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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参考文献 44

1	CHELI Y , OHANNA M , BALLOTTI R , et al. Fifteen-year quest for microphthalmia-associated transcription factor target genes[J]. Pigment Cell Melanoma Res, 2010, 23 (1): 27- 40. doi: 10.1111/j.1755-148X.2009.00653.x
2	李佳璐. 毛色相关miRNAs及其靶基因在不同品种、不同月龄山羊皮肤中的表达研究[D]. 重庆: 西南大学, 2020.
	LI J L. The research on the expression of coat color-related miRNAs and their target genes in the goats of different breeds and ages[D]. Chongqing: Southwest University, 2020. (in Chinese)
3	MERECZ-SADOWSKA A , SITAREK P , KOWALCZYK T , et al. The modulation of melanogenesis in B16 cells upon treatment with plant extracts and isolated plant compounds[J]. Molecules, 2022, 27 (14): 4360. doi: 10.3390/molecules27144360
4	孙学良. 川中黑山羊遗传多样性和毛色性状的全基因组关联分析[D]. 雅安: 四川农业大学, 2023.
	SUN X L. Genetic diversity and genome-wide association study of coat color in chuanzhong black goats[D]. Yaan: Sichuan Agricultural University, 2023. (in Chinese)
5	D'MELLO S A N , FINLAY G J , BAGULEY B C , et al. Signaling pathways in melanogenesis[J]. Int J Mol Sci, 2016, 17 (7): 1144. doi: 10.3390/ijms17071144
6	VAVRICKA C J , RAY K W , CHRISTENSEN B M , et al. Purification and N-glycosylation analysis of melanoma antigen dopachrome tautomerase[J]. Protein J, 2010, 29 (3): 204- 212. doi: 10.1007/s10930-010-9241-9
7	DOLINSKA M B , WOODS T , OSUNA I , et al. Protein biochemistry and molecular modeling of the intra-melanosomal domain of human recombinant Tyrp2 protein and OCA8-related mutant variants[J]. Int J Mol Sci, 2022, 23 (3): 1305. doi: 10.3390/ijms23031305
8	HUSHCHA Y , BLO I , OTON-GONZALEZ L , et al. microRNAs in the regulation of melanogenesis[J]. Int J Mol Sci, 2021, 22 (11): 6104. doi: 10.3390/ijms22116104
9	刘杰, 许香萍, 邓铭, 等. miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响[J]. 畜牧兽医学报, 2023, 54 (6): 2421- 2435. doi: 10.11843/j.issn.0366-6964.2023.06.021
	LIU J , XU X P , DENG M , et al. Effect of miR-144-5p targeting WNT5a on the proliferation and apoptosis of goat ovarian granulosa cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (6): 2421- 2435. doi: 10.11843/j.issn.0366-6964.2023.06.021
10	MILLER K J , BROWN D A , IBRAHIM M M , et al. MicroRNAs in skin tissue engineering[J]. Adv Drug Deliv Rev, 2015, 88, 16- 36. doi: 10.1016/j.addr.2015.04.018
11	张寒月, 赵丹, 梁煜, 等. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54 (8): 3262- 3274. doi: 10.11843/j.issn.0366-6964.2023.08.013
	ZHANG H Y , ZHAO D , LIANG Y , et al. miR-150 regulates ovine preadipocyte differentiation bytargeting AOC3[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (8): 3262- 3274. doi: 10.11843/j.issn.0366-6964.2023.08.013
12	XU Y Z , GUO B Y , LIU X Y , et al. miR-34a inhibits melanoma growth by targeting ZEB1[J]. Aging (Albany NY), 2021, 13 (11): 15538- 15547.
