Tumour biology: the Oncodevelopmental

high meat yield but poor meat quality [2][3]. Elucidating the relevant mechanism of the different formation of skeletal muscle between the two pig breeds will provide a theoretical basis for improving the porcine production performance and furtherly utilize the pig as medical models. Therefore, understanding the mechanism of porcine skeletal muscle growth will be of great signi cance to elucidate the mechanism of the formation of the differences between the BM and the LP.
The number of muscle bers is determined prenatally and remains constant throughout postnatal life, so the growth of skeletal muscle after birth is achieved through the hypertrophy of existing muscle bers [4][5][6]. Studies have shown that the hypertrophy of muscle bers is related to the increase of DNA content, but the myonuclei do not possess the ability to synthesize DNA [7][8]. Satellite cells are the postnatal source of DNA contributed to growing muscle bers. DNA accretion occurs through proliferation of satellite cells followed by differentiation and fusion with existing muscle bers [9]. The proliferation of skeletal muscle satellite cells not only maintain the muscle stem cell population, but also provide a large number of muscle derived cells [10][11]. Therefore, skeletal muscle satellite cells as muscle stem cells play an indispensable role in the growth of skeletal muscle after birth.
MicroRNAs (miRNAs) are conserved, non-coding and endogenous single-stranded RNA molecules involved in the regulation of gene expression in organisms, whose length is generally about 20-24nt. They inhibit translation or promote mRNA degradation generally by associating with the 3' UTR of their regulatory target mRNAs [12]. Previous studies have identi ed many miRNAs are the key regulators of skeletal muscle growth [13][14]. According to the expression levels in skeletal muscle and other tissues, miRNAs are divided into two categories. One is the skeletal muscle-speci c miRNAs, such as miR-1, miR-206 and miR-133, which play an important role in muscle augmentation and differentiation of skeletal muscle satellite cells and myoblasts [15][16]. The other is the non-skeletal muscle-speci c miRNA, such as miR-181, miR-34b, miR-26 [17][18][19]. Research has found that IGF-1 stimulated the up-regulation of miR-133, while miR-133 in turn repressed IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis [20]. Rui-Song Ye et al. acquired the miRNA and mRNA expression pro les of anterior pituitaries from the BM and the LP by miRNA microarrays and mRNA-seq.Among them, Y-56 is a newly discovered miRNA with high expression in the pig, and no relevant studies have been conducted on it [21].
According to the above analysis, the objective of this study is to assess the expression level of Y-56 in various porcine tissues and to investigate its in uences on porcine skeletal muscle growth. We will observe the effects of overexpression of Y-56 on proliferation of porcine skeletal muscle satellite cells and will explore the relevant mechanism, which will lay the foundation for understanding the mechanism of the different formation between the BM and the LP.

Animals and cells
The muscle, heart, liver, spleen, kindey and cartilage tissues were collected from the female Bama Xiang pig (BM) and the Landrace pig (LP) at 7-days age, respectively. The porcine skeletal muscle satellite cells (PSCs) were isolated from porcine leg muscle tissues at 7 days of age, following the method [22]. Brie y, the collected leg muscle tissues were washed with PBS supplement with 0.5% penicillin/streptomycin, then the sterile muscle tissues were minced into small pieces and were digested with collagenase II (Gibco, USA) at 37°C for 3-6 h. The suspension was ltered through a 70 µm cell strainer and centrifuged at 3000 rpm for 15 min at room temperature, then we resuspended precipitation by PBS, and ltered by 40 µm again, next centrifuged at 3000 rpm for 15 min. After removing the supernatant, we resuspended precipitation by complete culture medium, next the cells was transferred to the culture dish. To separate and purify satellite cells, after 2 h, the suspension was transferred to new culture dish. All animal experiments were performed in accordance with the rules and regulations of the Animal Care and Experimentation Committee of Jilin University (Changchun, China).

Immuno uorescence Staining
The PSCs cultured in 96-well plates were rst treated with 4% formaldehyde for 15 min and then permeabilized by 0.1% Triton X-100. Next, after being blocked in 10% FBS for 30 min, the cells were sequentially incubated overnight with anti-Pax7 (Abcom, US; 1:100). Thereafter, the cells were treated with an Alexa Fluor 594-conjugated anti-mouse IgG (Bioworld Technology, USA; 1:200) according to the manufacturer's instructions. Finally, the nuclei were stained with DAPI (Beyotime, Shanghai, China) for 5 min. Then the images were captured with an inverted luorescence microscope (Nikon, Japan) and the data were analyzed in Image J software (National Institutes of Health, Bethesda, MD, USA).

