| Mammals have three different types of muscle tissues,including cardiac muscle,skeletal muscle,and smooth muscle.Skeletal muscle represents half of the total body mass and has a critical role involuntary motion and support.The vertebrate skeletal muscle originates from the somites during embryonic development.MYOG is a complicated and orchestrated process,regulated by several genes,signalling pathways,and network regulation.For instance,the multiple specifics of MYOG regulatory transcription factors(MRFs)are important for the regulation of skeletal muscle development.Also,MRFs are a member of the basic helix-loop-helix(bHLH)family,including MYOG,MyoD,Myf5,and MRF4.MRFs that significantly expressed in skeletal muscle suggested that they have an essential role during the proliferation and differentiation of skeletal muscle.In addition,non-coding RNAs such as microRNAs(miRNAs),long non-coding RNAs(lincRNAs)and circular RNAs(circRNAs)have been reported to be involved in the development of skeletal muscle and play critical roles duringMYOG Although the molecular mechanism of muscle development has been a major research object on developmental biology.Nevertheless,to explore the molecular regulatory mechanisms during muscle development and meat quality need further in-depth experimentsCircular RNAs(circRNAs)are a class of endogenous non-coding RNAs(ncRNAs)that have a covalently closed circular structure with no 5' end cap and 3' end poly(A)tail,more stable compared with linear RNA molecules,produced by back-splicing from precursor mRNA.circRNAs have high expression in the cytoplasm of eukaryotic cells,and can be circRNAs can exist for a long time in various cell and tissue at specific developmental stages Recent studies have divided the circRNAs into three groups;1)exon-derived circRNAs(exonic circRNAs),which are formed by exon splicing of the coding gene,including one or Multiple exon sequences;2)Intron circRNA(circular intronic RNA,ciRNA),which without of exon sequences and produced by cyclization of the coding gene intron sequence;3)exon-intron circRNA(EIciRNA),which is a fusion circRNA that contains both an exon and an intron sequence.Furthermore,it is found that circRNAs are involved in the transcription and post-transcriptional regulation of gene through different modes of action.Previous studies noted that most circRNAs contain miRNA binding sites.Therefore,circRNA can act as a highly efficient competitive endogenous RNA(ceRNA),effectively sponging miRNAs and regulating miRNA target genes.Besides sponging miRNAs,circular RNA may also bind to RNA-binding proteins(RBPs)to form complexes of RNA proteins(RPCs).In addition,it was stated that(EIciRNAs)interact with small nuclear ribonucleoproteins of RNA polymerase II and U1 and cis-induce host-gene transcript in the nucleus.cirRNAs play critical roles during skeletal muscle proliferation and differentiation.MicroRNAs(miRNAs)are about 17 to 24 nucleotides in length and are a class of small single-stranded endogenous,non-coding RNAs.Usually,the sequence of miRNA is complementary to the 3' untranslated region(3'-UTR)of a gene.These complementary sequences down-regulate the expression of various genes through facilitating mRNA breakdown or down the translation process during the post-transcription level.miRNA involved in multiple physiological and biochemical processes,such as carcinogenesis,hematopoiesis,neurogenesis,cell proliferation,and cell apoptosis.Also,the translation of up to 30%genes is trigger by miRNAs in human.In addition,miRNAs play critical functions during muscle development by regulating MYOG proliferation and differentiation.In recent years,it has been found that miRNAs play an important role in muscle development,and the study of miRNA mainly focuses on the regulation of downstream target genes,only a few studies were performed on the factors that affect the expression of miRNAs.There is little research on muscle development in cattle and the regulation mechanism is still unclear.The main results are as follows:1.Circular RNA circMYL1 inhibit proliferation and promote differentiation of myoblasts by sponging miR-2400In this experiment,we have been tried to evaluate the effect of circMYLl and