mir 16 controls myoblast proliferation and apoptosis through directly suppressing bcl2 and foxo1 activities

Ờ Å ỊÙ× Ư Ờ miR-16 controls myoblast proliferation and apoptosis through directly suppressing Bcl2 and FOXO1 activities Xinzheng Jia, Hongjia Ouyang, Bahareldin Ali Abdalla, Haiping Xu, Qinghua Nie, Xiquan Zhang PII: DOI: Reference: S1874-9399(17)30079-2 doi:10.1016/j.bbagrm.2017.02.010 BBAGRM 1140 To appear in: BBA - Gene Regulatory Mechanisms Received date: Revised date: Accepted date: 25 June 2016 25 February 2017 27 February 2017 Please cite this article as: Xinzheng Jia, Hongjia Ouyang, Bahareldin Ali Abdalla, Haiping Xu, Qinghua Nie, Xiquan Zhang, miR-16 controls myoblast proliferation and apoptosis through directly suppressing Bcl2 and FOXO1 activities, BBA - Gene Regulatory Mechanisms (2017), doi:10.1016/j.bbagrm.2017.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT miR-16 controls myoblast proliferation and apoptosis through directly suppressing Bcl2 and FOXO1 activities IP T Xinzheng Jia1, 2, Hongjia Ouyang1, 2, Bahareldin Ali Abdalla1, 2, Haiping Xu1, 2, Department of Animal Genetics, Breeding and Reproduction, College of Animal NU SC R Qinghua Nie1, 2*, Xiquan Zhang1, Science, South China Agricultural University, Guangzhou, Guangdong 510642, MA China Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, D and Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of TE Agriculture, Guangzhou, Guangdong 510642, China CE P *To whom correspondence should be addressed Tel: 86-20-85285759; Fax: AC 86-20-85280740; Email: nqinghua@scau.edu.cn ACCEPTED MANUSCRIPT ABSTRACT Myogenesis mainly involves several steps including myoblast proliferation, T differentiation, apoptosis and fusion Except for muscle specific regulators, few IP miRNAs were proved to coordinate this complex process Here, we reported that SC R miR-16 inhibited myoblast proliferation and promoted myoblast apoptosis by directly targeting Bcl2 and FOXO1 The expression level of miR-16 was significantly NU decreased in the hypertrophic pectoral muscle compared to the normal pectoral muscle in chicken In vitro, elevating miR-16 significantly inhibited myoblast MA proliferation and promoted myoblast apoptosis, resulting in about 11.2% cells arrested in G1 phase and 12.3% apoptotic cells in the early stage Bioinformatic and D biochemical analyses revealed Bcl2 and FOXO1 as direct targets of miR-16 Consist TE to the effect of miR-16 on myogenesis, specific inhibition of Bcl2 or FOXO1 significantly suppressed myoblast proliferation and induced myoblast apoptosis, CE P indicating that both Bcl2 and FOXO1 contributed to miR-16 regulatory function in myogenesis Interestingly, FOXO1, as the core target, mediated multiple AC growth-related pathways induced by miR-16 such as PI3K-AKT-MAPK and PI3K-AKT-mTOR Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that 234 annotated genes bound by FOXO1 in the early-differentiated myoblasts, which were significantly enriched in myogenic proliferation, death and hypotrophy Altogether, we proposed that miR-16 acted as a coordinated mediator to suppress myogenesis in avian through the control of myoblast proliferation and apoptosis These findings have provided a novel mechanism whereby miR-16 represses Bcl2 and FOXO1 expression to maintain myoblast growth and skeletal muscle mass Keywords: miR-16, myoblast proliferation, apoptosis, FOXO1, Bcl2 ACCEPTED MANUSCRIPT Introduction The development of skeletal muscle mainly involves a coordinated regulation of T myogenic cell turnover, including myoblast proliferation, differentiation, apoptosis IP and fusion [1] Regularly, myoblast differentiation is always accompanied by SC R apoptosis cascade [2] During myogenesis process, the proliferated myoblast cells undergo either terminal differentiation or programed apoptosis For example, under NU the treatment of mitogen or serum deprivation, a fraction of proliferated myoblasts would differentiate into formatting myotube, while other cells would suffer MA programed cell-death [3] Once this coordinated balance of muscle maintenance is destroyed, it will directly lead to myogenic disorders Hence, to better understand the D cause of skeletal muscle atrophy or hypertrophy, it is critical to identify the molecular TE regulation mechanism for the balance of myoblast proliferation, differentiation and apoptosis during myogenesis CE P Many genetic regulators composed a complex regulation network to control muscle development Myogenic basic-helix-loop-helix (bHLH) transcription factors, AC including MyoD, MyoG, MSTN, MYF5, MRF4 and recently identified Myomaker were of the fundamental regulation pathway that determined the terminal differentiation of skeletal muscle cells through controlling muscle gene transcription [4-6] These muscle-specific functional genes have highlighted a primary regulation scenario of myoblast growth Another Forkhead box class O family (FOXO) were also reported to be widely participating in muscle growth and myogenic metabolism There are four conserved members (FOXO1, FOXO3, FOXO4, FOXO6) across human and avian were expressed in skeletal muscle [7] Two members of FOXO1 and FOXO3 were proved to be the key factors to control muscle energy homeostasis through regulating glycolytic and lipolytic flux, and mitochondrial metabolism ACCEPTED MANUSCRIPT Recent researches demonstrated that the non-coding RNAs were critical to control myogenesis, such as myoblast proliferation, differentiation and apoptosis, by T the interaction of these myogenic regulatory genes [8] MiRNAs, a class of 18-22nt IP small RNAs, were identified to be the most important non-coding RNAs, which might SC R negatively regulate 60% protein-coding genes at the posttranscriptional level, primarily by binding to their 3’ untranslated region (UTR) [9, 10] Growing evidence indicates that miRNAs, as the fine-regulators of gene expression, widely candidate in NU almost every biological processes, including cell development, cell proliferation, cell MA differentiation, and cell death [11, 12] Several muscle specific miRNAs, such as miR-1, 206 and 133, have been well characterized to control the key steps of skeletal D myogenesis, resulting in a core regulatory network between muscle regulatory factors TE and muscle specific miRNAs [13-16] The expression of these miRNAs is restricted in muscle tissue depending on the transcriptional and posttranscriptional control of CE P those myogenic factors While, they play their roles on myogenesis mainly through negative regulation of those key myogenic factors expression For example, AC overexpression of MRFs in chicken myogenic induced ectopic expression of miR-1 and miR-206, and inhibition of Myf5 resulted in a loss of miR-1 and miR-206 expression [17] Recently study showed that MyoD could initiate myoblast terminal differentiation and apoptosis by directly inhibition of muscle-specific miR-1/206 expression [18] However, except for these muscle specific miRNAs, few miRNAs have been proved to maintain the balance of myoblast proliferation, differentiation and apoptosis during myogenesis Also, little investigates in myogenesis focus on the regulatory function of other critical transcript factors such as FOXOs, whose family are always verified as crossroads for cellular metabolism, differentiation, apoptosis and transformation [18-20] ACCEPTED MANUSCRIPT In this study, we analysed