Plasmid Construction and Cell Transfection
The psiCHECK-2 dual-luciferase reporter vector: The 3'UTR fragment of IGF-1R containing the Y-56 binding site was cloned into the psiCHECK-2 dual-luciferase reporter vector and the fragment named IGF-1R-WT and IGF-1R-MT were synthesized by Genewiz (Beijing, China). The sequences are listed in Table 1.
The RNA oligonucleotides used in this study, including Y-56 mimics, mimics-NC, Y-56 inhibitor, inhibitor-NC, si-IGF-1R and si-NC, were obtained from Gene Pharma (Shanghai, China) and were shown in Table 1. The oligonucleotides and plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's protocol with at least three replications.

Quantitative real-time PCR
Total RNA was isolated from the collected tissues and the PSCs after transfection using RNAiso Plus (Takara, Ostu, Japan). cDNA synthesis for mRNA was carried out using the PrimeScript RT reagent Kit (Takara, Japan). The reverse transcription reaction for miRNA was performed with the PrimeScript RT reagent Kit (Takara, Japan) using speci c Bulge-loop miRNA qRT-PCR Primer for Y-56 and U6 that were synthesized by Genewiz (Beijing, China). qRT-PCR reactions were carried out with the SYBR Green PCR Master Mix (Thermo Fisher Scienti c, MA) according to the manufacturer's instructions on an ABI PRISM 7900HT thermocycler (Applied Biosystems, Foster City, CA, USA). The comparative Ct (2 -ΔCt ) method was used for qRT-PCR data analysis. All ampli cations were performed in triplicate, and the mRNA level of the target genes was normalized to the GAPDH gene as an internal reference. The primers used for qRT-PCR are listed in Table 2. . Brie y, the PSCs were seeded in a 96-well format at a density of 5000 cells/well and were then transfected with different miRNAs, vectors or siRNAs as described above. 10 μL of CCK-8 solution was then added to each well containing 100 μL of the culture medium and then the plate was incubated for 30 min at 37℃, and the absorbance was read at a wavelength of 450 nm using a microplate reader (TECAN, Switzerland).

EdU staining
The PSCs were seeded in 96-well plates at a concentration of 2 × 10 3 per well. Then the PSCs were treated with Y-56 mimics, Y-56inhibitor, pcDNA3.1+IGF1R and si-IGF-1R for 48 h and incubated with 50 μM EDU (RiboBio, Guangzhou, China) for 3 h. The cells were washed twice with PBS, then were xed with 4% paraformaldehyde for 15 min and were permeabilized with 0.5% Trixon-100 for 20 min. At the end of each step, the cells were washed twice with PBS for 5 min. According to the kit, the cells were incubated in a mixture of Reagents for 30 min. The nuclei were stained with DAPI for 5 min. The stained cells were nally observed on an inverted luorescence microscope (Nikon, Japan) and the data were analyzed in Image J (National Institutes of Health, Bethesda, MD, USA).

Flow cytometry
The PSCs were cultured in a 6-well culture plate at a density of 4 × 10 5 per well and were treated with Y-56 mimics, Y-56inhibitor, pcDNA3.1+IGF1R and si-IGF-1R for 48 h. Then, the cells were digested with 0.25% trypsin and terminated with DMEMF-12 containing 20% FBS, next the cells were collected and xed in cold 70% ethanol overnight at 4 °C. The cells were then washed twice and stained with 50mg/mL propidium iodide (PI) for 30 min. Finally, the cell cycles were analyzed by ow cytometry (Becton Dickinson, USA).

Western blotting analysis
The PSM cells 48 h after transfection were harvested to extract the proteins using the lysis buffer (KeyGen Biotech, China) complement with protease and protein phosphatase inhibitor on ice. Protein concentration was determined using BCA Protein Assay Kit (Beyotime, China) and 30 μg protein per sample was loaded and separated using a 5% stacking gel and a 12% separating gel. Separated proteins were transferred to PVDF membrane, which was then blocked in 5% BSA for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing thrice (10 min once) in TBST, membranes were incubated with HRP conjugated goat anti-rabbit IgG or HRP conjugated goat anti-mouse IgG (Bioworld Technology, USA, 1:2000) 2 h at room temperature.
The Western blot was visualized by Enhanced Chemiluminescence (ECL, Bioworld, USA) performed on Bio-Rad Gel Doc XR instrument (Bio-Rad, Hercules, CA, United States). Thereafter, the gray value of the target strip was analyzed using Geno Sens Analysis software and the expression of the target proteins was analyzed by the ratio of the gray value of the target strip to the gray value of β-actin.