miR-885 in regulating primary bovine myoblast proliferation and differentiation in cattle.A series of experimental approaches such as RT-qPCR,Western blot,cell cycle,cell counting kit-8 assay,and EdU have been used to find the potential role of circMYLl and miR-885 in the proliferation and differentiation of myoblast cells.At a local slaughterhouse in(Xi'an;China),all specimens from Qinchuan bovine embryonic phase(90 days)were collected,including skeletal muscle,stomach,intestine,spleen,heart,liver,lung,and kidney,and washed by diethylpyrocarbonate(DEPC)water,and stored at-80? until RNA extraction.Total cell or tissue RNAs were extracted using the manufacturer's instructions for TRIzol Reagent(Takara,Dalian,China).Next,using the PrimeScript RT reagent kit with gDNA Eraser(Takara,Dalian,China),RNA was reversed to complementary DNA(cDNA).The SYBR Green PCR master mix reagent kit(Takara,Dalian,China)was used to conduct RT-qPCR.GAPDH and U6(for miRNA)were used as an internal control for data normalization with three biological replicates.The relative expression level of mRNA was calculated using the 2-??Ct technique.To explore the possible functions of circMYL1 during bovine skeletal muscle development,we downloaded the sequencing data of circRNAs of bovine muscle tissue from NCBI:accession ID,GSE87908.For the construction of circMYLl overexpression plasmids,using PCR to amplify the linear sequences of circMYL1,and the cDNA template was produced from the bovine primary myoblasts' RNA by RT-PCR.The obtained linear sequences were then cloned into the KpnI and BamHI-restriction sites of the pCD2.1 vector(Invitrogen,Carlsbad,CA,USA)according to the manufacturer' s instruction,to produce the overexpression vector PCD2.1-circMYL1.The MYOG 3'UTR-WT fragment,including the miR-2400 binding site,was amplified and inserted into the psiCHECK-2 vector(MYOG-WT)(Promega,Madison,WI,USA)at the 3'end of the Renilla gene using the XhoI and NotI restriction enzymes(Takara,Dalian,China)and T4 DNA ligase.Mutant type psiCHECK-2-MYOG 3'UTR-MUT(MYOG-MUT)was created using overlapping PCR to mutate complementary to the miR-2400 seed region.MYOG-WT and MYOG-MUT are eventually incorporated in the same way into the psiCHECK-2 vector.A miR-2400 sensor(with a perfect miR-2400 binding site)was created by inserting two sequences that complement the mature miR-2400 sequence after the Rluc psiCHECK-2 vector.Consequently,the fluorescence activity alteration will react to miR-2400 reflecting.The PCK-circMYL1 vector was also obtained using the same procedure.The miR-885 mimics and inhibitor were compounded by(Ribobio,Guangzhou,China);the sequence was UCCAUUACACUACCCUGCCUCU.Wild-type and mutant 3'-UTR of MyoD1 containing the binding site of miR-885 were cloned into psi-CHECK-2 dual-luciferase reporter vector(Promega,Madison,WI,USA)at the 3'-end of the Renilla gene using XhoI and NotI restriction sites(TaKaRa,Dalian,China)and T4 DNA ligase(TaKaRa,Dalian,China).The psiCHECK2-Myod1-3'-UTR-MUT was generated by mutating the complementary seed region of the miR-885 binding site using a mutagenic primer.Sequencing was done to verify all constructed vector.The HEK293T cells(ATCC,USA)and bovine primary myoblasts were cultured in growth medium containing high-glucose Dulbecco's modified Eagle's medium(DMEM;Hyclone,USA)and supplemented with 10%or 20%fetal bovine serum(FBS)(Hyclone,USA)and 1%penicillin-streptomycin(Invitrogen,USA)and incubated at 37? with 5%CO2.The primary bovine myoblasts differentiation was induced by replacing growth medium on differentiation medium(DMEM containing 2%horse serum)when primary bovine myoblasts confluence reached nearly 70%?80%.The bovine primary myoblasts were cultured in six-well plates.When the confluence of the cells reached to 40%for proliferation or 70%?80%for differentiation the cells were transfected with PCD2.1-circMYL1(2 ?g/mL),100 nmol/L si-circMYL1(RiboBio,Guangzhou,China),50 nmol/L miR-2400 mimics(RiboBio,Guangzhou,China),miRNA-885 mimics,miR-885 negative control(NTC)at a concentration of 50 nmol/L,and miRNA-885 inhibitor(IN)and inhibitor negative control(INC)at a final concentration of 100 nmol/L using Lipofectamine 2000(Invitrogen,USA)according to the manufacturer's