| Myogenic Differentiation 1(MyoD1)belongs to a member of the Myogenic regulatory factors(MRFs)family,and is an important regulatory factor in the stages of cell myoblast Differentiation and muscle fiber formation.Myostatin(MSTN)is specifically expressed in skeletal muscle and plays an important role in muscle growth and fat deposition in humans and animals.DNA methylation,a form of epigenetic genetic regulation of DNA,is also one of the earliest methods of DNA modification.It changes the genetic characterization of DNA without changing the sequence of DNA,and is then stably passed on to offspring DNA under the action of DNA methyltransferase.In this study,Guanling cattle was used for tissue specific expression detection,single coupler amplified sequencing(high-throughput sequencing),primary cell culture,immunofluorescence cell identification,5-Aza-2 ’deoxycytidine,5-Aza-DC treatment,CCK8 detection,cell cycle detection,apoptosis detection and other methods were used to investigate the effects of DNA methylation on the expression of MyoD1 and MSTN genes and the proliferation of Guanling bovine myoblasts at the molecular and cellular levels.The test results are as follows:1.Expression of MSTN,MyoD1 and DNMT1 in different muscle tissuesThe expression levels of MSTN,MyoD1 and methyltransferase(DNMT1)in different muscle tissues were analyzed by qRT-PCR.MSTN gene in Guanling embryo cattle were psoas major muscle > longissimus dorsi muscle > foreleg muscle > hind muscle,respectively;In adult Guanling cattle,the psoas major is >,the hind leg is >,the longissimus dorsi is >,the foreleg is >;The expression level of MyoD1 gene was the highest in longissimus dorsi muscle,and its expression level was respectively in longissimus dorsi muscle >,foreleg muscle >,psoas major muscle >,and adult ganling cattle longissimus dorsi muscle >,hind leg muscle >,foreleg muscle >,psoas major muscle.The highest expression level of DNMT1 gene was found in the hind leg muscle,>,foreleg muscle,>,psoas major,>,and in adult Guanling cattle,the highest expression level was found in the foreleg muscle,>,longissimus dorsi,>,psoas major,>,and hindleg muscle.2.Detection of promoter methylation of MyoD1 gene and selection of methylation sitesThrough bioinformatics analysis,three Cp G islands of Cp G-1,Cp G-2 and Cp G-3were predicted in the promoter region of MyoD1 gene(-781bp— +67 bp),with the sizes of 120 bp,100bp and 104 bp respectively.Transcription factors include Zscan29,Zn F449,Zn F740,Zn F75 D,Zn F460 and Zscan4.The methylation level of MyoD1 gene promoter region in the longest dorsal muscle of embryonic cattle and adult cattle(24 months)was detected by single-junction library amplification sequencing(high-throughput sequencing),and it was found that methylation occurred in Cp G,CHG and CHH in embryonic cattle(15.4%,1.4% and 0.8%)and in adult cattle(20%,1.1% and 1.2%).The methylation level of MyoD1 gene in the longest dorsal muscle of embryonic cattle was extremely significantly lower than that of adult cattle(P<0.01),and there were three hypermethylated sites(P>0.49)for Cp G-1-1,Cp G-1-2,and Cp G-1-3,respectively.the methylation rates were 53.16%,49.36%,and 52.31% in embryonic cattle and 56.20%,54.24%,and 61.73% in adult cattle.54.24%,61.73%,and all belonged to Cp G-1.While a methylation site Else-2-1 is present in the non-Cp G island region Else-2,Else-1 has no methylation site.3.Effects of 5-Aza-DC on the proliferation of Guanling bovine myoblasts and the methylation level of MyoD1 gene promoter regionIn this experiment,after adding different concentrations of 5-aza-d C to bovine myocytes,the 0.1μmol/L 5-aza-d C treatment group was found to be suitable for subsequent experiments.The apoptosis rate was highly significantly increased(P<0.01)at0.1μmo L/L 5-aza-d C treatment for 48 h compared with the blank group,promoting apoptosis.To further test whether 5-aza-d C was related to apoptotic factors such as Bcl,Bax,Caspase-9,etc.,we detected by qRT-PCR that 0.1mol/L 5-aza-d C treatment of cells could highly significantly decrease the expression of the cellular anti-apoptotic factor Bcl(P<0.01),increase the expression of the pro-apoptotic factor Bax and significantly increase the expression of the pro-apoptotic factor Caspase-9 expression(P<0.05).The cells in the 5-aza-d C-treated group and the blank group were also detected by flow cytometry,and it was found that the G0/G1 phase cell ratio in the 0.1 mol/L 5-aza-d Ctreated group was higher than that in the blank group,but the difference was not significant(P>0.05),and the S phase and G2/M phase cells were lower than that in the blank group,but the difference was not significant(P>0.05).In order to further detect the effect of 5-Aza-DC on the expression of Cyclin B1,Cyclin A2 and Cyclin D,qRT-PCR was used to detect the expression of related cytokines,and it was found that 5-Aza-DC significantly increased the expression of Cyclin A2(P<0.05).Cyclin B1 and Cyclin D expression had no significant difference(P>0.05).At the same time,the methylation level was detected,and it was found that the Cp G methylation rate,CHG methylation rate and CHH methylation rate in the MyoD1 promoter region of the blank group were 28.0%,2.0%and 1.9%,while the Cp G methylation rate,CHG methylation rate and CHH methylation rate in the MyoD1 promoter region of the experimental group were 19.8%,1.3%and 1.8%respectively.0.1 mo L/L 5-Aza-DC treatment significantly decreased the methylation rate in the promoter region of MyoD1(P<0.01).In Cp G-1,the methylation rates of Cp G-1-1,Cp G-1-2,Cp G-1-3,and Cp G-1-4 were significantly reduced,and the methylation rates of Cp G-2 and Cp G-3 showed no significant difference before and after treatment and were all lower than 49%.The methylation levels of Else-1 and Else-2 methylation sites in nonCp G islands were all lower than 49%.Methylation sites were further screened out.4.Construction of MyoD1 promoterregion CpG-1 and ZnF 449 vectorCpG-1 was cloned by PCR and PGL-3-basic-Cp G-1 was constructed successfully.Eukaryotic expression vector pc DNA3.1(+)-Zn F449.In conclusion,the methylation level of the promoter region of MyoD1 in the longissimus dorsi muscle of Guanling cattle embryos was significantly lower than that of adult cattle(P<0.01),and the methylation of the promoter region of MyoD1 negatively regulated the expression of MyoD1 gene.5-aza-d C affects proliferation and apoptosis of bovine myogenic cells by regulating the expression levels of Caspase-9,Bcl,Bax and Cyclin A2 m RNA.0.1μmol/L 5-Aza-Dc significantly decreased the methylation level of the promoter region of MyoD1 in Guanling bovine myoblast cells(P<0.01),and significantly increased the expression level of MyoD1 m RNA,and significantly decreased the expression level of MSTN m RNA(P<0.05). |
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