Effect of miR-493-5p on proliferation and differentiation of myoblast by targeting ANKRD17

The hypertrophy and conversion of postnatal muscle fibers largely determine the yield and quality of meat, which is closely related to the economic value of pigs. MicroRNA (miRNA), as a kind of endogenous noncoding RNA molecule, is widely involved in myogenesis of livestock and poultry. The longissimus dorsi tissues of Lantang pigs at 1 and 90 days (LT1D and LT90D) were collected and profiled by miRNA-seq. We found 1871 and 1729 miRNA candidates in LT1D and LT90D samples, and 794 miRNAs were shared. We identified 16 differentially expressed miRNAs between two tested groups and explored the function of miR-493-5p inmyogenesis. The miR-493-5p promoted the proliferation and inhibited the differentiation of myoblasts. Using GO and KEGG analyses of 164 target genes of miR-493-5p, we found that ATP2A2, PPP3CA, KLF15, MED28, and ANKRD17 genes were related to muscle development. RT-qPCR detection showed that the expression level of ANKRD17 was highly expressed in LT1D libraries, and the double luciferase report test preliminarily proved that miR-493-5p and ANKRD17 have a directly targeting relationship. We established miRNA profiles for the longissimus dorsi tissues of 1-day-old and 90-day-old Lantang pigs and found that miR-493-5p was differentially expressed and associated with myogenesis by targeting ANKRD17 gene. Our results should serve as a reference for future studies on pork quality.


Introduction
The yield and quality of pork are the key indicators to measure the economic traits of pigs. Over the past few decades, the excessive pursuit of growth rate in pig breeding has led to the decline of meat quality (Schwab et al. 2006). In general, muscle fiber is the basic unit of muscle, whose number does not increase after birth (Moss 1968). The growth potential of postnatal muscle mainly focuses on the hypertrophy and conversion of muscle fibers (Paredes et al. 2013), which has an important impact on meat quality, such as meat tenderness, water power, and color (Ashmore 1974;Joo et al. 2013). Myofiber types are divided into oxidation and fermentation types (Lefaucheur 2010). Oxidation fibers include types I and IIa and mainly rely on aerobic metabolism, and the diameter is small (Peter et al. 1972). Oxidized muscle fibers are rich in oxidized myoglobin, thus making pork with bright red color (Karlsson et al. 1999). Fermentative muscle fibers include type IIx and type IIb fibers, and the diameter of fermentative fibers is large (Larzul et al. 1997); and the main breathing mode is anaerobic fermentation (Schiaffino and Reggiani 1996), which is related to water holding capacity of pork (Lawrie 1970;Bowker et al. 2005). Therefore, muscle fiber development is an important entry point to study muscle development.
The growth and development of muscle fibers involve complex molecular mechanism (Anakwe et al. 2002;Borycki et al. 1999;Swoap et al. 2000). With the progress of biological sequencing technology, the function of noncoding genes in muscle development has been gradually excavated. MicroRNA (miRNA) is a kind of endogenous noncoding small-RNA fragments containing 22-24 nucleotide bases (Ambros 2004). They mainly target 3′ untranslated region (UTR) of coding genes through base complementarity, affecting their translation and original biological function (Macfarlane and Murphy 2010;Bartel 2009;Filipowicz et al. 2008). It is reported that a number of miRNAs are widely involved in the proliferation and differentiation of livestock and poultry muscle fibers (Luo et al. 2014;Zhao et al. 2016a;Zhu et al. 2019).
In general, the conversion of muscle fiber types is mainly concentrated in the early 3 months after birth (Wank et al. 2006), and the proportion of type IIb fibers in the longissimus dorsi of pigs increased obviously (Lefaucheur and Vigneron 1986). In order to explore the potential miRNAs involved in this biological process, we selected the longissimus dorsi tissues of 1-day-old and 90-day-old Lantang pigs and screened the differentially expressed miRNA candidates through high-throughput sequencing. Among them, miR-493-5p was found to inhibit the proliferation of myoblasts and promote their differentiation by targeting ANKRD17 gene, revealing the potential mechanism and function of miR-493-5p in myogenesis. Our objective was to identify miRNA candidates regulating the development of muscle fibers and provide theoretical data for pig breeding.