the miRNA expression profiles between hypertrophic pectoral muscle and the normal pectoral muscle in chicken, and found that miR-16 T was obviously suppressed in the hypertrophic muscle tissues To investigate the IP regulatory function of miR-16 during myogenesis, avian myoblast cells were SC R performed to explore its effects on proliferation and apoptosis, and to identify its direct target genes Our results revealed that miR-16 inhibited myoblast proliferation NU and promoted myoblast apoptosis by targeting Bcl2 and FOXO1 MA Materials and methods 2.1 Ethics statement D All animal experiments in this study were conducted in accordance with Law of TE the People's Republic of China on Animal Protection and approved by the Animal CE P Care and Use Committee of the South China Agricultural University (approval ID: SCAU#0011, August, 2010) 2.2 Animals AC In this study, fast-growing commercial broilers with hypertrophic pectoral muscle (HPM) and slow-growing heritage chickens with normal pectoral muscle (NPM) were utilized to character miRNAs expression profiles associated with different muscle performance The two types of chicken breeds exhibited a highly significant difference in growth performance at weeks of age After seven-week feeding with the same diet, female birds (mean Body weight; in HPM and NPM were 1,241.43 ± 39.75 and 473.68 ± 26.81 grams, respectively) from each group were sacrificed to collect various tissue samples including pectoral muscle for the further analysis ACCEPTED MANUSCRIPT 2.3 Cell culture Primary embryonic myoblast cells were isolated from thigh muscles of T 11-day-old chicken embryos following to our previous methods [19] The primary IP cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) growth SC R medium with 20% fetal bovine serum (Gibco) and 0.2% penicillin/streptomycin (Invitrogen) in humidified air at 37°C with 5% CO2 To purify myoblasts, the cells were serially plated into 60mm cell culture dishes (Greiner) for three times with NU 40-minute intervals Chicken embryo myoblast cell differentiation was induced by MA culturing the cells in differentiation medium that consisted of DMEM (Gibco), supplemented with 2% horse serum (Hyclone) and 0.2% penicillin/streptomycin D (Invitrogen) in humidified air at 37°C with 5% CO2 Chicken fibroblast cell line TE (DF-1) was cultured in DMEM with 10% foetal bovine serum (Gibco) and 0.2% penicillin/streptomycin (Invitrogen) in humidified air at 37°C with 5% CO2 Quail CE P muscle cell (QM9), an ideal myoblast cell lines apart from mammalian, were cultured in Medium 199 with 10% foetal bovine serum (Gibco), 10% tryptose phosphate broth AC (Sigma) and 0.2% penicillin/streptomycin (Invitrogen) in humidified air at 37°C with 5% CO2 2.4 miRNA microarray analysis The total RNAs were isolated from six pectoral muscle tissues of HPM and NPM groups (n = 3) using Trizol (Invitrogen) according to the manufacturer’s instructions For each sample, 1μg of total RNA was treated with FlashTag Biotin RNA Labelling Kit (Genisphere), and hybridized to the GeneChip miRNA Array (Affymetrix), which contained 144 chicken miRNA probes Standard Affymetrix array cassette staining, washing and scanning were performed according to standard Affymetrix protocols using the post-hybridization kit (Affymetrix) and GeneChip Scanner 3000 Raw data ACCEPTED MANUSCRIPT were analysed by Partek Genomics SuiteTM (Partek GS), version 6.4, database (www.partek.com) and normalized with Robust Multichip Analyses (RMA) T (background correction and quantile normalization) After normalization, the Pearson IP correlation coefficients were calculated for replicates displayed a high level of SC R reproducibility (R2 > 0.92) in