protocol.Transcription was inhibited by adding 1 mM of Actinomycin D solution to the cell culture medium(Leagene,Beijing,China).The cell CCK-8 was used to assess the proliferation of cells.The bovine primary myoblasts were grown in 96-well plates with eight independent replicates for each treatment.After 24 h of cultured at 37?,we added 10 ?L of CCK-8 reagent to each well for 4 h.Then an automatic microplate reader(Molecular Devices,USA)was used to measure the absorbance value of all samples at 450 nm.Also,bovine primary myoblasts were seeded in 60 mm cell culture plates(2×106 cells/well).After 24 h of transfected,we collected cells and washed with PBS buffer,and resuspended with 1 mL of DNA staining solution and 10 ?L of permeabilization solution(Multisciences,Hangzhou,China).The suspension was then incubated for 30 min in the dark at room temperature.Flow Cytometry(FACS Canto TM ?,BD Biosciences,USA)measured the cell cycle and,for each treatment group,there were three independent replicates.For investigated the binding sites of circMYL1 with miR-2400,HEK293T cells were seeded in 96-well plates.Then,the miR-2400 mimics and PCK-circMYL1 or miR-2400 sensors and PCD2.1-circMYL1 were co-transfected with Lipofectamine 2000 into HEK293 T cells.The miR-2400 mimics and MYOG-WT or MYOG-MUT,miR-885 mimics or NTC,psiCHECK2-MyoD1-3' UTR-WT or psiCHECK2-MyoD1-3' UTR-MUT have also been co-transfected into cells.After 24 h,the cells were then washed by Phosphate Buffered Saline(PBS)and collected using 100 ?L Passive Lysis Buffer(PLB).Luciferase activity assay was performed using a MPPC luminescence analyzer(HAMAMATSU,Beijing,China),and Dual-Luciferase(?)Reporter(DLR)Assay Kit(Promega,Madison,WI,USA).The activities of Firefly luciferase were normalized in each well to Renilla luminescence.Total protein was extracted from the primary bovine myoblasts from different treatment groups by using RIPA lysis buffer containing 1mM PMSF(Solarbio;Beijing,China).Then the protein extract was boiled in 5×SDS-PAGE loading buffer for 10 min.Subsequently,the protein was separated by SDS-PAGE and transferred to a 0.2 ?m polyvinylidene fluoride membranes(PVDF).Then incubated for 2 h at room temperature with 5%skim milk in Tris Buffered Saline,with Tween(TBST)to block the PVDF membrane.Thereafter,4? overnight incubation with the primary antibodies that specific to anti-MYOD1(Dilution 1:1000;ab16148;Abcam,England),anti-MYOG(Dilution 1:1000;bs-3550R;Bioss,China),anti-MYH2(Dilution 1:1000;WL02785;Wanleibio,Shenyang,China),anti-CDK2(Dilution 1:1000;WL02028;Wanleibio,China),anti-PCNA(Dilution 1:500;WL03069;Wanleibio,China),anti-CCNB1(Dilution 1:1000;WL01760;Wanleibio,China),anti-P21(Dilution 1:1000;WL0362;Wanleibio,China),anti-FSTL1(Dilution 1:1000;ab223287;Abcam,England),anti-CDC6(Dilution 1:1000;bs-1390R;Bioss,China),anti-?-actin(Dilution 1:1500;WL01372;Wanleibio,China).PVDF membranes were washed five times with(TBST)then incubated for 2h at room temperature with anti-rabbit IgG-HRP secondary antibody(Dilution 1:1000;LK2001;Sungene Biotech,China).Finally,protein bands were detected by using ECL luminous fluid(Solarbio,China).Primary bovine myoblasts grown in 24-well culture plates,and the differentiation was induced for four days to form the myotubes.The differentiated cells were fixed with 4%paraformaldehyde for 30 min.Then,cells were washed with PBS and permeabilized with 0.5%Triton X-100 for 15 min.The cells were then incubated overnight at 4? with the MyHC-antibody(1:250;GTX20015,GeneTex,USA),diluted in 1%Bovine Serum Albumin.The cells were washed and incubated with secondary antibody(anti-mouse IgG H&L,Alexa flour 594)at 1:500 dilutions,at room temperature for 2 h.The nuclei were stained with DAPI for 15 min.The images were captured with a fluorescence microscope(DM5000B,Leica,Germany).For statistical analysis,all the quantitative data are presented as the mean ąSEM.Student's T-test procedure was used to analyze the statistical significance between groups.Statistical analysis was conducted using SPSS v20.0 software.P<0.05 was considered statistically significant and highly significant at P<0.01This study aimed to define the circMYL1 molecular mechanisms in the regulation of bovine