miR-137靶向MITF调控山羊黑色素细胞生成黑色素的机制研究

doi:10.11843/j.issn.0366-6964.2025.01.018

摘要/Abstract

摘要：

旨在探讨miR-137在黑山羊被毛颜色形成中的调控作用，为研究黑色素生成的分子机制提供新的视角。本研究选取4日龄黑山羊肩后约5 cm处皮肤组织分离黑色素细胞，通过免疫组化和免疫荧光试验对黑色素细胞定位，采用多巴染色和免疫荧光的方法鉴定山羊黑色素细胞，裂解黑色素细胞测定黑色素的含量，通过细胞转染、实时荧光定量PCR、Western blot等方法探究miR-137通过靶向黑素细胞诱导转录因子(melanocyte inducing transcription factor，MITF)对黑色素细胞生成黑色素的调控机制。结果显示，在毛乳头上方及外毛根鞘处有黑色素细胞的存在，黑色素细胞呈两极或三极状，多巴染色鉴定黑色素细胞呈棕黑色阳性，免疫荧光鉴定毛色生成有关的标记基因表达阳性。转染过表达载体后的山羊黑色素细胞与NC组相比，黑色素含量降低(P < 0.05)，同时，inhibitor组趋势相反(P < 0.05)。通过RT-qPCR与Western blot对黑色素细胞生成相关基因的表达水平进行分析，结果显示，过表达或抑制miR-137对靶基因MITF的mRNA表达水平影响并不显著(P＞0.05)。而过表达miR-137对黑色素细胞生成的标志基因TYR、TYRP-1、TYRP-2的mRNA表达起抑制作用(P＜0.05)，同时，inhibitor组趋势相反(P < 0.05)。MITF的蛋白质表达水平mimic组显著低于mimic NC组(P＜0.05)，inhibitor组的蛋白质表达水平显著高于inhibitor NC组(P＜0.05)，mimic组黑色素细胞生成的标志基因TYR、TYRP-1、TYRP-2的蛋白质表达水平显著低于mimic NC组(P＜0.05)，inhibitor组高于inhibitor NC组(P＜0.05)。本研究发现，miR-137不影响MITF的mRNA表达量，而是通过靶向MITF间接抑制山羊黑色素细胞黑色素的生成，这为研究miR-137在山羊肤色和毛色形成中的作用提供了理论依据。

关键词: miR-137, MITF, 黑色素细胞, 黑色素生成, 山羊

Abstract:

The aim of this study was to investigate the regulation role of miR-137 in coat color formation in black goats, in order to provide a new perspective in investigating the molecular mechanism of melanogenesis. Melanocytes were isolated from skin tissues of 4-day-old black goats at about 5 cm behind the shoulder, the melanocytes were localized by immunohistochemistry and immunofluorescence assays, the goat melanocytes were identified by Dopa staining and immunofluorescence, the content of melanin were determined after melanocytes lysed, and miR-137 regulation mechanism of melanogenesis by targeting melanocyte inducing transcription factor (MITF) was explored by cell transfection, real-time fluorescence quantitative PCR, and Western blot. The results demonstrated the presence of melanocytes above the dermal papilla and at the outer hair root sheath, melanocytes were bipolar or tripolar, Dopa staining identified the melanocytes as brownish-black positive, and immunofluorescence identified the expression of marker genes related to hair color generation as positive. The melanin content of transfected goat melanocytes was reduced compared with that of the NC group (P < 0.05), meanwhile the trend of the inhibitor group was opposite (P < 0.05). The expression levels of melanocyte generation-related genes were analysed by RT-qPCR and Western blot, and it was found that overexpression or inhibition of miR-137 did not have a significant effect on the mRNA expression levels of the target gene MITF (P>0.05). While overexpression of miR-137 inhibited the expression of mRNA of TYR, TYRP-1 and TYRP-2, which are marker genes for melanogenesis (P < 0.05), meanwhile, the trend was opposite in the inhibitor group (P < 0.05). Regarding the protein expression level, the protein expression level of MITF in the mimic group was significantly lower than that in the mimic NC group (P < 0.05), the protein expression level of MITF in the inhibitor group was significantly higher than that in the inhibitor NC group (P < 0.05).The protein expression levels of TYR, TYRP-1 and TYRP-2 were significantly lower in the mimic group than that in the mimic NC group (P < 0.05), and their expression in the inhibitor group was higher than that in the inhibitor NC group (P < 0.05). In this study, we found that miR-137 did not affect the mRNA expression of MITF, but indirectly inhibited melanin production in goat melanocytes by targeting MITF, which provides a theoretical basis for studying the role of miR-137 in the formation of skin and coat colour in goats.

Key words: miR-137, MITF, melanocytes, melanogenesis, goat

中图分类号:

S827.2

于凤姣, 刘开东, 宋伟杰, 柳楠, 李和刚, 赵金山, 高霄霄, 贺建宁. miR-137靶向MITF调控山羊黑色素细胞生成黑色素的机制研究[J]. 畜牧兽医学报, 2025, 56(1): 189-200.

YU Fengjiao, LIU Kaidong, SONG Weijie, LIU Nan, LI Hegang, ZHAO Jinshan, GAO Xiaoxiao, HE Jianning. Mechanism of miR-137 Targeting MITF Regulating Melanogenesis in Goat Melanocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 189-200.

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基因 Gene	引物序列(5′→3′) Primers sequence	基因ID Gene ID	产物长度/bp Length
TYR	F: CCTCGGCTGATGTGGAGTTT R: CTGGGACATCGTTCCGTTCA	102182281	188
TYRP-1	F: AACACCTGCGACATTTGCAC R: GTCCCCCTGTTCCATTCAGG	100861390	403
TYRP-2	F: TTCTCACACCAAGGACCTGC R: AGTAGGGCAAAGCGAAGGAC	102169882	109
MITF	F: AGGAGTTTCACGAAGAGCCC R: CTCCTCGGAACTGCTCTTCAA	102173423	71
GAPDH	F: GTCGGAGTGAACGGATTTGG R: CATTGATGACGAGCTTCCCG	100860872	196
U6	F: GGAACGATACAGAGAAGATTAGC R: TGGAACGCTTCACGAATTTGCG	102190828	68
miR-137	F: ACTCTCTTCGGTGACGGGTA R: CGCTGGTACTCTCCTCGACT	104795729	90

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