The Mechanism Of FTO-mediated M~6A Modification Of GADD45B And CCND1 Regulating Goat Primary Myoblast Proliferation And Differentiation

Skeletal muscle is not only an important organ for mammals to maintain body movement,but also the main component of meat products of livestock and poultry.Its development process not only affects the healthy growth of animals,but also is closely related to the productivity and quality of meat.Skeletal muscle development is complex-multistep processes,including muscle fiber formation,muscle fiber transformation and maturation,of which muscle fiber formation is the key stage that determine the subsequent growth and development potential of skeletal muscle.The muscle fibers were fully formed in about 100 days of gestation in sheep,and then transformed into muscle fiber hypertrophy.In the process of muscle fibers form,myogenic progenitor cells differentiated into myoblast and enter the cell cycle to produce proliferating myoblast,and the proliferated myoblast then exit the cell cycle to differentiate and fusion into muscle fibers.It is generally believed that the transcriptional regulation cascade composed of a series of myoblast regulators factors and myocyte enhancer factor genes is the main way to regulate myogenesis.However,previous studies have confirmed that epigenetics modification plays an important role in skeletal muscle development.N6-methyladenosine(m~6A)is dynamically regulated by m~6A methyltransferases and demethylases(FTO and ALKBH5),which regulate RNA processing and metabolism under the mediation of reader proteins.It is involved in various biological processes,such as brain development,spermatogenesis,and adipogenesis.In recent years,some studies have reported that m~6A modification is related to skeletal muscle development,but the potential role m~6A in muscle development and the underlying molecular mechanism in ruminant is still largely unknown,especially in ruminants.Therefore,this study takes haimen goat as the research object.Firstly,we analyzed the characteristics of m~6A modification in the skeletal muscle of fetuses(75 days gestation)and kids(1 day)using Me RIP-seq.Subsequently,the effects of FTO-mediated m~6A on the proliferation and differentiation of goat primary myoblast were further explored.It is not only enriching the molecular mechanism for the regulation of myogenesis,but also provides strong support for the promotion of mutton goat breeding and growth regulation.This study is mainly divided into the following five parts:1.Me RIP-Seq Reveal the RNA m~6A modification of goat skeletal muscle at different developmental stagesTo compare the myofibers development and m RNA m~6A modification level in goat skeletal muscle,we physiologically analyzed the longissimus dorsi muscle of fetuses and kids,and measured the expression of m~6A modification related enzyme and global m~6A modification level in muscle samples using H&E staining,western blot,and colorimetry.The results showed that the size and number of myofibers in newly born kid skeletal muscle was markedly higher compared with that of myofibers in the fetal skeletal muscle;FTO was more highly expressed in kid muscle samples than in fetal muscle samples,while the expression of METTL3 was lower in kid muscle samples than in fetal muscle samples.Consistently,fewer global m~6A modifications were observed in the muscles of kids compared to those of fetuses.These results suggest that FTO/METTL3-mediated m~6A modifications may be involved in goat skeletal muscle development.Subsequently,the characteristics of m~6A modification in the skeletal muscle from fetus and kid was analyzed using Me RIP-seq.The results showed that m~6A peaks mainly appeared in the coding sequence(CDS)and 3′UTR regions and were especially abundant close to the stop codon;most m~6A modification sites were conserved RRACH sequences;each gene had an average of 0.19 m~6A peak.A total of 937 common m~6A-modified genes from 3,270 to 2,227m~6A-modified transcripts in fetal and kid skeletal muscle were identified,among which286 genes with a significant change in both the m RNA and m~6A modification.Using gene ontology enrichment and KEGG pathway analysis,the m~6A-modified genes were found to be mainly involved in the biological