MYOG and to disclose its regulatory mechanism through miR-2400 interaction.We found that circMYL1 inhibits proliferation and promotes differentiation of bovine primary myoblasts by acting as a sponge of miR-2400 and activated MYOG gene.In order to explore the function of circMYL1 in the proliferation of skeletal muscle cells,we conducted overexpression and knocked down experiments by transfecting circMYL1 overexpression vector and small interfering RNAs(siRNAs)that was designed to target circMYL1 back-splicing(pCD2.1-circMYL1 and si-circMYL1)to bovine primary myoblasts.The relative expression of circMYL1 was detected by RT-qPCR after 48 h post-transfection.Furthermore,we detected the proliferation process of bovine primary myoblast by flow cytometry for cell cycle analysis,cell counting kit-8(CCK-8),5-ethynyl-2'-deoxyuridine(EdU)incorporation assays,RT-qPCR,and Western blot assays after transfecting with pCD2.1-circMYL1/NTC,or si-circMYL1/si-NC.Firstly,we detected the effect of circMYL1on expression of the proliferation marker genes(PCNA,CyclinD1,and CDK2)using RT-qPCR and Western blot and the results showed that the overexpression of circMYL1 significantly decreased the expression of these genes at the mRNA and protein levels.Then,the cell cycle analysis revealed that overexpression of circMYL1 decreased the number of cells in the S phase and increased the number of cells in the G0/G1 phase.The results were further confirmed by EdU staining.The results showed that the number of EdU-positive cells was less than that in the control group.Furthermore,the CCK-8 assay revealed that circMYL1 possessed lower proliferation vitality than the negative control.In addition,the bovine primary myoblasts were transfected with si-circMYL1,and we found that the knockdown of the circMYL1 increased the expression of the proliferation marker genes(PCNA,CyclinDl,and CDK2)at the mRNA and protein levels.The cell cycle analysis revealed that si-circMYL1 increased the number of cells in the S phase and decreased the number of cells in G0/G1.Moreover,knockdown of circMYL1 markedly increased the number of EdU labelled cells(p<0.05)compared with the negative control group.Furthermore,the cell counting assay(CCK8)demonstrated that transfected with si-circMYL1 possessed higher proliferation vitality than the negative control.These results revealed that circMYL1 inhibits the proliferation of bovine primary myoblastsTo investigate the possible roles of circMYL1 in the regulation of skeletal muscle differentiation,the bovine primary myoblasts were transfected by pCD2.1-circMYL1/NTC or si-circMYL1/si-NC and differentiated cells for four days.Then,the cells were harvested,and RT-qPCR and Western blot analysis were conducted to detect the expression level of the differentiation marker genes(MyoD,MYOG,and MYH2).The result of RT-qPCR demonstrated that the overexpression of circMYL1 increased the expression levels of MyoD,MYOG,and MYH2 compared with the control group.In contrast,the knockdown of circMYL1 decreased the expression level of MyoD,MYOG,and MYH2 mRNA compared with the si-NC group.Similarly,the Western blot analysis showed that the overexpression of circMYL1 increased the protein levels of MyoD,MYOG,and MYH2(P<0.001)compared with the control group,whereas,the knockdown of circMYL1 decreased the protein level of MyoD,MYOG,and MYH2 compared with the si-NC group.Collectively,these results indicated that circMYL1 might promote bovine myoblast differentiation.Furthermore,the results from the immunofluorescence experiment found that cirMYL1 overexpression enhanced myotube formation.Meanwhile,the differentiation index and fusion index were both increased after differentiation,whereas the knockdown of circMYL1 reduced the differentiation index and fusion index and the formation of myotube.To screen the potential of miRNAs binding with circMYL1,a RNAhybrid assay was used to conduct the putative complementarity site between circMYL1 and miRNA.Remarkably,we found that circMYL1 has potential miR-2400 binding site.The dual-luciferase assay was used to explore whether circMYL1 target miR-2400 or not.The result revealed that miR-2400 reduced the Rluc expression of psiCHECK-2-circMYL1(PCK-circMYL1)in