each group Differentially expressed miRNAs were identified by a combined filter based on fold change values (Fold change > 2) and P values (P < 0.01) were determined by ANOVA analysis and false-positive reduction NU 2.5 miRNA targets prediction and function analysis MA Targetscan7.1 system (www.targetscan.org/) was performed to predict miR-16 target genes Gene Ontology (GO) and KEGG pathway was utilized to analyse and Integrated Discovery system (DAVID 6.7; TE Visualization, D miR-16 regulatory function through the system of Database for Annotation, https://david.ncifcrf.gov/)[20] Gene network was constructed by Ingenuity Pathway CE P Analysis (IPA) software (http://www.ingenuity.com) which is based on the recently known biological response and regulatory network AC 2.6 qPCR RNA isolation and cDNA synthesis were carried out following to standard protocols qPCR was used to synthesise the first-strand cDNA of mRNA and miRNA by ReverTra Ace First Strand cDNA Synthesis Kit (Toyobo) and miScript Reverse Transcription kit (Qiagen), respectively The mRNA and miRNA relative expression were detected by qPCR using Bio-rad CFX96 instrument (Bio-Rad) with KAPA SYBR FAST qPCR Kit (KAPA Biosystems) and miScript SYBR Green PCR kit (Qiagen), respectively All assays were performed according to the manufacturer’s recommended protocols In order to assess the stability of host gene expression, 18S, 5S, U6 and β-actin were performed to analyse these genes’ expression pattern among ACCEPTED MANUSCRIPT each experimental group The results showed that there is no significant change between β-actin and 18S, and U6 and 5S, suggesting that U6 and β-actin are suitable T as a referenced gene to normalize the relative abundance of miRNA and mRNA, IP respectively Primers are listed in Table S4 The relative miRNA or mRNA gene) SC R expression level was calculated using the 2–∆Ct method (∆Ct = Cttarget gene – Ctreference Fold change values were calculated using the 2–∆∆Ct method (∆∆Ct = ∆Cttarget gene 2.7 Western blot analysis protein was extracted from the cells using ice-cold radio MA Total NU – ∆Ctcontrol gene) immunoprecipitation (RIPA) lysis buffer (Beyotime Institute of Biotechnology) D supplemented with 1mM phenylmethyl sulfonyl fluoride (Beyotime) The proteins TE were separated in 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore) and probed with antibodies following the standard procedures CE P The primary antibodies and their dilutions used in this assay were FOXO1 (ab39670; Abcam) 1:1000, Bcl2 (BS1031; Bioworld) 1:1000, H3 (Santa Cruz) 1:1000 and AC GAPDH (Santa Cruz) 1:2000 Finally, the blots were incubated with a secondary antibody containing horseradish peroxidase–linked (HRP) anti-Rabbit IgG antibody (Santa Cruz) at 1:5000 dilutions Proteins were scanned and visualized using the Clarity Western ECL Substrate (Bio-Rad), and the images were analyzed by ImageJ software (http://rsb.info.nih.gov/ij/) 2.8 Luciferase reporter assay To identify and validate the target sites for the gga-miR-16, 3’UTR fragment containing potential binding sites of FOXO1 and Bcl2 were cloned from WRR cDNA samples to generate 3’UTR-WT Then all the obtained sequences were inserted downstream of Renilla luciferase in the psiCHECK-2 vector (Promega) to generate ACCEPTED MANUSCRIPT the recombined dual reported vectors As another negative control, the mutated reporter vectors were constructed by replacing the seed regions of the miR-16 T targeting site in the 3’UTR of FOXO1 and Bcl2 After co-transfecting 0.5ug IP recombinant vector and 60nM mimic gga-miR-16 (Qiagen) per well of 24-well plates SC R into DF-1 cells for 36h by Lipofectamine 3000 (Invitrogen), luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions