circKIF27靶向miR-129-5p/TGIF2通路抑制山羊黑色素细胞增殖，降低黑色素生成

doi:10.1016/j.jia.2024.02.008

摘要/Abstract

摘要： 动物的被皮和毛发是经济动物十分重要的经济性状，其质量的好坏直接影响动物的经济价值。动物被毛的颜色是由皮肤毛囊中的黑色素细胞产生的黑色素决定，研究黑色素的生成规律不仅对指导动物育种具有重要的意义，同时对解析色素沉积和色素生成相关疾病的发病机理有重要的参考价值。Circular RNA-micro RNA (circRNA-miRNA) 网络广泛参与各种生物过程，但其在色素沉着中的作用尚未被解析。前期研究发现，circKIF27的表达水平在白色和棕色山羊皮肤分离的黑色素细胞中存在显著差异，其具体的功能尚不明确。我们通过原位杂交实验、PCR实验证实，circKIF27具有circRNA的结构特点，表达于黑色素细胞的细胞质。表型研究表明，黑色素细胞中过表达circKIF27可以显著降低黑色素产量(P<0.01)，抑制细胞增殖(P<0.0001)。通过生物信息学分析发现，circKIF27与miR-129-5p存在靶向结合位点，可能存在靶向吸附关系。通过荧光素酶报告实验、qRT-PCR实验证实，circKIF27靶向吸附miR-129-5p抑制其表达。MiR-129-5p功能研究发现，黑色素细胞中过表达miR-129-5p可以提高黑色素产量(P<0.01)，促进细胞增殖(P<0.0001)。通过生物信息学分析发现，TGIF2存在两个潜在的miR-129-5p结合位点，通过qRT-PCR实验证实miR-129-5p可以抑制TGIF2表达。为进一步研究TGIF2在黑色素细胞中的功能，通过构建siRNA-TGIF2，转染黑色素细胞降低TGIF2表达水平，黑色素的产量(P<0.01)和细胞增殖(P<0.0001)速度都显著提升。以上结果表明，circKIF27通过靶向miR-129-5p/TGIF2通路抑制山羊皮肤黑色素生成。本研究为circRNA-miRNA网络调控动物黑色素形成的分子机制提供了新的见解，这有助于了解哺乳动物皮肤和被毛色素沉着的复杂过程，并为指导动物育种提供参考。

Abstract: Skin and hair pigmentation in animals involve intricate regulatory processes. Circular RNA-microRNA (circRNA-miRNA) networks play vital roles in various biological processes, although their involvement in pigmentation has been underexplored. This study focused on circKIF27 expression, which differs significantly in melanocytes isolated from white and brown Boer coat-colored skin, yet its function remains unclear. Here, we investigated the roles of circKIF27 in melanocytes. In situ hybridization assays demonstrated that circKIF27 is expressed in the cytoplasm of melanocytes. qRT-PCR results revealed differential expression levels of circKIF27 in various tissues of male and female goats. Functional analysis showed that circKIF27 overexpression in melanocytes significantly reduces melanin production (P<0.01) and inhibits cell proliferation (P<0.0001). Bioinformatics analysis identified a putative miR-129-5p binding site on circKIF27, and luciferase reporter assays confirmed their interaction. Overexpression of miR-129-5p in melanocytes enhances melanin production (P<0.01) and promotes cell proliferation (P<0.05). Further analysis revealed that TGIF2 possesses two potential miR-129-5p binding sites, and miR-129-5p overexpression in melanocytes significantly inhibits TGIF2 expression (P<0.0001), suggesting a targeted regulatory relationship between these two molecules. Silencing TGIF2 expression via siRNA-TGIF2 transfection leads to increased melanocyte proliferation (P<0.0001) and increased melanin production (P<0.01). These findings highlight the involvement of the circRNA-miRNA network in pigmentation, offering new insights into the molecular mechanisms underlying pigmentation and guiding animal hair color breeding strategies.