miR-493-5p promotes C2C12 proliferation
We synthesized miR-493-5p mimics and inhibitor and verified the overexpression and knockdown efficiency of miR-493-5p by RT-qPCR (Fig. 2a). Compared with the control group, miR-493-5p mimics and inhibitor significantly increased and knocked down the expression level of miR-493-5p (P < 0.01), indicating the effect of miR-493-5p mimics and inhibitor that can be used for subsequent experiments. Then, C 2 C 12 cells were transfected with miR-493-5p mimics and inhibitor, and the relative expression levels of cell proliferation-related genes such as PCNA, cyclin D1 and cyclin E were evaluated by RT-qPCR. The expression levels of PCNA (P < 0.05) and cyclin D1 (P < 0.01) increased significantly in over-expressed cells, while the expression level of cyclin E did not change significantly (P > 0.05). After knockdown of miR-493-5p, the expression levels of PCNA (P < 0.05) and cyclin D1 (P < 0.05) in C 2 C 12 cells decreased significantly, and the decrease in level of cyclin E was not significant (P > 0.05) (Fig. 2b). After transfection with miR-493-5p mimics, the protein expression levels of PCNA (P < 0.05) and cyclin E (P < 0.05) increased significantly, while the increased level of cyclin D1 was not significant (P > 0.05). When the expression of miR-493-5p was downregulated, the protein expression levels of PCNA (P < 0.05) and cyclin D1 (P < 0.05) decreased significantly, while the protein expression level of cyclin E also decreased but not significantly (P > 0.05) (Fig. 2c, c′, and d). The trends were consistent with the results of RT-qPCR, indicating that miR-493-5p promoted the proliferation of muscle cells. CCK-8 assay was performed on C 2 C 12 cells that were transfected with miR-493-5p mimics and inhibitor ( Fig. 2e and f).
Compared with the control group, the absorbance (450 nm) of miR-493-5p mimic treatments increased significantly at 24 h (P < 0.05), and the absorbance (450 nm) of inhibitor group decreased significantly at 36 h (P < 0.05), indicating that miR-493-5p improved the proliferation efficiency of C 2 C 12 cells and promoted cell proliferation. When miR-493-5p was highly expressed, the number of cells entering S phase is significantly higher than that in the control group (P < 0.01; Fig. 3a, a′, and 3b), indicating that cells are rich in DNA replication phase. When the expression of miR-493-5p was knocked down, the cells were blocked in G0/G1 phase and G2/M phase, and the cells in DNA replication phase decreased significantly (P < 0.05, Fig. 3c, c′, and d).

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The results of EdU analysis are shown in Fig. 4. The rate of cells with DNA replication activity in C 2 C 12 cells transfected with miR-493-5p mimics was significantly higher than that in the control group (P < 0.05, Fig. 4a-a′′′′′), while the rate of cells with DNA replication activity in C 2 C 12 cells transfected with miR-493-5p inhibitor was significantly lower than that in the control group (P < 0.05, Fig. 4b-b′′′′′). Quantitative analysis of proportion of C 2 C 12

miR-493-5p inhibits differentiation
To further study the effect of miR-493-5p on C 2 C 12 differentiation, the relative expression levels of differentiation marker genes Myf5, MyoD1, MyoG, and MyHC were detected by RT-qPCR analysis. The miR-493-5p mimics significantly decreased the expression level of MyHC (P < 0.05), while Myf5, MyoD1, and MyoG decreased but not significantly (P > 0.05). miR-493-5p inhibitor significantly promoted the expression level of MyoG (P < 0.05), and the levels of Myf5, MyoD1, and MyoG were increased but not significantly (P < 0.05) (Fig. 5a). The effects of miR-493-5p mimics and inhibitor on the expression levels of MyoD1, MyoG, and MyHC proteins were detected by Western blot (Fig. 5b, b′), and the results showed the same trend as the results of RT-qPCR. Compared with the control group, miR-493-5p mimics significantly inhibited the protein expression levels of MyoD1 and MyoG (P < 0.05, Fig. 5c), and miR-493-5p inhibitor significantly promoted the protein expression levels of MyoD1 and MyoG (P < 0.05, Fig. 5c); MyHC showed the same trend as MyoD1 and MyoG, but the result was not significant. In addition, we also evaluated the RNA expression levels of MyHC I and MyHC IIb. The miR-493-5p mimics significantly inhibited the expression of MyHC IIb (P < 0.05), while miR-493-5p inhibitor significantly promoted the expression of MyHC I (P < 0.01, Fig. 5d). The results of Western blot showed that the protein expression levels of MyHC I (P < 0.05) and MyHC IIb (P < 0.01) decreased significantly when miR-493-5p was highly expressed; when the expression of miR-493-5p was inhibited, the protein expression level of MyHC I increased significantly (P < 0.05), and MyHC IIb showed no significant difference (P > 0.05) (Fig. 5e, e′, and 5f), indicating that miR-493-5p inhibited the expression of MyHC I and MyHC IIb. MyHC immunofluorescence staining was performed.

