Effects Of Promoter Methylation On Transcription Regulation Of Bovine FGFR1 Gene And Its Effect On Myoblast Proliferation And Differentiation

Muscle development is a highly coordinated biological process,and at present there is a in-depth understanding of the genetic basis of muscle development.This process is involved by the PAX gene(Paired-homeobox transcription factor)family,the muscle-generating regulatory factor(myogenic regulatory Factors,MRFs)family,and the muscular cell enhancement factor(the Myocyte Enhancer factor 2,MEF-2)family and a variety of complex signaling pathways.Although the genetic hierarchy of skeletal muscle formation has been intensely studied,a large proportion of the regulatory factors involved in this process are still unknown due to the complexity of the regulatory networks involved in muscle generation.Therefore,the discovery of new regulatory factors and the further understanding of the molecular mechanisms during skeletal muscle development are still urgently needed.In recent years,with the continuous deepening of the genomics study,the very important role of FGFR1 in muscle development is beginning to be realized.To date,there is a small amount of reports on domestic animals,but its regulatory role in cattle muscle development has not been reported yet.Therefore,this paper used vector construction,cell culture,dualfluorescein enzyme reporting system,bisulfite sequencing PCR,CCK-8,EdU,quantitative PCR(qPCR),RNA interference and Western Blot and other techniques to carry out an in-depth study on the molecular mechanism of FGFR1 gene in the proliferation and differentiation of myoblast cells.The main findings are as follows:1.Screening of the active promoter region of FGFR1 gene in muscle.We designed primers in different regions of the 2000 bp genomic region upstream the TSS,and cloned into vector of pGL3-Basic,and then transfected the C2C12 and 293 T cell lines.The active promoter region of the FGFR1 gene was identified as 202 bp to 509 bp and 794 bp to 1295 bp in the upstream of the FGFR1 gene TSS.Using the online software JASPAR2018 to predict transcription factors in active area 794 bp to 1295 bp,it was found that there were multiple transcription factor binding sites associated with cell differentiation in the active region,such as Sp1,EGR1 and KLF5 transcription factors.2.Study on the methylation in the promoter region of the FGFR1 gene in Qinchuan cattle.In order to understand whether there was a negative correlation between methylation in the core promoter region of FGFR1 gene and the expression of mRNA in muscle tissue in different growth periods of Qinchuan cattle.The methylation status of FGFR1 gene promoter region in muscle tissue of three growth periods(fetal,calf and adult)was detected by bisulfite sequencing PCR.Besides,the corresponding expression level of FGFR1 mRNA in these muscle tissues was detected by qPCR.The methylation level of fetal bovine(0.595%)in the promoter region of FGFR1 gene was significantly lower than that of calf bovine(2.14%)(P < 0.05),while the mRNA expression level of FGFR1 gene in muscle tissue of fetal bovine was significantly higher than that in calf bovine(P < 0.05).Therefore,there is a different methylation level in the promoter region of FGFR1 gene in the muscle tissue of Qinchuan cattle,and the higher the methylation degree,the lower the FGFR1 gene expression.3.Study on proliferation and differentiation effects of FGFR1 in myocytes.In order to study the effect of FGFR1 gene on proliferation and differentiation of cattle myoblasts,the cell proliferation was detected by EdU and CCK-8 methods,and it was found that the cell number of si FGFR1 decreased significantly compared with that of NC control group.The cell cycle was detected by flow cytometry,and it was found that the number of cells in the G1 phase and the S phase of siFGFR1 gene increased and decreased significantly respectively compared with that of NC control group.And after siFGFR1,the mRNA and protein levels of the proliferative marker gene were detected,and the mRNA and protein abundance of CylincD1,CDK2 were found to be significantly lower than that of NC control group.The mRNA and protein levels of the differentiation marker gene were tested.MyoD and MyHC had an elevated trend and MyoG had a falling trend,which was not significant,so these data were not enough to prove that its differentiation functions.The above results show that FGFR1 has a significant effect on the proliferation of bovine myoblasts.In this paper,the regulation mechanism of FGFR1 gene on bovine myoblasts was probed,which will provide a new idea and basis for the study of genes related to muscle development of cattle,and provide theoretical support for the cultivation of local cattle in China.