山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究

doi:10.11843/j.issn.0366-6964.2024.03.015

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (3): 1019-1029.doi: 10.11843/j.issn.0366-6964.2024.03.015

山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究

肖敏¹, 赵威¹, 孙武^1,2, 娜日苏^1,3, 赵乐¹, 刘陶禄¹, 张继攀^1*, 赵永聚^1*

1. 西南大学动物科学技术学院, 草食动物科学重庆市重点实验室, 重庆市牧草与草食家畜重点实验室, 重庆 400715;
2. 青海大学畜牧兽医科学院, 西宁 810016;
3. 内蒙古农业大学动物科学学院, 呼和浩特 010018

收稿日期:2023-08-29 出版日期:2024-03-23 发布日期:2024-03-27
通讯作者: 张继攀,主要从事山羊遗传育种研究,E-mail:jpanzhang@live.com;赵永聚,主要从事山羊遗传育种研究,E-mail:zyongju@163.com
作者简介:肖敏(1998-),女,回族,贵州盘州人,硕士生,主要从事山羊被毛颜色分子调控机制研究,E-mail:minxiao1698@163.com;赵威(1997-),男,白族,云南昆明人,硕士生,主要从事山羊被毛颜色分子调控机制研究,E-mail:2859588007@qq.com
基金资助:
重庆市博士后科学基金项目（CSTB2023NSCQ-BHX0147）；重庆市技术创新与应用发展专项重点项目（CSTB2022TIAD-CUX0013）；重庆市现代农业产业技术体系（草食牲畜：CQMAITS202313）

The Skin Tissue miRNA-seq and Regulation Mechanism of miR-129-5p on Melanogenesis in Goat (Capra hircus)

XIAO Min¹, ZHAO Wei¹, SUN Wu^1,2, NA Risu^1,3, ZHAO Le¹, LIU Taolu¹, ZHANG Jipan^1*, ZHAO Yongju^1*

1. Chongqing Key Laboratory of Forage & Herbivore, Chongqing Key Laboratory of Herbivore Science, College of Animal Science and Technology, Southwest University, Chongqing 400715, China;
2. Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, China;
3. College of Animal Science, Inner Mongolia Agricultural University, Huhhot 010018, China

Received:2023-08-29 Online:2024-03-23 Published:2024-03-27

摘要/Abstract

摘要： 旨在筛选对黑色素生成起调节作用的小RNA，并探究其对山羊肤色及毛色的调控机制。本研究采集了健康酉州乌羊（Youzhou dark goat，YZDG）、川东白山羊（Chuandong white goat，CDWG）100日龄胎羊皮肤样本（n=3），和健康2~3周岁大足黑山羊（Dazu black goat，DBG）、内蒙古绒山羊（Inner Mongolia cashmere goat，IMCG）个体皮肤样本（n=3），利用组织切片染色技术观察皮肤中黑色素沉积情况；通过小RNA测序技术筛选差异miRNAs；培养B16-F10皮肤黑色素瘤细胞，利用细胞转染、qPCR、Western Blot、黑色素含量检测等技术验证miR-129-5p对黑色素生成的影响。结果显示，黑色素颗粒明显在YZDG胎羊皮肤和DBG毛囊的毛球、毛干、外根鞘等部位沉积，而在CDWG胎羊皮肤和IMCG表皮、毛囊中没有被观察到。经测序分析，在肤色差异的YZDG和CDWG中筛选到62个差异表达miRNAs，其中31个在乌皮山羊中上调，31个下调。在毛色差异的DBG和IMCG中，筛选到38个差异表达miRNAs，其中10个在黑色被毛山羊中表达上调，28个表达下调。两组测序结果均显示miR-129-5p在乌皮和黑色被毛山羊皮肤中高表达（P<0.05）。在细胞中过表达miR-129-5p后，相比于对照组，mimics组细胞黑色素沉积量提高了18.9%（P<0.05），TYR、TYRP1基因表达量分别上调57.3%和16.5%（P<0.05），蛋白表达量分别显著上调49.2%和40.2%（P<0.05）；但MITF基因及其蛋白表达量无显著变化（P>0.05）。在抑制miR-129-5p后，inhibitor组TYR基因mRNA表达下调38.9%、蛋白表达水平下调21.1%（P<0.05）；TYRP1、MITF蛋白表达水平分别下调25.3%及28.4%（P<0.05）。本研究发现，miR-129-5p在不同肤色及毛色的山羊皮肤中差异表达，且可通过调控TYR、TYRP1等关键基因的表达影响黑色素的生成，是山羊肤色和毛色形成过程的重要调节因子。