Statistical Analysis
All experimental results are presented as the mean ± SEM, with at least three independent replications. The statistically signi cant difference between groups was tested by one-way ANOVA using GraphPad Prism version 6.01. We considered P < 0.05 to be statistically signi cant. * P < 0.05; ** P < 0.01; *** P < 0.001.

Results
Y-56 is highly expressed in muscle and is upregulated in the BM compared with LP Rui-Song Ye et al. compared the miRNA expression pro les in anterior pituitary between the BM and the LP [21], and the Table S3 data -"All miRNAs expression" were used in this study. For an overview of the differentially expressed miRNAs, a volcano plot was drawn where the fold-change of miRNA expression was plotted on the x-axis, versus the signi cance of difference in miRNA expression between pools on the y-axis (Fig.1A). Differential expression analysis revealed that 41 miRNAs (32 up and 9 down) were regulated signi cantly in the BM after FDR correction ( fold change ≥1.5, P < 0.001). Among the 32 upregulated miRNAs, Y-56 was a new miRNA. Then we examined the expression of Y-56 in porcine other tissues and found that Y-56 highly expressed in muscle tissues (Fig.1B). Furthermore, we compared the expression level of Y-56 in leg muscle tissues between the LP and the BM, then the results showed that the Y-56 expression in the BM was signi cantly higher than that in the LP (Fig.1C)  Y-56 suppresses proliferation of the PSCs Firstly, we isolated the PSCs according to the above description and the PSCs were identi ed by pax7 (Fig.S1). To reveal the effect of Y-56 on proliferation of the PSCs, we transfected the Y-56 mimics into the PSCs ( Fig.2A). The EdU staining assay showed that the number of EdU labeled positive cells reduced in the Y-56 mimics group, unlike in the mimics-NC group (Fig. 2B-C) (P < 0.01). The CCK-8 assay also found that the Y-56 overexpression hampered the proliferation of the PSCs (Fig. 2D). In addition, ow cytometer assays showed that the PSCs numbers were lower in G1-phase (P < 0.01), whereas the proportion of the PSCs was increased in the S-phase (Fig.2E-F) (P < 0.01). The proteins encoded by CDK4, PCNA and Cyclin D1 have been identi ed to perform critical functions in G1, G2 and S phases during cell cycle progression [23][24][25]. When Y-56 was overexpressed, the results of qRT-PCR and western blotting showed that the expressions of CDK4, PCNA and Cyclin D1 were signi cantly decreased (Fig. 2G-I).
A. The transfection e ciency of Y-56 mimics; B. EdU staining after the transfection of Y-56 mimic in the PSCs; C. The statistical results of EdU staining; D. The CCK-8 assay after transfecting Y-56 mimics into the PSCs; E. Flow cytometry raw data of cell cycle analysis in the PSCs after overexpression of Y-56; F. The statistical results of cell cycle analysis; G. The mRNA expression levels of the cell cycle marker genes; H-I. The protein levels of the cell cycle marker genes. Results are shown as mean ± SEM and the data are representative of at least three independent assays. Independent sample t-test was used to analyze the statistical differences between groups. (* P < 0.05; ** P < 0.01, *** P < 0.001).
To further explore the effect of Y-56 on the PSCs proliferation, we next treated the cells with Y-56 inhibitor and inhibitor-NC, and the expression of Y-56 in the Y-56 inhibitor group was dramatically reduced below the inhibitor-NC group (Fig.3A) (P < 0.001). The number of EdU positive cells markedly increased in the Y-56 inhibitor group (Fig. 3B-C) (P < 0.001). Our CCK-8 assay also veri ed the knock-down of Y-56 induced cell viability (Fig. 3D). The ow cytometry assay results indicated down-regulation of the G1-phase and G2-phase cells and up-regulation the S-phase cells after suppressing the expression of Y-56 (Fig. 3E-F).
qRT-PCR and western blotting data showed that inhibition of Y-56 depressed the expression of CDK4, PCNA and Cyclin D1 (Fig. 3G-I). Therefore, we suggested that the overexpression of Y-56 suppressed the PSCs proliferation, while knock-down of Y-56 promoted the PSCs proliferation.
A. The transfection e ciency of Y-56 inhibitor; B. EdU staining after the transfection of Y-56 inhibitor in the PSCs; C. The statistical results of EdU staining; D. The CCK-8 assay after transfecting Y-56 inhibitor into the PSCs; E. Flow cytometry raw data of cell cycle analysis in the PSCs after inhibition of Y-56; F. The statistical results of cell cycle analysis; G. The mRNA expression levels of the cell cycle marker genes; H-I. The protein levels of the cell cycle marker genes. Results are shown as mean ± SEM and the data are representative of at least three independent assays. Independent sample t-test was used to analyze the statistical differences between groups. (* P < 0.05; ** P < 0.01, *** P < 0.001).

