Ji Kai Yuan, Zhang Xue Qing, Zhao Yi Wei, Liang Chun E, Yuan Xin, Zhang Yun Hai
Anhui province Key Laboratory of Genetic Resources Protection and Biological Breeding for Livestock and Poultry, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.
Linquan Comprehensive Experimental Station of Anhui Agricultural University, Anhui Agricultural University, Linquan 236400, China.
Anim Biosci. 2025 Feb;38(2):255-266. doi: 10.5713/ab.24.0316. Epub 2024 Aug 26.
This study investigated the effects of circRNA18_46222157_46248185 (named circRNA18) on goat melanogenesis, which differs significantly in goat skins isolated from white and brown coat-colored skins.
Expression patterns of circRNA18 in goat skin and melanocytes were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. The circRNA18 interference vector was designed and synthesized to transfect melanocytes and detect the effect of circRNA18 interference on melanin production. Bioinformatics software was used to predict the targeted adsorption miRNAs of circRNA18, verified by luciferase assay. A miRNA expression vector was constructed and transfected into melanocytes to detect the effect of miRNA on melanin production, and the targeted regulatory genes were detected by luciferase assay. Target gene interference vector was constructed to detect the influence of target gene interference on melanin production.
qRT-PCR results unveiled distinct expression patterns of circRNA18 in diverse tissues of male and female goats, while in situ hybridization assays showed that circRNA18 is expressed in the cytoplasm of melanocytes. Functional analysis demonstrated that the downregulation of circRNA18 in melanocytes leads to a significant increase (p<0.01) in melanin production. Bioinformatics analysis identified a potential miR-211 binding site on circRNA18, and luciferase assay confirmed their interaction. Overexpression of miR-211 in melanocytes significantly augmented (p<0.01) melanin production. There were two potential miR-211 binding sites on adenoviral E1A-binding protein (EP300), and the overexpression of miR-211 in melanocytes significantly decreased (p<0.001) EP300 expression, with luciferase assay confirming their interaction. Downregulation of EP300 expression in melanocytes through siRNA-EP300 transfection results in a substantial increase (p<0.05) in melanin production. qRT-PCR results indicated that overexpression of mimics-circRNA18 in melanocytes markedly suppressed (p<0.0001) miR-211 expression, significantly elevated (p<0.01) EP300 expression, and significantly inhibited (p<0.001) melanin production.
circRNA18_46222157_46248185 acted as a negative regulator of melanogenesis in goat melanocytes by targeting the miR-211/EP300 pathway, and guiding animal hair color breeding strategies.
本研究探讨了circRNA18_46222157_46248185（命名为circRNA18）对山羊黑色素生成的影响，该circRNA在白色和棕色被毛山羊皮肤中存在显著差异。
通过定量实时聚合酶链反应（qRT-PCR）和原位杂交确定circRNA18在山羊皮肤和黑色素细胞中的表达模式。设计并合成circRNA18干扰载体转染黑色素细胞，检测circRNA18干扰对黑色素生成的影响。利用生物信息学软件预测circRNA18靶向吸附的miRNA，并通过荧光素酶报告基因检测进行验证。构建miRNA表达载体并转染黑色素细胞，检测miRNA对黑色素生成的影响，并通过荧光素酶报告基因检测靶向调控基因。构建靶基因干扰载体，检测靶基因干扰对黑色素生成的影响。
qRT-PCR结果揭示了circRNA18在雄性和雌性山羊不同组织中的不同表达模式，而原位杂交分析表明circRNA18在黑色素细胞的细胞质中表达。功能分析表明，黑色素细胞中circRNA18的下调导致黑色素生成显著增加（p<0.01）。生物信息学分析在circRNA18上鉴定出一个潜在的miR-211结合位点，荧光素酶报告基因检测证实了它们之间的相互作用。黑色素细胞中miR-211的过表达显著增加（p<0.01）黑色素生成。腺病毒E1A结合蛋白（EP300）上有两个潜在的miR-211结合位点，黑色素细胞中miR-211的过表达显著降低（p<0.001）EP300表达，荧光素酶报告基因检测证实了它们之间的相互作用。通过siRNA-EP300转染下调黑色素细胞中EP300的表达导致黑色素生成显著增加（p<0.05）。qRT-PCR结果表明，黑色素细胞中mimics-circRNA18的过表达显著抑制（p<0.0001）miR-211表达，显著升高（p<0.01）EP300表达，并显著抑制（p<0.001）黑色素生成。
circRNA18_46222157_46248185通过靶向miR-211/EP300途径，作为山羊黑色素细胞黑色素生成的负调节因子，为动物毛色育种策略提供指导。