miR-493-5p has a target relationship with ANKRD17
To identify the potential function of miR-493-5p, target prediction was performed using miRanda software, and a total of 184 putative target sites were identified (Table S1C). GO and KEGG pathway analyses showed that potential targets of miR-493-5p were mainly related to muscle development such as cardiac muscle hypertrophy and smooth muscle cell differentiation (Table S1D and S1E, Fig. 7a and 7b). RT-qPCR detection of myogenic targets showed that the expression level of MED28 and ANKRD17 increased significantly in LT90D compared with LT1D, and that there were no significant differences in the expression levels of ATP2A2, PPP3CA, and KLF15 (Fig. 7c). The interaction target between ANKRD17 and miR-493-5p was predicted successfully (Fig. 7d), and we also designed three double luciferase vectors for ANKRD17 target sequences, namely, ANKRD17pmirGLO-Wt, pmirGLO-Mut, and pmirGLO-Del. We co-transfected the above three vectors with miR-493-5p mimics into 293 T cells and detected the changes in double luciferase activity. By co-transfection of miR-493-5p and pmirGLO-Wt, the ratio of Firefly/Renilla luciferase activity decreased significantly compared with the control group (P < 0.05), while the pmirGLO-Mut and pmirGLO-Del groups did not change significantly, indicating that there was direct interaction between miR-493-5p and ANKRD17. Fig. 4 Enhancement of the C 2 C 12 cell proliferation activity by miR-493-5p. a-a′′′′′ Edu analysis of C 2 C 12 cell DNA replication activity following miR-493-5p mimics, b-b′′′′′ Edu analysis of C 2 C 12 cell DNA replication activity following miR-493-5p inhibitor, and c quan-titative analysis of proportion of C 2 C 12 cells with replication activity following miR-493-5p mimics and inhibitor. Note: data are represented as mean ± SEM. n = 6; *, P < 0.05; **, P < 0.01