functions and processes related to muscle development,as well as in some signaling pathways related with skeletal muscle development,including the MAPK,Wnt,and Notch signaling pathway.This study is the first to ananlyse m~6A modification in skeletal muscle of goat at different developmental stages,and preliminarily forecast the role of m~6A modification in goat muscle development,which provides materials for further studies of the molecular mechanism for the regulation of goat muscle development via m~6A modification.2.Regulation of FTO on myogenic differentiation of goat primary myoblastIn this study,we isolated and cultured goat primary myoblast to measure the expression of m~6A modification related enzyme and global m~6A modification level in differentiating goat primary myoblast,and to explore the function of FTO in regulating the myogenic differentiation of goat primary myoblast using RNAi and gene overexpression technology.The results showed that the expression of FTO increased,along with the myogenic differentiation of goat primary myoblast,whereas the level of ALKBH5 and METTL3 decreased after differentiation initiation;consistently,the level of global m~6A modifications decreased markedly,along with the differentiation of goat primary myoblast,which indicate that m~6A modifications may negatively regulate myogenesis.In addition,FTO knockdown significantly increased the level of m~6A modifications and inhibited myotube formation,as well as decreased the expression of My HC and myogenin;enforced FTO expression significantly reduced the level of m~6A modifications,but did not influence myogenic differentiation of goat primary myoblast.After transfection with FTO si RNA,the differentiation of goat primary myoblast was induced for 3 days.Mitochondrial content,autophagy,and apoptosis during differentiation was detected.As a result,FTO knockdown significantly reduced the expression of PPARGC1A and OPA1,repressed mitochondrial biogenesis,and reduced mt DNA content.In addition,FTO knockdown resulted in the downregulation of mitochondrial encoded genes(mt Cytb,mt Co1,mt Co2,and mt Nd1)in goat primary myoblast,as well as in a reduction in the ATP levels during differentiation.ROS production,ATG7 and ATG5 expression,and the LC3B-II/LC3B-I ratio were elevated in FTO-knockdown cells than in control cells.Meanwhile,the expression of SQSTM1 was reduced.Meanwhile,FTO knockdown significantly increased the BAX protein level,and decreased the pro-survival protein BCL2 content in goat primary myoblast,thus increased the apotosis rate of goat primary myoblast at day 3 of differentiation.In conclusion,abnormal FTO expression can impair mitochondrial biogenesis and inhibit myogenic differentiation,as well as promote autophagy and apoptosis during differentiation of goat primary myoblast.3.FTO-mediated m~6A modification of GADD45B regulates myogenic differentiation of goat primary myoblastTo determine the key target genes of FTO-mediated m~6A modified regulating myogenic differentiation,we performed RNA-seq on goat primary myoblast with FTO knockdown,and conducted associated analysis of differentially expressed genes in goat primary myoblast caused FTO knockdown and m~6A modified genes in goat skeletal muscel at different developmental stages.GADD45B was preliminarily seletected as the target gene of FTO-mediated m~6A modification regulating myogenic differentiation of goat primary myoblast.To verify the target relationship between FTO and its target gene GADD45B,these study methods of q RT-PCR,Western blot,m~6A-q PCR and dual luciferase reporting system were performed.The results showed that FTO knockdown significantly increased the m~6A levels in GADD45B m RNA,promoted the degradation rate of GADD45B m RNA,and inhibited the expression of GADD45B.The dual luciferase reporting system confirmed that FTO control the m RNA stability and expression of GADD45B by regulating the m~6A modification in the GADD45B-3′UTR.The m RNA and protein expressions of GADD45B were significantly increased during the differentiation of goat myoblast.The function of GADD45B on differentiation of goat myoblast was investigated by interfering with and overexpression of GADD45B.The results showed that GADD45B knockdown significantly