Effects Of Promoter Methylation Of The Guanling Bovine FOXO1 Gene On Transcriptional Regulation And Its Effect On Proliferation And Differentiation Of Myoblasts

Forkhead box transcription factor O1(FOXO1)is a member of the O subfamily of the forkhead box transcription factor(FOX)family,which is involved in several signaling pathways in the nucleus in the form of transcription factors.In skeletal muscle,FOXO1 is involved in regulating the expression of MRFs family members to influence the differentiation process of myoblasts.FOXO1 is an indispensable regulator of animal growth and development,and an important candidate gene in livestock and poultry breeding.In this experiment,we investigated the effects of FOXO1 promoter methylation on transcriptional regulation and myogenic cell proliferation and differentiation in Guanling cattle by molecular cloning,bioinformatics analysis,q RT-PCR,bisulfite sequencing PCR(BSP),indirect immunofluorescence assay(IFA),Western blotting,flow cytometry,CCK-8 and other techniques.The main findings are as follows:1 Molecular characterization and tissue expression analysis of FOXO1 gene in Guanyin cattleThe CDS region of Guanling bovine FOXO1 gene was successfully cloned.Bioinformatics analysis showed that the CDS sequence of Guanling bovine FOXO1gene was 1998 bp long,encoding 665 amino acids,without transmembrane structural domain and signal peptide shear site,which is a hydrophilic and unstable protein,and predicted three protein kinase B phosphorylation regulatory sites:Thr24,Ser267 and Ser330.Homology analysis revealed that the FOXO1 gene in Guanling cattle is relatively stable in mammals,while it shows genetic differences among species.The q RT-PCR showed that FOXO1 was expressed in all tissues of Guanling and crossbred cattle.2 Analysis of transcriptional regulation by FOXO1 promoter methylation in Guanling cattleThe presence of two Cp G islands in the promoter region of FOXO1 gene(-1077to-366 bp)was predicted by Meth Primer online software,and 43 sites were found to exist in Cp G1 and 29 sites in Cp G2,for a total of 72 Cp G sites.The average methylation rates of FOXO1 promoter in heart,kidney and longissimus muscle tissues of Guanling cattle were 0.740%,0.138%and 0.510%,respectively,and correlation analysis with the transcript levels of each tissue revealed that the methylation levels of promoter region and m RNA expression of Guanling cattle FOXO1 gene were negatively correlated(R~2=0.9961,P=0.040).In the study of Guanling cattle at different ages,it was found that the methylation rate of FOXO1 promoter region in calves was significantly higher than that in adults(P<0.05),while the relative expression of FOXO1 gene in the longissimus muscle tissue of adult Guanling cattle was significantly higher than that in calves(P<0.05),showing a trend that the higher the methylation rate of FOXO1 gene promoter region,the lower its m RNA expression level.3 Effect of FOXO1 on the proliferation of Guanling bovine adult myoblastsThe Guanling bovine myoblasts were isolated by enzymatic digestion,and the purity of myogenic cells was analyzed by IFA.Subsequently,recombinant plasmids were transfected into the cells,and the effects of Guanling bovine FOXO1 on the proliferation of myoblasts were investigated by q RT-PCR,Western blotting,flow cytometry,and CCK-8 techniques.The results revealed that interference with FOXO1significantly upregulated the expression of proliferation-related genes(CDK2,PCNA,CCND1)at the m RNA and protein levels(P<0.05).And overexpression of FOXO1 was found to significantly decrease the expression of CDK2,PCNA and CCND1 at the m RNA and protein levels(P<0.05).CCK-8 results showed that cells growth ware significantly(P<0.05)slower in the control group(sh NC)compared to the interfering group(sh FOXO1)12 h after transfection.Flow-through results showed that interfering with the FOXO1 gene significantly reduced the number of cells in the G1 phase of the cell cycle,significantly increased the number of cells in the S phase(P<0.05),and significantly elevated the number of cells in the G2/M phase compared with the sh NC group,with a highly significant difference(P<0.01).Taken together,the study suggests that the FOXO1 gene negatively regulates the proliferation of myoblasts.4 Effect of FOXO1 on myoblasts differentiation in Guanyin cattleThe effects of FOXO1 on myoblasts differentiation in Guanyin cattle were examined by q RT-PCR and Western blotting.The results showed that interference with FOXO1 significantly upregulated the expression of differentiation-related genes(MYOD,MYOG,MYHC)at the m RNA and protein levels(P<0.05).It was also found that overexpression of FOXO1 significantly decreased the expression of MYOD,MYOG and MYHC at the m RNA and protein levels(P<0.05).The changes of FOXO1expression during myoblasts differentiation were examined by q RT-PCR.The results showed that FOXO1 gene expression increased gradually with the increase of differentiation time induced by myoblasts.In conclusion,FOXO1 is involved in the process of myoblasts differentiation and plays a negative regulatory role.The above study showed that the level of FOXO1 promoter methylation negatively regulated transcriptional expression in Guanling cattle,and found that FOXO1 was involved in regulating the expression of genes related to the proliferation differentiation of Guanling cattle myoblasts and inhibited the proliferation differentiation of myoblasts.