In each case, the constitutively expressed firefly NU luciferase activity in the psiCHECK-2 vector served as an internal control for MA evaluating transfection efficiency 2.9 Over-expression and knock-down of gene in myoblast D The over-expression of miR-16 was performed by mimic miR-16 with negative TE mimics as control group Briefly, mimic miR-16 or negative mimics (Qiagen) was CE P transfected into myoblast at the concentration of 60nM AS1842856 (FOXO1 inhibitor; Selleck Chemicals) and ABT199 (Bcl2 inhibitor; Selleck Chemicals) were utilized for specific knockdown of FOXO1 and Bcl2 activities in myoblast for 24h at AC the concentration of 10nM with equal volume of DMSO as control groups 2.10 Cell proliferation and apoptosis assay Flow cytometry assay was utilized to assess the effects of miR-16, Bcl2 and FOXO1 on myoblast proliferation and apoptosis progress For proliferation assay, myoblasts were pre-treated by 80 nM nocodazole for 12h, then released, synchronized and treated with mimic miRNA or inhibitors of Bcl2 and FOXO1 in 24-well plates Then, the cells were fixed in 75% precooling ethanol at 4°C for 12h, and then incubated in 50 μg/ml propidium iodide (Sigma) containing 10 μg/ml RNase A (Takara) and 0.2% Triton X-100 (Sigma) for 30 min at 4 °C The characters of cell ACCEPTED MANUSCRIPT skeletal muscle-derived cells in vitro: differential modulation of myoblast markers by TGF-beta2, J Cell Physiol, 196 (2003) 70-78 T [47] Y Leshem, I Gitelman, C Ponzetto, O Halevy, Preferential binding of Grb2 or IP phosphatidylinositol 3-kinase to the met receptor has opposite effects on SC R HGF-induced myoblast proliferation, Exp Cell Res, 274 (2002) 288-298 [48] T.H.t Reynolds, S.C Bodine, J.C Lawrence, Jr., Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal NU muscle load, J Biol Chem, 277 (2002) 17657-17662 MA [49] F An, B Gong, H Wang, D Yu, G Zhao, L Lin, W Tang, H Yu, S Bao, Q Xie, miR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via D BCL2 in acute liver failure, Apoptosis, 17 (2012) 702-716 TE [50] J.E Davies, D.C Rubinsztein, Over-expression of BCL2 rescues muscle weakness in a mouse model of oculopharyngeal muscular dystrophy, Hum Mol CE P Genet, 20 (2011) 1154-1163 [51] C He, M.C Bassik, V Moresi, K Sun, Y Wei, Z Zou, Z An, J Loh, J Fisher, AC Q Sun, S Korsmeyer, M Packer, H.I May, J.A Hill, H.W Virgin, C Gilpin, G Xiao, R Bassel-Duby, P.E Scherer, B Levine, Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis, Nature, 481 (2012) 511-515 [52] M Saito, U Novak, E Piovan, K Basso, P Sumazin, C Schneider, M Crespo, Q Shen, G Bhagat, A Califano, A Chadburn, L Pasqualucci, R Dalla-Favera, BCL6 suppression of BCL2 via Miz1 and its disruption in diffuse large B cell lymphoma, Proc Natl Acad Sci U S A, 106 (2009) 11294-11299 29 ACCEPTED MANUSCRIPT Figure legends Fig The expression of miR-16 was negatively associated to pectoral muscle size T (A) The heatmap of the differentially expressed miRNAs between hypertrophic IP pectoral muscle (HPM) and normal pectoral muscle (NPM) These two types muscle SC R tissues were collected from two chicken breeds with a highly significant difference in pectoral muscle size about 3-fold difference at weeks of age (B) Validation of NU downregulated miRNAs between HPM and NPM by qPCR (C) Validation of upregulated miRNAs between HPM and NPM by qPCR (D) miR-16 expression MA pattern during primary myoblast differentiation 2% horse serum was used for myoblast differentiation induction after culturing in growth medium with 20% fetal D bovine serum for 36h GM and DM indicated growth medium and differentiation TE medium, respectively U6 was used as the referenced gene to estimate miRNA