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

Abstract

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

CLC Number:

S827.2

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.

Figures/Tables 8

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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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Mechanism of miR-137 Targeting MITF Regulating Melanogenesis in Goat Melanocytes

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