Key words: Capra hircus , pigmentation , melanocyte , circRNA , miRNA

. circKIF27靶向miR-129-5p/TGIF2通路抑制山羊黑色素细胞增殖，降低黑色素生成[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.02.008.

JI Kai-yuan, ZHAO Yi-we, YUAN Xin, LIANG Chun-e, ZHANG Xue-qing, TIAN Wen-li, YU Tong, MA Yang-yang, LING Ying-hui, ZHANG Yun-hai. circKIF27 inhibits melanogenesis and proliferation by targeting miR-129-5p/TGIF2 pathway in goat melanocytes[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.02.008.

参考文献

Aoki H, Moro O. 2002. Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R). Life Sciences, 71(18), 2171-2179.

Bartel D P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297.

Cecchi T, Valbonesi A, Passamonti P, Gonzale M, Antonini M, and Renieri C. 2011. Quantitative variation of melanins in alpaca (Lama pacos L.). Italian Journal of Animal Science, 10, e30.

Diao Y L, Jin B Z, Huang L Y, Zou W K. 2018. MiR-129-5p inhibits glioma cell progression in vitro and in vivo by targeting TGIF2. Journal of Cellular and Molecular Medicine, 22, 2357-2367.

Hansen T B, Jensen T I, Clausen B H, Bramsen J B, Finsen B, Damgaard C K, Kjems J. 2013. Natural RNA circles function as efficient microRNA sponges. Nature, 495(7441), 384-388.

Huang W, Wu Y, Qiao M X, Xie Z, Cen X, Huang X, Zhao Z. 2022. CircRNA-miRNA networks in regulating bone disease. Journal of Cellular Physiology, 237(2), 1225-1244.

Igarashi M, Hirata A, Yamaguch H, Sugae N, Tominaga M. 2003. Mechanism of an inhibitory effect of nipradilol on rat vascular smooth muscle cell growth. Journal of Atherosclerosis and Thrombosis, 10(4), 226.

Ji K Y, Wen R J, Wang Z Z, Tian Q Q, Zhang W, Zhang Y H. 2023. MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes. Journal of Integrative Agriculture, 22, 2-13.

Ji K Y, Zhao Y W, Wen R J, Khan I M, Zhang Y H. 2022. A genome-wide integrated analysis of lncRNA-mRNA in melanocytes from white and brown skin hair boer goats (Capra aegagrus hircus). Frontiers in Veterinary Science, 9, 1009174.

Ji K Y, Zhang P Q, Zhang J Z, Fan R W, Liu Y, Yang S S, Hu S P, Liu X X, Dong C S. 2018. MicroRNA 143-5p regulates alpaca melanocyte migration, proliferation, and melanogenesis. Experimental Dermatology, 27(2), 166-171.

Jin X, Feng C Y, Xiang Z, Chen Y P, Li Y M. 2016. CircRNA expression pattern and circRNA-miRNA-mRNA network in the pathogenesis of nonalcoholic steatohepatitis. Oncotarget, 7, 66455-66467.

Juan A F, Jesús M-P, Adolfo T-D, Julián S-M, José A D. 2007. Sex and testosterone effects on growth, immunity and melanin coloration of nestling Eurasian kestrels. Journal of Animal Ecology, 76(1), 201-209.

Kim M O, Park Y S, Nho Y H, Yun S K, Kim Y, Jung E, Paik J K, Kim M, Cho I H, Lee J. 2016. Emodin isolated from Polygoni Multiflori Ramulus inhibits melanogenesis through the liver X receptor-mediated pathway. Chemico-Biological Interactions, 250, 78-84.

Lin J Y, Fisher D E. 2007. Melanocyte biology and skin pigmentation. Nature, 445(7130), 843-850.

Liu H, Lan T, Li H, Xu L, Chen X, Liao H, Chen X, Du J, Cai Y, Wang J, Li X, Huang J, Yuan K, Zeng Y. 2021. Circular RNA circDLC1 inhibits MMP1-mediated liver cancer progression via interaction with HuR. Theranostics, 11(3), 1396-1411.

