| Deer antler is the only mammalian appendage that has a complete regenerative mechanism.There is a unique biological characteristic which is rapid growth without ‘tumor carcinogenesis’ in antler.Especially the molecular biological functions of growth factors of antler growth and development have been widely concerned in the field of human tumor biology.COL1A1,OPN and S100A4 genes are the important regulators in growth and development of animals that are all involved in the occurrence and development of tumors.DNA methylation,an important epigenetics,affects the activities of animals by regulating the transcription and expression of target genes.The abnormal of DNA methylation in tumor tissues can be used as a candidate for cancer detection and diagnosis.Studies have shown that the antler top tissue is the antler ‘growth center’ after the antler regenerated.Therefore,in order to explore the effect of COL1A1,OPN and S100A4 genes in the growth and development of different top tissues in different growth stages of antler,in this study,three healthy 5-year-old adult male sika deers were selected.The experimental materials were dermis,mesenchyme,precartilage and cartilage tissues of about 5 cm antler tissue from the top of the antler main stem at about 30 d,60 d and 90 d after the antler shed,and the three periods were early stage(small saddle),middle stage(two poles)and late stage(three bifurcation)The BSP technology was used to study the DNA methylation patterns of the three growth regulating genes promoter region and analyze the DNA methylation difference among different tissues and different periods,exploring the regulatory mechanism that leaded to its DNA methylation difference.The results are as follows:1.COL1A1,OPN and S100A4 genes in the same tissue and the same gene in different tissues had different DNA methylation patterns,and the methylation rate ranged from(1.33±0.23)% to(40.48±0.82)%.The methylation rate of OPN gene was relatively high,ranging from(24.29±1.43)% to(40.48±0.82)%,while the methylation rate of COL1A1 and S100A4 genes were relatively low,ranging from(1.33±0.23)% to(9.73±0.92)% and from(3.06±0.48)% to(11.94±0.96)%.2.The methylation rates of COL1A1 gene in the dermis tissues of the early stage,middle stage and late stage were(9.73±0.92)%,(7.60±0.69)% and(3.73±0.23)%;the methylation rates in the mesenchyme tissues were(3.20±0.40)%,(1.33±0.23)% and(1.60±0.69)%;the methylation rates in the precartilage tissues were(4.67±0.83)%,(2.53±0.46)% and(2.67±0.23)%;the methylation rates in the cartilage tissues were(5.60±0.40)%,(2.80±0.40)%and(2.27±0.61)%.There were 25 CpG sites in the promoter region of COL1A1 gene,and these 17 CpG sites at 39 bp,56 bp,118 bp,190 bp,221 bp,226 bp,233 bp,242 bp,262 bp,278 bp,291 bp,299 bp,303 bp,393 bp,400 bp,411 bp and 415 bp were all methylated to varying degrees.In the same period,the methylation rate of dermis tissue of COL1A1 gene was the highest,the methylation rate of mesenchyme,precartilage,and cartilage tissues were lower,and the methylation rate of mesenchyme tissue was the lowest,and the methylation difference between dermis tissue and the other three tissues was extremely significant(P<0.01).In the same tissue,the methylation rate of the middle stage of COL1A1 gene was significantly down-regulated compared with the early stage(P<0.05),and there was no significant difference in mesenchyme,precartilage,and cartilage tissues(P>0.05)and was extremely significant in mesenchyme tissue between the middle stage and the late stage(P<0.01).3.The methylation rates of OPN gene in the dermis tissues of the early stage,middle stage and late stage were(40.48±0.82)%,(40.00±1.43)% and(39.05±0.82)%;the methylation rates in the mesenchyme tissues were(37.62±0.82)%,(34.76±2.18)% and(38.57±1.43)%;the methylation rates in the precartilage tissues were(36.67±0.28)%,(29.52±1.65)% and(28.10±2.18)%;the methylation rates in the cartilage tissues were(31.90±1.65)%,(26.67±1.65)% and(24.29±1.43)%.There were 7 CpG sites in the promoter region of OPN gene,and these 3 CpG sites at 141 bp,263 bp and 477 bp were all methylated to varying degrees.In the same period,the methylation rates of the cartilage and precartilage tissues of OPN gene were significantly lower than those of dermis and mesenchyme tissues,and the methylation rate of precartilage tissue was significantly different from that of dermis and mesenchyme tissues in the middle and late stages(P<0.05),and the methylation rate of cartilage tissue was significantly different from that of dermis and mesenchyme tissues in three periods(P<0.01).In the same tissue,the methylation rate of the middle stage of OPN gene was down-regulated compared with the early stage,and the difference between the early stage and the middle stage in precartilage and cartilage tissues was extremely significant(P<0.01).There was no significant difference between the middle stage and the late stage(P>0.05).4.The methylation rates of S100A4 gene in the dermis tissues of the early stage,middle stage and late stage were(11.11±1.27)%,(10.83±1.44)% and(11.94±0.96)%;the methylation rates in the mesenchyme tissues were(4.72±0.48)%,(3.06±0.48)% and(3.33±0.83)%;the methylation rates in the precartilage tissues were(5.00±1.44)%,(4.72±0.96)% and(5.28±1.73)%;the methylation rates in the cartilage tissues were(6.67±0.83)%,(4.72±0.96)%and(6.39±1.73)%.There were 12 CpG sites in the promoter region of S100A4 gene,and these7 CpG sites at 67 bp,238 bp,279 bp,327 bp,359 bp,422 bp and 553 bp were all methylated to varying degrees.In the same period,the methylation rate of dermis tissue of S100A4 gene was the highest and was extremely significant with the other three tissues(P<0.01),while mesenchyme,precartilage,and cartilage tissues had no significant difference which had lower methylation levels(P>0.05),while the methylation level of mesenchyme tissue was the lowest.In the same tissue,the methylation rate of the middle stage of S100A4 gene was downregulated compared with the early stage,and the methylation rate of the late stage was upregulated compared with the middle stage,but there was no significant difference(P>0.05).5.COL1A1,OPN and S100A4 genes might be involved in the regulation of antler growth and development during the rapid growth phase and the ossification phase,as well as mesenchyme,precartilage and cartilage tissues.COL1A1 and S100A4 genes might play an important role in promoting antler growth and cell proliferation in the mesenchyme which was a region of rapid cell proliferation of antler.OPN gene might have an important regulatory role in the process of antler osteogenesis.In conclusion,COL1A1,OPN and S100A4 genes were all important regulators in the antler growth.Gene DNA methylation was closely related to the regulation of the gene.This study had scientific significance for clarifying the epigenetic differences between the deer antler top tissues of different growth stages at the molecular level,and to provide a theoretical basis for further revealing the regeneration,rapid growth and ossification mechanism of deer antler,and to provide reference ideas and scientific references for the research of cancer biology,tissue regeneration and organ repair of mammalian and other medical problems. |