13	任晓丽, 范玉营, 皇甫和平, 等. miR-502调控犬乳腺肿瘤细胞增殖、迁移和EMT的作用机制[J]. 畜牧兽医学报, 2023, 54 (10): 4379- 4388. doi: 10.11843/j.issn.0366-6964.2023.10.034
	REN X L , FAN Y Y , HUANGFU H P , et al. Mechanism of miR-502 regulating proliferation, migration, invasion and EMT of canine mammary tumor cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (10): 4379- 4388. doi: 10.11843/j.issn.0366-6964.2023.10.034
14	CHEN W J , LAI Y J , LEE J L , et al. CREB/ATF3 signaling mediates indoxyl sulfate-induced vascular smooth muscle cell proliferation and neointimal formation in uremia[J]. Atherosclerosis, 2020, 315, 43- 54. doi: 10.1016/j.atherosclerosis.2020.11.009
15	MAHMOUDI E , CAIRNS M J . MiR-137:an important player in neural development and neoplastic transformation[J]. Mol Psychiatry, 2017, 22 (1): 44- 55. doi: 10.1038/mp.2016.150
16	VAVRICKA C J , RAY K W , CHRISTENSEN B M , et al. Purification and N-glycosylation analysis of melanoma antigen dopachrome tautomerase[J]. Protein J, 2010, 29 (3): 204- 212. doi: 10.1007/s10930-010-9241-9
17	LAGOS-QUINTANA M , RAUHUT R , YALCIN A , et al. Identification of tissue-specific microRNAs from mouse[J]. Curr Biol, 2002, 12 (9): 735- 739. doi: 10.1016/S0960-9822(02)00809-6
18	STAFFORD M Y C , MCKENNA D J . MiR-182 is upregulated in prostate cancer and contributes to tumor progression by targeting MITF[J]. Int J Mol Sci, 2023, 24 (3): 1824. doi: 10.3390/ijms24031824
19	WRIGHT C , TURNER J A , CALHOUN V D , et al. Potential impact of miR-137 and its targets in schizophrenia[J]. Front Genet, 2013, 4, 58.
20	肖敏, 赵威, 孙武, 等. 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. doi: 10.11843/j.issn.0366-6964.2024.03.015
	XIAO M , ZHAO W , SUN W , et al. 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. doi: 10.11843/j.issn.0366-6964.2024.03.015
21	BEMIS L T , CHEN R , AMATO C M , et al. MicroRNA-137 targets microphthalmia-associated transcription factor in melanoma cell lines[J]. Cancer Res, 2008, 68 (5): 1362- 1368. doi: 10.1158/0008-5472.CAN-07-2912
22	BANZHAF-STRATHMANN J , EDBAUER D . Good guy or bad guy: the opposing roles of microRNA 125b in cancer[J]. Cell Commun Signal, 2014, 12, 30. doi: 10.1186/1478-811X-12-30
23	CHEN X , BA Y , MA L J , et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases[J]. Cell Res, 2008, 18 (10): 997- 1006. doi: 10.1038/cr.2008.282
24	MITCHELL P S , PARKIN R K , KROH E M , et al. Circulating microRNAs as stable blood-based markers for cancer detection[J]. Proc Natl Acad Sci U S A, 2008, 105 (30): 10513- 10518. doi: 10.1073/pnas.0804549105
25	CHEN W J , LAI Y J , LEE J L , et al. CREB/ATF3 signaling mediates indoxyl sulfate-induced vascular smooth muscle cell proliferation and neointimal formation in uremia[J]. Atherosclerosis, 2020, 315, 43- 54. doi: 10.1016/j.atherosclerosis.2020.11.009
26	FILIPOWICZ W , BHATTACHARYYA S N , SONENBERG N , et al. Mechanisms of post transcriptional regulation by microRNAs: are the answers in sight?[J]. Nat Rev Genet, 2008, 9 (2): 102- 114. doi: 10.1038/nrg2290
27	LEI C , JING G , JICHAO W , et al. MiR-137's tumor suppression on prolactinomas by Targeting MITF and modulating Wnt signaling pathway[J]. J Clin Endocrinol Metab, 2019, 104 (12): 6391- 6402. doi: 10.1210/jc.2018-02544
28	BEMIS L T , CHEN R , AMATO C M , et al. MicroRNA-137 targets microphthalmia-associated transcription factor in melanoma cell lines[J]. Cancer Res, 2008, 68 (5): 1362- 1368. doi: 10.1158/0008-5472.CAN-07-2912