Melhuish T A, Gallo C M, Wotton D. 2001. TGIF2 Interacts with Histone Deacetylase 1 and Represses Transcription. The Journal of Biological Chemistry, 276(34), 32109-32114.

Nishimura E K. 2011. Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation. Pigment Cell & Melanoma Research, 24(3), 401-410.

Nishimura E K, Jordan S A, Oshima H, Yoshida H, Osawa M, Moriyama M, Jackson I J, Barrandon Y, Miyachi Y, Nishikawa S. 2002. Dominant role of the niche in melanocyte stem-cell fate 410 determination. Nature, 416, 854-860.

Niu C, Aisa H A. 2017. Upregulation of Melanogenesis and Tyrosinase Activity: Potential Agents for Vitiligo. Molecules, 22(8), 1303.

O’ Sullivan M, Scott S D, McCarthy N, Figg Nicola, Shapiro Leonard M, Kirkpatrick Peter, Bennett Martin R. 2003. Differential cyclin E expression in human in-stent stenosis smooth muscle cells identifies targets for selective anti-restenosis therapy. Cardiovascular Research, 60(3), 673.

Rybak-Wolf A, Stottmeister C, Glažar P, Jens M, Pino N, Giusti S, Hanan M, Behm M, Bartok O, Ashwal-Fluss R, Herzog M, Schreyer L, Papavasileiou P, Ivanov A, Öhman M, Refojo D, Kadener S, Rajewsky N. 2015. Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Molecular Cell, 58(5), 870-885.

Samir S T, Guilherme G, Adam S K, David F P. 2009. Prostate cancer detection using a novel computerized three-dimensional prostate biopsy template (Targetscan ™): results of a multicenter prospective data registry. The Journal of Urology, 181, 712.

Singh D, Kesharwani P, Alhakamy N A, Siddique H R. 2022. Accentuating CircRNA-miRNA-Transcription Factors Axis: A Conundrum in Cancer Research. Frontiers in Pharmacology, 12, 3904-3904.

Suzuki H, Tsukahara T. 2014. A view of pre-mRNA splicing from RNase R resistant RNAs. International Journal of Molecular Sciences, 15(6), 9331-9342.

Wang X. 2008. miRDB: A microRNA target prediction and functional annotation database with a wiki interface. RNA, 14, 1012-1017.

Wang R, Zhang S, Chen X, Li N, Li J, Jia R, Pan Y Q, Liang H Q. 2018. CircNT5E acts as a sponge of microRNA-422a to promote glioblastoma tumorigenesis. Cancer Research, 78(17): 4812-4825.

Widlund H R, and Fisher D E. 2003. Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival. Oncogene, 22, 3035-3041.

Wotton D, Lo R S, Lee S, Massagué J. 1999. A Smad transcriptional corepressor. Cell. 97(1), 29-39.

Yang S, Liu B, Ji K, Fan R, Dong C. 2018. MicroRNA-5110 regulates pigmentation by cotargeting melanophilin and WNT family member 1. FASEB Journal, 32(10), 5405-5412.

Zhang Y, Shi D, Yang J, Chen X, Huang W. 2020. Identification of miR-146a is Associated with the Aggressiveness and Suppresses Proliferation via Targeting CDKN2A in Breast Cancer. Pathology & Oncology Research, 26(1): 245-251.

Zou D P, Chen Y M, Zhang L Z, Yuan X H, Zhang Y J, Inggawati A, Kieu Nguyet P T, Gao T W, Chen J. 2020. SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling. Genes and Diseases, 8(5), 677-688.

Zhu Z, Ma Y, Li Y, Cheng Z, Li H, Zhang L, Xu D, Li P. 2019. Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation. BMC Molecular and Cell Biology, 20(1), 19.

Zhu Z, Ma Y, Li Y, Li P, Tang Z. 2020. The comprehensive detection of miRNA, lncRNA, and circRNA in regulation of mouse melanocyte and skin development. Biological Research, 53(1), 4.