IGF-1R is a direct target gene of Y-56
The experiments described above showed that Y-56 can promote the PSCs proliferation ( Fig.2 and Fig.3). To better understand the regulatory mechanism of this process, we used TargetScan7.2 and miRTar Base to predict potential target genes. We detected IGF-1R as a candidate target gene and the seed sequence of Y-56 was presented in Fig. 4A. The secondary structures formed by Y-56 binding to the 3'UTR of IGF-1R was shown in Fig. 4B, as well as the corresponding minimum free energy formed by Y-56 and IGF-1R was -27.4 kcal/mol. Then we constructed wild-type IGF-1R 3'UTR (IGF-1R-WT) and mutant IGF-1R 3'UTR (IGF-1R-MT) dual luciferase reporter vectors accordingly. Then the mRNA expression levels of IGF-1R in porcine different tissues were detected, and the results showed that IGF-1R mRNA expression level was negatively correlated with Y-56 ( Fig. 4C and Fig. 1B). Next, we found that the dual-luciferase activity of IGF-1R-WT and Y-56 mimics co-transfected into the 293T cells was higher than that of co-transfected IGF-1R-WT and mimics-NC, while the dual-luciferase activity of IGF-1R-MT with Y-56 mimics and mimics-NC appeared to have no effect (Fig. 4D) (P < 0.01). To further verify that Y-56 targets the 3'UTR of endogenous IGF-1R, Y-56 mimics were transfected into the PSCs. The results of qRT-PCR and western blotting data suggested that IGF-1R mRNA and protein levels were reduced and increased, respectively, in Y-56 mimics group and mimics-NC group (Fig. 4E-G) (P < 0.01).
A. The Y-56 binding site in the IGF-1R mRNA 3'UTR; B. The potential secondary structures formed by Y-56 binding to IGF-1R mRNA 3'UTR; C. The relative mRNA expression of IGF-1R in porcine different tissues; D. The dual-luciferase reporter assay was performed by co-transfecting a wild-type or mutant IGF-1R 3'UTR with Y-56 mimic or mimic NC in 293T cells; E-G. The mRNA and protein levels of IGF-1R in the PSCs after being transfected with Y-56 mimic or Y-56 inhibitor. Results are shown as mean ± SEM and the data are representative of at least three independent assays. Independent sample t-test was used to analyze the statistical differences between groups. (* P < 0.05; ** P < 0.01).

