Xing Haihua, Zhang Furui, Han Ruobing, Li Heping
College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China.
Gene. 2023 Jun 5;868:147382. doi: 10.1016/j.gene.2023.147382. Epub 2023 Mar 21.
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.
为探究骨桥蛋白（OPN）基因在梅花鹿鹿茸生长过程中的生物学作用，对处于鹿茸早期（呈现鞍状外观，30天）、鹿茸快速生长且有两个分支期（60天）以及鹿茸最终有三个分支期（90天）的梅花鹿鹿茸顶端的真皮、间充质、软骨前组织和软骨组织进行了分析。采用亚硫酸氢盐测序PCR（BSP）和定量实时PCR（qRT-PCR）技术，从时空角度探究梅花鹿鹿茸中OPN的DNA启动子甲基化和mRNA表达情况。试验结果表明：1）真皮组织早期、中期和晚期OPN启动子的甲基化率分别为（40.48±0.82）%、（40.00±1.43）%和（39.05±0.82）%；间充质组织中的甲基化率分别为（37.62±0.82）%、（34.76±2.18）%和（38.57±1.43）%；软骨前组织中的甲基化率分别为（36.67±0.28）%、（29.52±1.65）%、（28.10±2.18）%；软骨组织中的甲基化率分别为（31.90±1.65）%、（26.67±1.65）%、（24.29±1.43）%。2）OPN启动子区域有7个CpG位点，其中-367bp、-245bp和-31bp的3个CpG位点均有不同程度的甲基化。3）在同一生长时期，真皮、间充质、软骨前和软骨组织中OPN的甲基化水平依次降低。在中期和晚期，软骨前组织启动子区域的甲基化水平与真皮和间充质组织的甲基化水平差异显著（P<0.05）；在不同生长时期，软骨组织启动子区域的甲基化水平与真皮和间充质组织的甲基化水平差异极显著（P<0.01）；在同一组织中，中期启动子区域的甲基化水平较早期下调，在软骨前和软骨组织中，早期和中期启动子区域的甲基化水平差异极显著（P<0.01）。4）OPN mRNA在软骨前和软骨组织中高表达。5）OPN启动子的甲基化水平与mRNA表达水平呈负相关。综上所述，推测OPN基因可能受启动子DNA甲基化水平调控，主要通过调节软骨前和软骨组织的生长来促进鹿茸的生长发育。