29	SALEH A A , RASHAD A M A , HASSANINE N N A M , et al. Assessment of hair and cashmere properties and their genetic background of several goat breeds in Southwest China[J]. Sci Rep, 2022, 12 (1): 11135. doi: 10.1038/s41598-022-14441-1
30	DONG C S , WANG H D , XUE L L , et al. Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model[J]. RNA, 2012, 18 (9): 1679- 1686. doi: 10.1261/rna.033977.112
31	ZHU Z , HE J , JIA X , et al. MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in Alpaca (Lama pacos) skin melanocytes[J]. Domest Anim Endocrinol, 2010, 38 (3): 200- 209. doi: 10.1016/j.domaniend.2009.10.004
32	WANG P , LI Y , HONG W S , et al. The changes of microRNA expression profiles and tyrosinase related proteins in MITF knocked down melanocytes[J]. Mol Biosyst, 2012, 8 (11): 2924- 2931. doi: 10.1039/c2mb25228g
33	LEE H D , LEE W H , ROH E , et al. Manassantin A inhibits cAMP-induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes[J]. Exp Dermatol, 2011, 20 (9): 761- 763. doi: 10.1111/j.1600-0625.2011.01296.x
34	YUN J Y , ROH E , SON J K , et al. Effect of saucerneol D on melanin production in cAMP-elevated melanocytes[J]. Arch Pharm Res, 2011, 34 (8): 1339- 1345. doi: 10.1007/s12272-011-0814-8
35	KIM A , YANG Y N , LEE M S , et al. NDRG2 gene expression in B16F10 melanoma cells restrains melanogenesis via inhibition of Mitf expression[J]. Pigment Cell Melanoma Res, 2008, 21 (6): 653- 664. doi: 10.1111/j.1755-148X.2008.00503.x
36	CENTENO P P , PAVET V , MARAIS R . The journey from melanocytes to melanoma[J]. Nat Rev Cancer, 2023, 23 (6): 372- 390. doi: 10.1038/s41568-023-00565-7
37	ZHOU S H , ZENG H L , HUANG J H , et al. Epigenetic regulation of melanogenesis[J]. Ageing Res Rev, 2021, 69, 101349. doi: 10.1016/j.arr.2021.101349
38	黄晶. 鹅色素相关基因TYR和MITF的单核苷酸多态性与羽色性状的相关性研究[D]. 雅安: 四川农业大学, 2012.
	HUANG J. Study on the association between single nucleotide polymorphisms of melanogenic TYR gene and MITF gene and the plumage color traits of Goose[D]. Yaan: Sichuan Agricultural University, 2012. (in Chinese)
39	马淑慧, 薛霖莉, 徐刚, 等. 黑色素细胞中过量表达miR-137对TYRP-1和TYRP-2的影响[J]. 中国农业科学, 2013, 46 (16): 3452- 3459. doi: 10.3864/j.issn.0578-1752.2013.16.016
	MA S H , XUE L L , XU G , et al. The influences of over-expressing miR-137 on TYRP-1 and TYRP-2 in melanocytes[J]. Scientia Agricultura Sinica, 2013, 46 (16): 3452- 3459. doi: 10.3864/j.issn.0578-1752.2013.16.016
40	许冬梅, 唐中伟, 朱芷葳, 等. MiR-186-5p对绵羊黑色素细胞黑色素生成的调控[J]. 中国生物化学与分子生物学报, 2020, 36 (8): 961- 969.
	XU D M , TANG Z W , ZHU Z W , et al. Regulation of miR-186-5p on melanogenesis in sheep melanocytes[J]. Chinese Journal of Biochemistry and Molecular Biology, 2020, 36 (8): 961- 969.
41	LUO C L , MERZ P R , CHEN Y W , et al. MiR-101 inhibits melanoma cell invasion and proliferation by targeting MITF and EZH2[J]. Cancer Lett, 2013, 341 (2): 240- 247. doi: 10.1016/j.canlet.2013.08.021
42	LI J L , LIU L B , ZHANG J P , et al. The expression of miR-129-5p and its target genes in the skin of goats[J]. Anim Biotechnol, 2021, 32 (5): 573- 579. doi: 10.1080/10495398.2020.1730392
43	CHEN W Y , WANG H , DONG B , et al. Molecular cloning and expression analysis of tyrosinase gene in the skin of Jining gray goat (Capra hircus)[J]. Mol Cell Biochem, 2012, 366 (1-2): 11- 20. doi: 10.1007/s11010-012-1275-1
44	JIANG S , YU X J , DONG C S . MiR-137 affects melanin synthesis in mouse melanocyte by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway[J]. Biosci Biotechnol Biochem, 2016, 80 (11): 2115- 2121. doi: 10.1080/09168451.2016.1200455

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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