关键词: 山羊, 肤色, miR-129-5p, 黑色素生成, TYR

Abstract: This work aimed to screen key miRNA(s) that regulate skin melanin deposition in goats, and to explore its regulation mechanism on skin color or coat color. The 100-day-old fetal skin samples from the Youzhou dark goat (YZDG, n=3) and Chuandong white goat (CDWG, n=3), and skin samples from the healthy 2-3-years-old Dazu black goats (DBG, n=3), Inner Mongolia cashmere goat (IMCG, n=3) were collected to observe skin melanin deposition by section staining technology, and to explore differential miRNAs by miRNA-seq technology. Otherwise, B16-F10 cutaneous melanoma cells were cultured. The effect of miR-129-5p on melanin production was verified by cell transfection, qPCR, Western blot and melanin content detection. The results showed that melanin granules were obviously deposited in the hair bulb, hair shaft, and outer root sheath of DBG hair follicles and YZDG fetal goat skin, whereas they were not observed in the skin of CDWG fetal goats and the epidermis, hair follicles of IMCG. The sequencing results showed that 62 miRNAs were differentially expressed between YZDG and CDWG, of which 31 were up-regulated and 31 down-regulated in dark-skin goats. A total of 38 differentially expressed miRNAs were found between DBG and IMCG, 10 were up-regulated and 28 down-regulated in black-coated goats. The result showed miR-129-5p was significantly highly expressed in dark-skin and black-coated goats (P<0.05). After over-expression of miR-129-5p, the cell melanin deposition in the mimics group was increased by 18.9% (P<0.05), the expression of the TYR and TYRP1 genes was up-regulated by 57.3% and 16.5%, respectively (P<0.05), and the protein expression was significantly up-regulated by 49.2% and 40.2% (P<0.05); but the mRNA and protein expressions of MITF were not significantly changed (P>0.05). After inhibition of miR-129-5p, the mRNA expression of TYR gene and its protein expression level in the inhibitor group were significantly down-regulated by 38.9% and 21.1%, respectively (P<0.05). And TYRP1, MITF protein expression levels were down-regulated by 25.3% and 28.4%, respectively (P<0.05). The results indicated that the miR-129-5p was differentially expressed in different skin colors or coat colors of goats, and it could affect melanin deposition by regulating the transcript and protein abundance of melanin-related genes (such as TYR and TYRP1). These results demonstrate that miR-129-5p is an important regulator involved in the skin melanogenesis of goats.

Key words: Capra hircus, skin color, miR-129-5p, melanogenesis, TYR

中图分类号:

S827.2

肖敏, 赵威, 孙武, 娜日苏, 赵乐, 刘陶禄, 张继攀, 赵永聚. 山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究[J]. 畜牧兽医学报, 2024, 55(3): 1019-1029.

XIAO Min, ZHAO Wei, SUN Wu, NA Risu, ZHAO Le, LIU Taolu, ZHANG Jipan, ZHAO Yongju. The Skin Tissue miRNA-seq and Regulation Mechanism of miR-129-5p on Melanogenesis in Goat (Capra hircus)[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1019-1029.

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山羊皮肤组织miRNA测序与miR-129-5p调控黑色素生成的功能研究

The Skin Tissue miRNA-seq and Regulation Mechanism of miR-129-5p on Melanogenesis in Goat (Capra hircus)

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