IGF-1R promotes the cell proliferation of the PSCs
To address the function of IGF-1R in the regulation of the PSCs proliferation, the pcDNA3.1+IGF-1R expression vector was constructed, and the mRNA expression in the pcDNA3.1+IGF-1R group was higher than the pcDNA3.1 group (Fig. 5A) (P < 0.001). The EdU staining and CCK-8 assays were carried out to evaluate the PSCs proliferation, and the results showed that the number of cells in the proliferative phase was signi cantly increased due to the overexpression of IGF-1R (Fig. 5B-C and Fig. 5D) (P < 0.001). The number of cells in G1-phase and G2-phase were less than that in the control group after IGF-1R overexpression (P < 0.01), while the S-phase cells number were higher (Fig. 5E-F) (P < 0.01). Furthermore, the analysis of q-RT-PCR and western blotting found that overexpression of IGF-1R also signi cantly increased CDK4, PCNA and Cyclin D1 expression at both mRNA and protein levels ( Fig. 5G-I) (P < 0.01).
A. The mRNA level of IGF-1R after IGF-1R overexpression; B-C. The proliferation rate of the PSCs after overexpression of IGF-1R by EdU assay; D. The CCK-8 assay after overexpression of IGF-1R in the PSCs; E-F. Flow cytometry raw data and statistical results of cell cycle analysis after IGF-1R overexpression; G-H. The mRNA and protein expression of the cell cycle marker genes. Results are shown as mean ± SEM and the data are representative of at least three independent assays. Independent sample t-test was used to analyze the statistical differences between groups. (* P < 0.05; ** P < 0.01; *** P < 0.001).
Next, we employed siRNA technology to con rm the role of IGF-1R in the PSCs. It showed that si-IGF-1R down-regulated the expression of IGF-1R e ciently at the mRNA (Fig. 6A) (P < 0.001). The EdU assay found that the inhibition of IGF-1R could hamper the proliferation of the PSCs (Fig. 6B-C) and the CCK-8 assay showed the same results (Fig. 6D). In addition, the cells in G1-phase were signi cantly increased after being transfected with si-IGF-1R, but the number cells of the S-phase and G2-phase were noteworthy decreased ( Fig. 6E-F) (P < 0.01). What's more, when the si-IGF-1R was transfected into the PSCs, the expression of CDK4, PCNA and Cyclin D1 were suppressed at both mRNA and protein levels ( Results are shown as mean ± SEM and the data are representative of at least three independent assays. Independent sample t-test was used to analyze the statistical differences between groups. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Discussion
The BM (Bama Xiang pig) has obvious growth difference with the LP (Landrace pig). As a local breed in China, the BM has short size, slow growth rate but better meat quality, and it is an important model for medical experiment [1][2][3]. Uncovering the relevant mechanism of the differences between the two breeds will provide favorable basic evidence for the future porcine industry and even the medical industry. Skeletal muscle, an important tissue that accounts for 40-60% of the animal body, plays an indispensable role in the growth and development of animals after birth [26]. miRNAs have been proved to be the key factor to regulate the growth and development of skeletal muscle [27]. In the prenatal skeletal muscles of Landrace, Tongcheng and Wuzhishan pigs, miRNA-133b and miRNA-206 had different expression patterns, which indicated that the same miRNAs would play regulatory mechanisms in different genotype pigs [28]. Skeletal muscle satellite cells, as muscle stem cells, are an important cell source of muscle development after birth [8][9]. Skeletal muscle satellite cells bear on the formation of muscle in postpartum animals, they go through the cell proliferation, differentiation and fusion to form muscle, nally [11,29]. Therefore, its proliferation ability will directly affect its differentiation and fusion. This is the reason why this paper focused on exploring the effect of a novel miRNA on the proliferation of skeletal muscle satellite cells during the development of porcine.
Many miRNAs were proved to be involved in the regulation of skeletal myogenesis by binding to its target genes, for examples, miR-34c inhibits cell proliferation of the PSCs but promotes the differentiation by targeting Notch1 [30], and miR-199b has an inhibitory effect on the proliferation of muscle satellite cells in Large White pigs by targeting the JAG1 gene [31], as well as miR-27b promotes muscle development by inhibiting MDFI expression [32]. Similar as previously reported, Our EdU staining and CCK-8 assays showed that the novel miRNA Y-56 had the ability to inhibit the proliferation of skeletal muscle satellite cells by targeting IGF-1R gene (Fig 2-4). However, the effect of Y-56 on the differentiation ability of skeletal muscle satellite cells remains to be further explored.
In addition, the cell proliferation of PSCs is dependent on an active cell cycle [31]. The results of ow cytometry showed that high expression levels of Y-56 also increased the percentage of cells in the G1phase and reduced the number of cells in S-phase by inhibiting the expression of IGF-1R ( Fig. 2E and Fig.  5E). In the cell cycle, CDK4 is a kinase that regulates the transition from the G1 to S phases of the cell cycle [33], and PCNA is a key factor in DNA replication and cell cycle regulation [34], as well as Cyclin D1 is proposed to serve as an active switch in the regulation of continued cell cycle progression [35].
Consistently, the mRNA and protein expression levels of CDK4, PCNA and Cyclin D1 were all shown to be downregulated when overexpressing Y-56, while these marker genes were upregulated when the expression of IGF-1R was increased in the PSCs. Thus, Y-56 inhibit the expression levels of cell cycle factors: CDK4, PCNA and Cyclin D1 by targeting IGF-1R gene to suppress the cycle process of the PSCs.
IGF-1R is an important receptor of IGF-1, while IGF-1 has been proved to have a positive effect on the growth and development of the skeletal muscle [36][37]. Meanwhile, IGF-1R is required for cell growth and development and to maintain the cell cycle [38][39][40]. Substantial pieces of direct evidence have emerged that the biological function of IGF-1R is generally achieved by activating two of the following most important intracellular signaling pathways: MAPK/ERK and PI3K/AKT after binding to the ligand IGF-1 [41][42]. In our previous study, we found that IGF-1R promotes the cell proliferation on the PK-15 cells and IGF-1R promotes C2C12 proliferation through the AKT and ERK signaling pathways [43][44]. There have been no such results regarding the function of IGF-1R on the PSCs published previously, however, our results demonstrate the proliferation-promoting function of IGF-1R on the PSCs, as well as, overexpression of IGF-1R increases the cell cycle and up-regulates the marker genes (CDK4, PCNA and Cyclin D1) expression levels ( Fig. 5-6).

Consent for publication
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