Discussion
Myofiber hyperplasia in pigs is basically completed at 90 days of pregnancy (Wigmore and Stickland 1983), and the number of postnatal muscle fibers basically does not increase (Moss 1968). Therefore, the hypertrophy and transformation of postnatal muscle fibers are the main research fields (Schiaffino et al. 2013;Pearson 1990;Brummer et al. 2013). In the early stage after birth, the proportion of oxidized muscle fibers gradually decreased and the proportion of enzymatic muscle fibers increased (Wank et al. 2006). This process was accompanied by the increase in muscle volume and the decrease in meat quality (Chang et al. 2003). In details, Pax7 gene regulates early muscle development (Lagha et al. 2005); MyoD family controls myogenic differentiation (Hasty et al. 1993); myostatin can inhibit muscle development (Gao et al. 2020). In addition to the above transcription factors, noncoding miRNAs are also widely involved in the regulation of muscle development (Horak et al. 2016;Zhuang et al. 2022). In this paper, the muscles of Lantang were selected to study the miRNA expression from birth to early postnatal period. We found that the expression levels of ssc-miR-1, ssc-miR-206, ssc-miR-10b, ssc-miR-378, ssc-miR-26a, ssc-let-7a, ssc-let-7f-5p, and ssc-let-7i-5p were highly expressed in LT1D and LT90D libraries. Among them, miR-1 and miR-206 are muscle-specific miRNA molecules (Mitchelson and Qin 2015) which play a regulatory role in the differentiation of skeletal muscle cells (Sun et al. 2010;Kim et al. 2006). MiR-26a is abundant in skeletal muscle and promotes myogenic differentiation and skeletal muscle regeneration (Dey et al. 2012). miR-378 can regulate autophagy and apoptosis of muscle cells and maintain muscle homeostasis (Li et al. 2018). miR-10b plays a regulatory role in the proliferation of vascular smooth muscle (Yu et al. 2015). Tewari et al. found that miR-10b is highly expressed in 10-month-old bovine skeletal muscle, indicating the potential biological function of miR-10b in skeletal muscle (Tewari et al. 2021). Let-7 family is involved in cell differentiation (Tolonen et al. 2014), in which let-7a regulates myocardial hypertrophy (Zhou et al. 2017) and smooth muscle cell proliferation (Lin et al. 2020). Hu et al. reported that let-7i-5p is involved in regulating cardiomyocyte proliferation and affecting the process of cardiomyocyte cycle .
We excavated 16 differentially expressed miRNAs, and the expression of miR-432-5p was significantly down-regulated at 90 days, which was similar to previous study (Chen et al. 2020). They compared the miRNA Note: data are represented as mean ± SEM. n = 6; *, P < 0.05; **, P < 0.01 expression in the longissimus dorsi of Rongchang pigs between 35 and 287 days after birth and found that the expression of miR-432-5p was down-regulated in adulthood (Chen et al. 2020). miR-novel-1656 is homologous with hsa-mir-378a-3p, and it is involved in myoblast proliferation and differentiation in skeletal muscle development (Wei et al. 2016). miR-novel-2117 is homologous with hsa-miR-377-3p, and miR-377-3p is mainly related to cancer progression (Zhang et al. 2020) and vascular smooth muscle proliferation and migration ). This study explored the function of miR-493-5p in skeletal muscle. Several studies have indicated that miR-493-5p has antitumor properties in various cancer types. For instance, epigenetic silencing of miR-493-5p was regarded as a marker of some advanced cancers, and miR-493-5p upregulation can effectively impede the growth and migration of tumor cells (Zhao et al. 2016b). Although miR-493-5p has been widely studied in inhibiting the proliferation of cancer cells such as liver cancer and breast cancer (Zhao et al. 2016b;Gailhouste et al. 2019), a recent study found that the overexpression of miR-493-5p promoted the growth of cholangiocarcinoma tumor cells in vitro (Toshida et al. 2022). It shows that miR-493-5p has more biological functions that need further exploration. Our data imply that miR-493-5p may act as a regulator of muscle development.

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In general, PCNA, cyclin D1 and cyclin E are marker genes of cell proliferation, and PCNA is involved in DNA synthesis (Kumari et al. 2021) and cell cycle regulation (Zhang et al. 1993). Cyclin D1 is an important regulator of the early to metaphase process of cell G1 (Fu et al. 2004). Cyclin E mainly controls the transformation of G1 and S phases of cell cycle and promotes the process of cell cycle (Möröy and Geisen 2004). MiR-493-5p mimics increased the expression levels of PCNA, cyclin D1 gene, and protein in C 2 C 12 cells. Knocking down of miR-493-5p showed the opposite trend, indicating that miR-493-5p can promote the proliferation of C 2 C 12 cells. CCK-8 experiment, flow cytometry, and Edu results further proved the promoting effect of miR-493-5p on cell proliferation. Myogenic regulatory factors (MRFs), including Myf5, MyoD1, MyoG, and MRF4, play myogenic regulatory roles in myoblasts (Asfour et al. 2018). MRF promotes differentiation and sarcomere assembly by blocking the cell cycle of precursor cells (Buckingham and Rigby 2014). In the early stage of myoblastic differentiation, Myf5 and MyoD1 can jointly Fig. 7 Analysis of candidate target genes downstream of miR-493-5p. a GO analysis of candidate target genes downstream of miR-493-5p, b KEGG analysis of candidate target genes downstream of miR-493-5p, c expression of candidate target genes downstream of miR-493-5p in longissimus dorsi muscle of LT1D and LT90D, and d targeting sites and double luciferase test results between miR-493-5p and ANKRD17. Note: LT1D, Lantang pig 1 day after birth; LT90D, Lantang pig 90 days after birth; *, P < 0.05; **, P < 0.01 promote the early differentiation of myoblasts (Braun et al. 1992). MyoG is an advanced factor regulating the terminal differentiation of myoblasts, participates in the regulation of myocyte fusion, and is very important for muscle fiber growth and myonuclear proliferation (Ganassi et al. 2020). Myosin heavy chain (MyHC) is the basic unit of myosin, and the expression level of MyHC indicates the differentiation process of myoblasts (Parry 2001). In this study, miR-493-5p mimics inhibited the levels of Myf5, MyoD1, MyoG, and MyHC, and the down-regulation of miR-493-5p showed the opposite results, which proved that miR-493-5p could inhibit the differentiation of C 2 C 12 cells. The results of cellular immunofluorescence experiment on MyHC gene further proved that miR-493-5p could inhibit C 2 C 12 muscle fiber differentiation. This experiment also detected the effect of miR-493-5p on the gene and protein expression of MyHC I and IIb. MiR-493-5p inhibited the expression of MyHC I and IIb proteins, indicating that miR-493-5p inhibited the formation of myotubes (Parry 2001).
In general, miRNA negatively regulates the expression of downstream target genes by binding with 3′ UTR (Filipowicz 2005). We therefore predicted the downstream target genes of miR-493-5p and conducted function enrichment analysis. Through target prediction, we found 184 potential target genes interacting with miR-493-5p. GO functional annotation found that ATP2A2, PPP3CA, KLF15, MED28, and ANKRD17 were related to muscle development. RT-qPCR found that MED28 and ANKRD17, as potential target genes of miR-493-5p, were also significantly differentially expressed between LT1D and LT90D. The double luciferase experiment finally verified the target relationship between ANKRD17 and miR-493-5p. It is reported that ANKRD17 is involved in the development of mouse liver (Watt et al. 2001), promotes the differentiation of vascular smooth muscle cells (Hou et al. 2009), and is also a downstream effector of cyclin E, which is directly involved in the cell cycle regulation of DNA replication (Deng et al. 2009). Therefore, our paper preliminarily proves that there is a target relationship between miR-493-5p and ANKRD17 in myogenesis.

