Wu Min, Su Dan, Li Xinxin, Xu Junjie, Fan Yihao, Wu Xinglin, Liu Xuelu, Su Bofei, Wang Dehe, Chen Yifan, Hao Erying, Bai Hao, Chen Hui, Chen Jilan, Shi Lei
College of Animal Science and Technology, Hebei Agricultural University, Baoding Hebei 071001, China.
BeijingHuadu Yukou Poultry Industry Co., Ltd. Beijing 101200, China.
Poult Sci. 2025 May 28;104(8):105373. doi: 10.1016/j.psj.2025.105373.
Crossed beak is a beak deformity that reduces feeding and drinking in affected chickens, thereby diminishing chicken production performance. Previous studies have shown that long non-coding RNA CA13 (lncRNA CA13), as an upstream regulator of carbonic anhydrase 13 (CA13), exhibited down-regulated expression in the lateral mandibular condyles of the short bone branch in cross-beaked chickens along with CA13. The study investigated the expression characteristics of lncRNA CA13 and its regulatory relationship with CA13 and elongation factor Tu GTP-binding domain-containing protein 2 (EFTUD2), laying the groundwork for the molecular mechanisms of lncRNA CA13 in chicken mandibular mineralization. The length of lncRNA CA13, amplified by 5' and 3' rapid amplification of cDNA ends (RACE) technique, was 2380 bp. Expression of lncRNA CA13 was significantly higher in mandibular condylar tissues compared to other tissues (P < 0.01). Using a successfully constructed osteoblast-induced differentiation model, orange calcified nodules were observed under the microscope after alizarin red staining, and the expressions of CA13, lncRNA CA13, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) were also detected to be significantly higher than those before induction (P < 0.01; P < 0.05). Integrated RNA affinity pull-down assay (RNA pull-down) combined with multiple bioinformatic analysis methods, as well as RNA binding protein immunoprecipitation assay (RIP), identified EFTUD2 as a transcription factor involved in the cellular mineralization process. The oe-lncRNA CA13 vector was constructed with an overexpression efficiency of 95.85 %, and the sh-lncRNA CA13 vector had an interference efficiency of 54.58 %. Overexpression of lncRNA CA13 significantly upregulated EFTUD2 and downregulated CA13; simultaneous interference with lncRNA CA13 significantly downregulated EFTUD2 and upregulated CA13. Thus, the lncRNA CA13/EFTUD2/CA13 axis plays a key role in the development of crossed beak by regulating the calcification process in the chicken mandible.
交叉喙是一种喙部畸形，会降低患病鸡的采食和饮水能力，从而降低鸡肉生产性能。先前的研究表明，长链非编码RNA CA13（lncRNA CA13）作为碳酸酐酶13（CA13）的上游调节因子，在交叉喙鸡短骨分支的外侧下颌髁中与CA13一起表现出表达下调。本研究调查了lncRNA CA13的表达特征及其与CA13和含延伸因子Tu GTP结合结构域蛋白2（EFTUD2）的调控关系，为lncRNA CA13在鸡下颌骨矿化中的分子机制奠定了基础。通过5'和3' cDNA末端快速扩增（RACE）技术扩增得到的lncRNA CA13长度为2380 bp。与其他组织相比，lncRNA CA13在下颌髁组织中的表达显著更高（P < 0.01）。利用成功构建的成骨细胞诱导分化模型，茜素红染色后在显微镜下观察到橙色钙化结节，同时检测到CA13、lncRNA CA13、 runt相关转录因子2（RUNX2）和碱性磷酸酶（ALP）的表达也显著高于诱导前（P < 0.01；P < 0.05）。综合RNA亲和拉下试验（RNA pull-down）结合多种生物信息学分析方法以及RNA结合蛋白免疫沉淀试验（RIP），确定EFTUD2是参与细胞矿化过程的转录因子。构建的oe-lncRNA CA13载体过表达效率为95.85%，sh-lncRNA CA13载体干扰效率为54.58%。lncRNA CA13的过表达显著上调EFTUD2并下调CA13；同时干扰lncRNA CA13显著下调EFTUD2并上调CA13。因此，lncRNA CA13/EFTUD2/CA13轴通过调节鸡下颌骨的钙化过程在交叉喙的发育中起关键作用。