HEK293T cells.Furthermore,we generated a sensor for miR-2400 by inserting two copies of the miR-2400 complementary sequence into the psiCHECK-2 vector.The result showed that miR-2400 markedly reduced the Rluc activity of the miR-2400 sensor in HEK293T cells,on the other hand,the overexpression of the circMYL1 partially returned the decreased Rluc activity that made by binding miR-2400 in a dose-dependent manner.Furthermore,RNA immunoprecipitation(RIP)assay was conducted in bovine primary myoblasts to confirm the interaction between circMYL1 and miR-2400,and RT-qPCR results showed positive enrichment of miR-2400 and circMYL1 in Argonaute 2(Ago2)pull-down samples as compared to the negative control(IgG),indicating that circMYL1 binds to miR-2400 through the Ago2 protein.Moreover,the transfection with circMYL1 inhibits the relative expression level of miR-2400 in bovine primary myoblasts.Additionally,we analyzed the expression level of miR-2400 during MYOG in bovine primary myoblasts,and the result showed that the expression of miR-2400 was increased during the proliferation period and decreased during differentiation.From these results,the expression levels of miR-2400 were opposite to circMYL1 expression during MYOG.Altogether,these finding indicated that circMYL1 regulate proliferation and differentiation of bovine primary myoblast by sponging miR-2400.To explore the role of miR-2400 during myoblast proliferation,the bovine primary myoblasts at 40%density were transfected,by 50 nmol/L miR-2400 mimic and 50 nmol/L mimic negative control and pCD2.1-circMYL1/NTC for 48 h.Various methods,such as RT-qPCR,Western blot,cell counting kit-8 assay,and EdU,were used to detect the potential role of miR-2400 in the proliferation of myoblast.The RT-qPCR results demonstrated that the transfected by miR-2400 mimic increased the relative expression levels of proliferation marker genes(PCNA,CyclinDl,and CDK2)at mRNA level Similarly,the Western blot analysis showed that the miR-2400 overexpression increased the protein expression level of(PCNA,CyclinDl,and CDK2)genes.Analysis of the cell cycle showed that the miR-2400 mimic increased the number of cells in the S phase and decreased the proportion of cells in the G0/G1 phase,indicating that miR-2400 may promote the proliferation of bovine myoblast.Moreover,EdU analysis and CCK-8 and showed that overexpression of miR-2400 significantly up-regulated the cell proliferation.Potential roles of miR-2400 in the regulation of skeletal muscle differentiation were analyzed through transfecting bovine primary myoblasts with miR-2400 mimic or PCD2.1-circMYL1 at 70%-80%density and differentiated cells for four days.The cells were collected,RT-qPCR and Western blot analysis were conducted for the expression analysis of the differentiation marker genes(MyoD,MYOG,and MYH2).The result of RT-qPCR demonstrated that the miR-2400 mimic decreased the expression levels of MyoD,MYOG,and MYH2 at mRNA compared with the control group.Furthermore,the Western blot analysis showed that the miR-2400 mimic decreased the protein levels of MyoD,MYOG,and MYH2 compared with the control group.Immunofluorescence assay showed that miR-2400 reduced the differentiation index and fusion index and myotube formation.Collectively,these results confirmed that miR-2400 inhibits the differentiation of bovine primary myoblasts.The findings show a new mechanism involved in muscle development and could be helpful for cattle breeding programs.Also,the results found that miR-885 may have a critical role in cell development and regeneration,particularly in the proliferation and differentiation by directly targeting the 3'-UTR of MyoDl in myoblast cells2.MiR-885 promotes proliferation and inhibits differentiation of myoblasts by targeting MyoD1The primary bovine myoblasts were selected to investigate the potential role of miR-885 on MYOG.Cells were transfected by miR-885 mimic,negative control(NTC),to detect an over-expression effect.The RT-qPCR based results indicated that the expression of miR-885 in miR-885 mimics-treated cells was triggered more than 200 folds compared with the(NTC).Various experimental approaches,including RT-qPCR,Western blot,cell cycle,CCK-8,and EdU