Sample collection
The pigs were obtained from Banling farm (Xinfeng town, Shaoguan City, Guangdong Province, China). Five male pigs of Lantang breed balanced with body weight were humanely slaughtered at birth (LT1D) and 3 months (LT90D). A total of 10 longissimus samples were immediately collected and stored in liquid nitrogen until further analysis.

Library preparation and miRNA-seq analysis
For total RNA extraction, three longissimusdorsi tissues were randomly selected in each growth stage and homogenized in TRIzol reagents (Takara, Dalian, China), and the RNA quantity and purity were further determined by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) and RNA 6000Nano LabChip Kit (Agilent, Santa Clara, CA, USA) with RNA integrity number (RIN) value of > 7.0. Next, the RNA fragments with the length of 18-30nt were separated and enriched by 15% polyacrylamide gel electrophoresis (PAGE), and the proprietary indexed adapters were then ligated to 5′ and 3′ termini, respectively. A reverse transcription reaction followed by low cycle PCR was performed to obtain sufficient products for Illumina sequencing. The preliminary quality control analysis of Fastq files was carried out using FastQC software (https:// github. com/s-andre ws/ FastQC). After trimming adaptor sequences and removal of reads with poly A and reads smaller than 18 nt by FASTX-Toolkit (http:// hanno nlab. cshl. edu/ fastx_ toolk it), clean reads were further mapped with Sscrofa11.1 reference genome and counted by miRDeep2 package v2.0.0.8 with a Perl script "miRDeep2. pl" (Friedländer et al. 2012). Then, the edgeR package v3.30.3 (https:// bioco nduct or. org/ packa ges/ edgeR/) was used to find the differentially expressed miRNAs with FDR < 0.05. A search for miRNA target genes was performed using the miRanda software (John et al. 2004), and gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID software (https:// david. ncifc rf. gov/).

RNA extraction and RT-qPCR
TRIzol (Takara, Dalian, China) was used to extract the total RNA of the samples, and the reverse transcription was carried out using PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, China). Quantitative reverse transcription PCR (RT-qPCR) was performed using Bio-Rad CFX 96™ Real Time Detection System (Agilent Technologies, Santa Clara, CA, USA) and SYBR Green PCR Master Mix (Takara, Dalian, China) in a 20-μl reaction. The cycle conditions were as follows: 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 68 °C for 20 s. The primer sequences are listed in Table S1F, and the specificity and efficiency of primers were checked with melt curve analysis. The tested miRNA and mRNA used U6 and GAPDH as internal parameters control, respectively, and 2 −ΔΔCt method was used to process the data in the experiment.

