DNA methylation pattern and mRNA expression of OPN promoter in sika deer antler tip tissues.

ABSTRACT

In order to explore the biological role of OPN gene during the growth of sika deer antler, the dermis, mesenchyme, precartilage and cartilage tissues of sika deer antler tip at the early period of the antler with a saddle-like appearance (30 days), the rapid growth period of the antler with two branches (60 days), and the final period of the antler with three branches (90 days) were analyzed. Bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR) were used to explore the DNA promoter methylation and mRNA expression of OPN in sika deer antler from the perspective of space and time. The test results showed that 1) The methylation rates of OPN promoter at the early, middle and late periods of dermis tissue were (40.48 ± 0.82)%, (40.00 ± 1.43)%, and (39.05 ± 0.82)%; The methylation rates in mesenchyme tissue were (37.62 ± 0.82)%, (34.76 ± 2.18)%, and (38.57 ± 1.43)%; The methylation rates in precartilage tissue were (36.67 ± 0.28)%, (29.52 ± 1.65)%, (28.10 ± 2.18)%; The methylation rates in cartilage tissue were (31.90 ± 1.65)%, (26.67 ± 1.65)%, (24.29 ± 1.43)%. 2) There are 7 CpG sites in the OPN promoter region, and the 3 CpG sites of -367 bp, -245 bp and -31 bp are all methylated to different level. 3) The methylation level of OPN in the dermis, mesenchyme, precartilage and cartilage tissues decreased in sequence at the same growth period. At the middle and late periods, the methylation level of the promoter region of the precartilage tissue was significantly different from that of the dermis and mesenchyme tissues (P < 0.05); At different growth periods, the methylation level of the promoter region of cartilage tissue was extremely significantly different from that of dermis and mesenchyme tissues (P < 0.01); In the same tissue, the methylation level of the promoter region at the middle period was down-regulated compared with the early period, and the methylation level of the promoter region at the early period and the middle period was extremely significantly different in the precartilage and cartilage (P < 0.01). 4) OPN mRNA is highly expressed in precartilage and cartilage tissues. 5) The methylation level of OPN promoter was negatively correlated with mRNA expression level. In summary, it is speculated that the OPN gene, which may be regulated by the DNA methylation level of the promoter, promotes the growth and development of deer antler mainly by regulating the growth of precartilage and cartilage tissues.