have been used to investigate the potential function of miR-885 in myoblast proliferation.To investigate the expression of various marker genes(PCNA,CDK2,and CCNB1)that play an important role in cell proliferation,we transfected the myoblast cells by miR-885 mimics and(NTC)when the confluence reached to 40%.The results found that the expression of PCNA,CDK2,and CCNB1 genes were high in miR-885 mimic myoblast cells compared with negative control myoblast cells.We also confirmed the high expression of PCNA,CDK2,and CCNB1 marker genes at the protein level through western blot analysis.We further evaluated the cell cycle distribution through flow cytometry in various phases,such as G1/G0,S,and G2 phases in the infected myoblast cells.The results noticed that miR-885 mimic decreased the cells number in G1 phase and increased the number of cells in S-phase,suggesting the potential role of miR-885 in the various cell proliferation stages.The results were further confirmed through EdU assay.In EdU analysis,we detected that the number of EdU-positive cells was more in the miR-885 mimic myoblast cells as compared with negative control myoblast cells.Moreover,we also performed cell counting assay to further get insight into the contribution of miR-885 in cell growth and division.The analysis observed that miR-885 showed a high number of cells absorptivity at 490 nm compared with the NTC group.However,miR-885's knockdown by its inhibitor significantly decreased miR-885's expression level.Also,the results demonstrated that the miR-885 inhibitor significantly decreased the expression of proliferation marker genes PCNA,CDK2,and CCNB1 at mRNA and protein levels.Also,the Flow cytometry assay noted that knockdown of miR-885 inhibited cell distribution in S-phase and significantly inhibited cell cycle.Moreover,knockdown of miR-885 markedly decreases the number of EdU labelled cells compared with the inhibitor negative control group.Furthermore,the cell counting assay(CCK8)observed that transfected with miR-885 inhibitor possessed lower proliferation vitality than inhibitor negative control.Based on these results,is essential for cell development and may promote cell proliferation in cattle.Also,we detected the function of miR-885 during MYOG differentiation by introducing miR-885 mimics,negative control(NTC),miR-885 inhibitors or inhibitor negative control(INC)into primary bovine myoblasts.Moreover,the primary bovine myoblast cells were transfected by miR-885 mimics or inhibitors at 70%?80%densities in the growth medium and then changed the medium to differentiation medium to induce cells differentiation.The cells were collected for RT-qPCR and Western blot analysis to detect the expression of myoblast differentiation marker genes,including MyoD and MYOG,on the 3rd day of differentiation.The overexpression of miR-885 down-regulated the MyoD and MYOG genes at mRNA levels in DM.In contrast,knockdown of miR-885 increased the mRNA expression of MYOG differentiation markers(MyoD and MYOG)in DM.Similarly,the western blot analysis also noted a significant decrease for MyoD and MYOG on differentiation after transfected by miR-885 mimics at the protein level,whereas miR-885 inhibitors enhanced the protein expression of MYOG differentiation marker genes.Furthermore,the results of immunofluorescence showed in the miR-885-mimics group,the amount of MyHC-positive myotube was smaller than that of the control group.In contrast,the amount of MyHC-positive myotube in the miR-885-inhibitor group was higher than that of the control group Taken together,these results revealed that miR-885 plays a negative role during primary bovine myoblasts differentiationTo investigate the mechanism of miR-885 in myoblast differentiation,we predicted MyoD1 as a probable target gene of miR-885 by using Target Scan Human 7.1 and DAVID,MyoD1 was among the target genes of miR-885.The result found that MyoDl 3'-UTR possessed a binding site that complementary to the miR-885 seed sequence.It has been noted that MyoDl is a MYOG transcriptional factor that plays an essential role during muscle differentiation.We then tested whether MyoD1 is a direct targeting gene of miR-885 or not by Dual luciferase assay.We cloned