decreased the expression of My HC and myogenin and inhibited myogenic differentiation;the overexpression of GADD45B increased the expression of My HC and myogenin,and promoted myogenic differentiation.Similar to the effects of the knockdown of FTO on mitochondrial biogenesis and function,as well as autophagy and apoptosis,the knockdown of GADD45B also impaired mitochondrial biogenesis and function,and enhanced autophagy and apoptosis.Finally,we performed the rescue experiment of co-transfection with si FTO and p EX3-GADD45B goat primary myoblast.It was found that the forced expression of GADD45B CDS alone rescued the inhibition of GADD45B expression caused by FTO knockdown,subsequently improving myotube formation in FTO-knockdown cells.In summary,this study revealed that FTO-mediated m~6A modification of GADD45B regulates myogenic differentiation of goat primary myoblast.4.FTO regulates myogenic differentiation of goat primary myoblast by activating the GADD45B-p38 MAPK pathwayTo further explore the molecular meachanism of FTO-mediated m~6A modification of GADD45B,we investigated whether the function of FTO in myogenesis depended on GADD45B-mediated p38 MAPK pathway activation.The results showed that p38 MAPK was activated during myogenic differentiation.However,the inhibition of p38 MAPK down-regulates the expression of Myf5,PPARGC1A,myogenin,and My HC,thereby inhibiting myoblast differentiation.The knockdown of GADD45B significantly decreased the level of phos-p38 MAPK and the expression of its downstream genes including Myo D1,Myf5,and PPARGC1A;whereas the overexpression of GADD45B significantly increased the protein levels of phos-p38 MAPK,Myo D1,Myf5,and PPARGC1A.To better understand the correlation between GADD45B,p38 MAPK,MRFs,and PPARGC1A,the p38 MAPK inhibitor SB203580 and p38 MAPK activator hesperetin were used to block and activate p38 MAPK,respectively.The results showed that Hesperetin treatment restored the decrease in myotubes formation and increased the expression of Myo D1,Myf5,and PPARGC1A in GADD45B-knockdown cells,while SB203580 treatment dramatically antagonized the increase in myotubes formation in GADD45B-overexpressing cells,and attenuated the upregulation effect of GADD45B overexpression on the expression of Myo D1,Myf5,and PPARGC1A.In addition,the protein levels of phos-p38 MAPK,Myo D1,Myf5,and PPARGC1A were found to be deamatically downregulated in FTO-knockdown cells,but GADD45B overexpression restored the reduced them expression levels in FTO-knockdown cells.Consistently,hesperetin treatment achieved a similar restoration effect regarding myotube formation,phosphorylation levels of p38MAPK,MRFs,and PPARGC1A expression levels,as observed upon GADD45B overexpression in FTO-knockdown cells.Taken together,our findings indicate that FTO regulates myogenic differentiation via the GADD45B-p38 MAPK pathway.5.FTO regulates myoblast proliferation by controlling CCND1 expression in an m~6A-YTHDF2-dependent mannerThe aim of this study was to explore the role and underlying mechanism of FTO in regulating goat primary myoblast proliferation.The effects of FTO knockdown and FTO inhibitor FB23-2 on the proliferation and the cell cycle progression of goat primary myoblast,as detected by CCK8 assays,Ed U proliferation assays,western blot and flow cytometry.The results showed both knockdown of FTO and inhibition of FTO demethylase activity induced the impairment of cell proliferation and the decrease in the protein expression of PCNA,as well as the delay of G1/S transition.In order to further explore the mechanism of FTO in regulating the proliferation of goat primary myoblast,the downstream target genes CDK6 and CCND1 involved in FTO-mediated regulation of cell cycle were screened from the differentially expressed genes induced by FTO deficiency.It was verified that FTO knockdown decreased the stability of CDK6 and CCND1,thereby down-regulating their expression.Moreover,FTO mediates CCND1 expression in an m~6A-YTHDF2-dependent manner.Consistently,loss of YTHDF2 recovered cell proliferation and cell cycle progression in FTO deficient cells.Taken together,these results demonstrated that FTO mediates CCND1 expression in an m~6A-YTHDF2-dependent manner,further regulates cell cycle progression.