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MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes
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JI Kai-yuan1, 2, WEN Ru-jun1, WANG Zheng-zhou1, TIAN Qian-qian1, ZHANG Wei1, ZHANG Yun-hai2, 3#
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1 Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P.R.China
2 Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resources Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P.R.China
3 Linquan Comprehensive Experimental Station, Anhui Agricultural University, Linquan 236400, P.R.China
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摘要
microRNAs（miRNAs）是广泛存在于哺乳动物中的一种微小、非编码RNA，可以通过靶向吸附调节下游基因的表达，参与多种生物学过程。虽然关于 miRNAs 调控哺乳动物毛色的研究取得了一定的成果，但其调控网络尚不完善，仍需要不断深入研究。前期测序发现，miR-370-5p在白色绵羊皮肤中的表达水平显著高于黑色绵羊，推测其可能参与绵羊皮肤黑色素生成。本研究以绵羊黑色素细胞为研究对象，探究miR-370-5p在绵羊黑色素细胞中的作用。表型检测发现，高表达的miR-370-5p可以显著抑制（P=0.001）酪氨酸酶活性，从而显著降低（P<0.001）黑色素的产生；CCK 8实验检测发现，黑色素细胞转染miR-370-5p后的第4天至第5天，细胞的增殖速率显著降低（P<0.01）。靶基因预测发现，丝裂原活化蛋白激酶8（Mitogen-activated protein kinases, MAP3K8）的3'非翻译区（Untranslated Region, UTR）存在miR-370-5p的靶向结合位点，推测两者可能存在靶向调控关系。双荧光素酶报告载体实验结果显示，miR-370-5p可以靶向吸附MAP3K8-3’UTR。原位杂交实验显示，MAP3K8广泛表达于黑素细胞的细胞质。定量RT-PCR和Western blot结果显示，miR-370-5p显著抑制（P<0.01）MAP3K8的表达。以上结果表明，miR-370-5p可以靶向结合MAP3K8-3’UTR，抑制其表达。siRNA干扰结果显示，黑素细胞中干扰MAP3K8的表达可以显著抑制（P<0.01）细胞增殖，降低（P<0.001）黑色素生成，影响趋势与过表达miR-370-5p一致。靶基因拯救实验结果显示，黑色素细胞中共转染miR-370-5p和MAP3K8-cDNA（含有miR-370-5p靶向结合位点）载体，可以显著上调（P≤0.001）MAP3K8的表达，显著促进细胞增殖（P<0.001）和黑色素产生（P<0.01）。以上结果表明， miR-370-5p通过靶向抑制MAP3K8表达，抑制绵羊黑色素细胞增殖、降低黑色素产量。本研究通过miRNA过表达探明了miR-370-5p对黑色素细胞增殖、酪氨酸酶活性及黑色素产量的影响；通过靶基因干扰、拯救实验解析了miR-370-5p抑制黑色素细胞增殖、酪氨酸酶活性及黑色素生成的分子机制，有助于丰富miRNAs参与毛色形成的调控机制，为后续毛用动物毛色改良提供参考。
Abstract
In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes. Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear. In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production. Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays. In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes. The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR. Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p. Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01). Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.
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Received: 29 October 2021
Accepted: 29 December 2022
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| Fund:
This research was funded by the Natural Science Foundation of Anhui Province, China (2008085QC158) and the University Natural Science Research Project of Anhui Province (KJ2019A0165).
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| About author: JI Kai-yuan, E-mail: 18234487253@163.com; #Correspondence ZHANG Yun-hai, E-mail: yunhaizhang@ahau.edu.cn |
Cite this article:
JI Kai-yuan, WEN Ru-jun, WANG Zheng-zhou, TIAN Qian-qian, ZHANG Wei, ZHANG Yun-hai.
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(4): 1131-1141.
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