the miR-885 binding seed regions of MyoD1 3'-UTR with or without mutation into the Renilla luciferase coding sequence of the psiCHECK-2 vector.The miR-885 mimics or negative control(NTC)were co-transfected with psiCHECK-2-MyoD1-3'-UTR-WT or psiCHECK-2-MyoD1-3'-UTR-MUT into HEK-293T cells.As predicted,dual luciferase found that the overexpression of miR-885 significantly reduced the Renilla luciferase activity in co-transfect with miR-885 mimic and psiCHECK-2-MyoDl-3'-UTR-WT compared with NC in myoblast cells.However,the luciferase assay found no significant decrease in the psiCHECK-2-MyoD1-3'-UTR-MUT treatment group.Furthermore,we investigated the MyoD1 is a direct target gene of miR-885 by transfecting the miR-885 mimics,NC,miR-885 inhibitors into primary bovine myoblasts The expression of MyoD1 at mRNA and protein level was detected.The results showed that the overexpression of miR-885 significantly decreased MyoD1 expression at mRNA and protein levels during primary bovine myoblasts differentiation.Conversely,knockdown of miR-885 markedly increased MyoD1 expression at mRNA and protein levels expression These results revealed that the overexpression n of miR-885 inhibited MyoD1 mRNA and protein expression in primary bovine myoblast.Taken together,these findings showed that miR-885 regulates the proliferation and differentiation of bovine myoblast cells by targeting MyoD1 genePreviously,it has been investigated that MyoD1 transcription factor activates and suppresses various skeletal muscle genes.Several studies reported that MyoD1 induced P21 expressions in vitro.Besides,knockdown of MyoD1 reduces the potential of C2C12 cells to express Cdc6 gene.Furthermore,MyoD1 stimulating transcription of miR-206 by inhibiting the expression of Fstl1 gene.It was noted that the MyoD1 downstream genes expression affects by alteration in MyoD1 expression.Here,in the present report,we further study the mechanism of miR-885 in regulating MYOG in primary bovine myoblast cells by targeting the MyoD1 gene.The expression levels of P21,Cdc6,and Fstl1 genes were detected in primary bovine myoblasts cells transfected with miR-885 mimics,negative control(NTC),miR-885 inhibitors or inhibitor negative control(INC)into primary bovine myoblast cells The present study found that p21 and Cdc6 mRNA expression significantly decreased in miR-885 mimics treated group,whereas knockdown of miR-885 significantly increased the expression of p21 and Cdc6 at mRNA levels.Similarly,p21 and Cdc6 protein levels significantly decreased in the miR-885 mimics-treated cells,while increased considerably by knockdown of miR-885.The mRNA levels of Fstll gene markedly triggered in miR-885 mimics-treated group but substantially down-regulated with miR-885 inhibitors treatment in DM.Similarly,Fstll protein levels significantly increased in the miR-885 mimics-treated cells,however,significantly reduced by knockdown of miR-885,suggesting that miR-885 might affect MyoD1 expressions,thus altering the expressions of MyoD1 downstream genes The findings of the present investigation would be fruitful for the understanding of the miRNA-885 role in MYOG,and will also provide a platform for circular RNA and miRNAs-based experiments to study the cell proliferation and differentiation in MYOGWe can conclude that,miR-2400 is not recognizable in other mammalian genomes Therefore,it is a specific bovine miRNA,MYOG is an important target gene of miR-2400,miR-2400 can regulate the expression level of target gene MYOG,and play a regulatory role in muscle cell proliferation and differentiation.And circMYLlis highly expressed in muscle tissue.There is a miR-2400 binding site in the circMYL1 sequence.It can down-regulate the expression level of miR-2400 in muscle cells by competitively binding miR-2400,and relieve the target gene.The expression MYOG is inhibited,thereby inhibiting the proliferation of myoblasts and promoting the differentiation of myoblasts.The MyoDl is a direct target gene of miR-885,the mechanism study found that miR-885 regulates MyoD1 through post-transcription.MiR-885 positively regulate proliferation and inhibit differentiation primary bovine myoblasts. |
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