relative expression In all panels, data are presented as mean ± SD (n = for each CE P group) * P < 0.05; ** P < 0.01 AC Fig miR-16 inhibited myoblast proliferation and promoted myoblast apoptosis (A) The effect of miR-16 on myoblast proliferation For proliferation assay, myoblasts were pre-treated by 80 nM nocodazole for 12h, then released, synchronized and treated with mimic miR-16 for 24h Propidium iodide (PI) was used to mark the cell DNA Cell cycle analysis was performed by flow cytometry to calculate the cells in G1, S or G2 phases (B) The effect of miR-16 on myoblast apoptosis AnnexinV-FITC-PI detection method was employed for the detection of apoptotic cells by flow cytometry PI was used as a counterstain to discriminate the dead cells from apoptotic ones The ratio of apoptotic cells was calculated based on AnnexinV and PI straining Q1 represents the necrotic cell potion, Q2: apoptotic cells 30 ACCEPTED MANUSCRIPT in the late stage; Q3: apoptotic cells in the early stage; Q4: normal cells (C) The effect of elevating miR-16 in the myoblasts on myoblast differentiation Four marker T genes were detected (MyoD, MyoG, MHC and Myomaker) with β-actin as referenced IP gene Mimic miRNAs were used to overexpress miR-16 in myoblast, with negative SC R mimics as control group (n=3) In all panels, data are presented as mean ± SD * P < 0.05; ** P < 0.01 NU Fig Identification of FOXO1 and Bcl2 as direct targets of miR-16 (A) KEGG MA pathway analysis of miR-16 putative targets Database for Annotation, Visualization, and Integrated Discovery system (DAVID 6.7; https://david.ncifcrf.gov/) was used for gene function enrichment analysis (FDR < 0.05) (B) The conserved binding sites in TE D the 3’UTRs of target gene FOXO1 and Bcl2 across most species miR-16 seed sequences were well complemented with FOXO1 and Bcl2 (C) Confirmation of the CE P regulation relationship between 3’UTR of target genes and miR-16 by dual luciferase reporter assay The 3’UTRs of target gene FOXO1 and Bcl2 were cloned into AC psiCHECK-2 Dual Luciferase Reporter vectors for validation in vitro (D) The effect of miR-16 on mutated binding sites in the 3’UTR Two mutated vectors (mu) including a mutated target cleavage site at the 3’UTRs of FOXO1 and Bcl2 were constructed (E) The mRNA expression changes of FOXO1 and Bcl2 induced by overexpression of miR-16 in myoblasts β-actin was used as referenced gene (E) The protein expression changes of FOXO1 induced by overexpression of miR-16 in myoblasts In panels C-F, data are presented as mean ± SD (n = for each group) * P < 0.05; ** P < 0.01 31 ACCEPTED MANUSCRIPT Fig FOXO1 and Bcl2 supressed myoblast proliferation and promoted myoblast apoptosis (A) The effects of inactivating FOXO1 and Bcl2 on myoblast T proliferation Propidium iodide (PI) was used to mark the cell DNA Cell cycle IP analysis were performed by flow cytometry to calculate the cells in G1, S or G2 phase SC R (B) The effects of inactivating FOXO1 and Bcl2 on myoblast apoptosis AnnexinV-FITC-PI detection method was employed for the detection of apoptotic cells by flow cytometry PI was used as a counterstain to discriminate dead cells from NU apoptotic ones The ratio of apoptotic cells was calculated based on AnnexinV and PI MA straining Q1 represents the necrotic cell potion, Q2: apoptotic cells in the late stage; Q3: apoptotic cells in the early stage; Q4: normal cells (C) The plot of cell D proliferation analysis in different cell cycles (D) The plot of cell apoptosis analysis in TE different stages AS1842856 (FOXO1 inhibitor) and ABT199 (Bcl2 inhibitor) were utilized for specific knockdown of FOXO1 and Bcl2 activities in myoblast at the CE P concentration of 10nM with equal