Cell culture and transfection
The myoblast cell line C 2 C 12 used in this experiment was purchased from the Chinese Collection of Authenticated Cell Cultures (Beijing, China). The cells were cultured in humidified incubators at 37 °C and 5% CO 2 . In the proliferation stage, the cells grew in growth medium (GM, DMEM; GIBCO, Grand Island, NY, USA) + 10% fetal bovine serum (GIBCO, Grand Island, NY, USA) + 1% penicillin streptomycin (Invitrogen, Carlsbad, CA, USA). In the differentiation stage, the cells were induced by differentiation medium (DM, DMEM; GIBCO, Grand Island, NY, USA) + 2% horse serum (GIBCO, Grand Island, NY, USA) + 1% penicillin streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were transfected when cell confluence reached approximately 60%, and miR-493-5p mimics and inhibitor were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were harvested for protein and RNA analysis after 48 h to study cell proliferation or switched to DM culturing for 96 h to study cell differentiation.

Western blot
The cells were lysed in RIPA lysis buffer (Solarbio, Beijing, China) to collect cell total protein.

Flow cytometry
C 2 C 12 cells transfected with miR-493-5p mimics and inhibitor for 48 h were collected, fixed in 70% ethanol, and stored at -20 °C. The cells were resuspended with PI/RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) and cultured in dark at 37 °C for 30 min. Finally, the cell cycles were analyzed by BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FACSDiVa software (BD Biosciences, Franklin Lakes, NJ, USA).

Edu experiment
The cells were inoculated into 48-well plates and transfected with miR-493-5p mimics and inhibitor. After 48 h, Beyo-click™ Edu-488 Kit (Biyuntian Biotechnology, Shanghai, China) was used for cell proliferation detection. Finally, the fluorescence inverted microscope (Nikon, Tokyo, Japan) was used to take photos, and the image was analyzed by Image J software (Media Cybernetics, Bethesda, MD, USA).

Cellular immunofluorescence
C 2 C 12 cells were inoculated on 12-well plates, transfected with miR-493-5p mimics and inhibitor, and then cultured until 96 h of differentiation. The cells were fixed with 4% paraformaldehyde for 60 min incubated with 0.4% TritonX-100 at room temperature for 30 min, and finally blocked with 5% goat serum for 60 min. MyHC primary antibody (cat.: MAB4470, dilution ratio: 1/2000) (R&D Systems, Minneapolis, USA) was incubated at 4 °C overnight, the corresponding fluorescent labeled secondary antibody was incubated at room temperature for 1 h, and DAPI was incubated at room temperature for 5 min. Finally, the fluorescence inverted microscope (Nikon, Tokyo, Japan) equipped with an RGB digital camera was used for microscopic examination, while the images were taken at × 20 objectives. The ImageJ software (Media Cybernetics, Bethesda, MD, USA) was used to analyze images and statistics.

Data statistics
The SPSS v17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the data. Independent sample 1 3 t-test was used for the numerical comparison between the two treatment groups. P < 0.05 indicated that the difference was significant and marked with *; P < 0.01 indicated that the difference was very significant and marked with **.

Conclusions
We established the miRNA expression profiles of porcine longissimus dorsi tissues at 1 day old and 90 days old, and screened differentially expressed miRNA candidates. We also identified a DE miRNA candidate, miR-493-5p, which was found to promote the proliferation of myoblasts and inhibit their differentiation by targeting ANKRD17gene, revealing the potential mechanism and function of miR-493-5p in muscle development.
Author contribution XZ and JS gathered samples, conceived the study, participated in its design, performed data analysis, and drafted the manuscript; FX and ZL performed the experiments; JL, TC, QX, and YZ provided guidance and funding. All authors have read and approved the final version of the manuscript. The sponsors had no role in the study design, in the collection, analysis, and interpretation of the data, in the writing of the report, and in the decision to submit the article for publication.
Availability of data and materials All data are contained in the manuscript.

Declarations
Ethics approval and consent to participate All procedures were performed in accordance with the procedures approved by the Institutional Animal Care and Use Committee of South China Agricultural University (ethics approval code: SCAU2018F006, March 13, 2018). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org) for the reporting of animal experiments.

Competing interests
The authors declare no conflict of interest. The funding sponsors had no role in the design of the study, in the collection, analyses, and interpretation of data, in the writing of the manuscript, or in the decision to publish the results.