volume of DMSO as control groups.FOXO1 * P < 0.05; ** P < 0.01 AC inhibitor (10uM) treatment for 24 hours (n = 4) Fig Global analysis of FOXO1 binding features by ChIP-seq in differentiated myoblast cells (A) Genomic distribution of FOXO1-occupied sites (B) Annotated genes of FOXO1-occupied peaks and overlapping with ChIP-seq data in mouse T cells (C) Cellular functional enriched by IPA analysis using FOXO1 putative targets from ChIP-seq data (P < 0.01) Red indicated the gene paly a special role in corresponded regulation function, while grey meant no effect * was used to show whether the gene was validated to be bound by FOXO1 in the corresponding cells (D) Several conserved genes confirmed in both avian and mouse ChIP-seq investigates (E) KEGG pathway analysis of FOXO1-occupied targets The number indicated that the 32 ACCEPTED MANUSCRIPT genes were involved in the corresponded pathway All 234 annotated targets were T used for pathway enrichment by DAVID system (P < 0.01) IP Fig The regulation pathway of miR-16 on myoblast proliferation and SC R apoptosis Briefly, miR-16 directly inhibits myoblasts proliferation and promotes apoptosis by down-regulating FOXO1 and Bcl2; while both FOXO1 and Bcl2 manipulated myoblast proliferation and apoptosis independently Meanwhile, FOXO1 AC CE P TE D MA NU could mediate multiple genes transcription to control myoblast growth 33 ACCEPTED MANUSCRIPT Table legends Table Upregulated or downregulated miRNAs in hypertrophic pectoral T muscle Fold change P value gga-miR-193b-5p 39.9 3.90E-05 gga-miR-92-5p 7.18E-05 12.9 3.76E-03 gga-miR-181b-5p 12.2 1.51E-03 gga-miR-456-5p 11.9 1.78E-03 9.2 4.08E-06 gga-miR-199-3p 4.9 9.85E-04 gga-miR-107 4.3 4.70E-06 gga-miR-181a 2.7 4.25E-03 gga-let-7b 2.4 3.40E-04 gga-miR-140-3p 2.3 9.37E-03 gga-miR-16 -7.9 1.19E-05 gga-miR-19b -3.7 1.66E-03 gga-let-7j -2.7 1.88E-03 gga-miR-20a -2.6 2.19E-05 gga-let-7a -2.5 3.63E-03 gga-miR-126 -2.4 6.35E-04 gga-miR-30c -2.2 8.98E-03 MA D TE CE P AC 15.4 3.17E-06 gga-let-7d downregulated 2.68E-06 14.6 gga-miR-133b-5p upregulated 38.9 NU gga-miR-221-5p SC R gga-miR-222-5p IP miRNA 34 ACCEPTED MANUSCRIPT Differently expressed miRNAs were performed by miRNA microarray detecting between hypertrophic pectoral muscle (HPM) and normal pectoral muscle (NPM) T from the different two chicken breeds The fold-change value is given as the IP HPM/NPM ratio of normalized signals The miRNAs with P value (< 0.01) and fold SC R change (> 2.0) were considered to be differentially expressed ones Thirteen miRNAs AC CE P TE D MA NU were found to be upregulated Seven miRNAs were found to be downregulated 35 MA NU SC R IP T ACCEPTED MANUSCRIPT AC CE P TE D Fig 36 AC Fig CE P TE D MA NU SC R IP T ACCEPTED MANUSCRIPT 37 AC Fig CE P TE D MA NU SC R IP T ACCEPTED MANUSCRIPT 38 AC Fig CE P TE D MA NU SC R IP T ACCEPTED MANUSCRIPT 39 AC Fig CE P TE D MA NU SC R IP T ACCEPTED MANUSCRIPT 40 D MA NU SC R IP T ACCEPTED MANUSCRIPT AC CE P TE Fig 41 ACCEPTED MANUSCRIPT Competing interests AC CE P TE D MA NU SC R IP T No competing interests declared 42 ACCEPTED MANUSCRIPT T Highlights IP We found that: the expression of miR-16 was negatively associated to muscle size SC R with significantly down-regulated in the hypertrophic muscle; elevating miR-16 significantly inhibited myoblast proliferation and promoted myoblast apoptosis; Bcl2 and FOXO1 were identified as direct targets of miR-16; Both Bcl2 or FOXO1 could NU significantly suppress myoblast proliferation and induced myoblast apoptosis; MA ChIP-seq for FOXO1 indicated that FOXO1 participated myogenic growth and development through regulating a series of functional targets and multiple AC CE P TE D muscle-growth-related pathways 43