Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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MiRNA-24 downregulates KLF6 affecting STAT3 protein expression and phosphorylation regulating melanogenesis in cashmere goat coat

  • Baoyu Zhang (College of Veterinary Medicine, Jilin University) ;
  • Runlai Liu (College of Veterinary Medicine, Jilin University) ;
  • Yuxin Zhao (College of Veterinary Medicine, Jilin University) ;
  • Xinyu Li (College of Veterinary Medicine, Jilin University) ;
  • Huixian Su (College of Veterinary Medicine, Jilin University) ;
  • Shufang Li (College of Veterinary Medicine, Jilin University) ;
  • Jianping Li (College of Animal Science and Technology, Jilin Agricultural Science and Technology University) ;
  • Huaizhi Jiang (College of Animal Science and Technology, Jilin Agricultural University) ;
  • Qiaoling Zhang (College of Veterinary Medicine, Jilin University)
  • Received : 2024.11.25
  • Accepted : 2025.05.20
  • Published : 2025.09.01
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Abstract

Objective: This study revealed that miRNA-24 downregulates KLF6, regulating melanogenesis in the coat of Cashmere goats. Methods: The correlation between miRNA-24 and coat color was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). GO/KEGG analysis and bioinformatics tools were used to screen for target genes. Targeted interactions were confirmed using the dual luciferase assay. The expressions of miRNA-24 and KLF6 on different colored skins of Cashmere goats, and the KLF6 expression in B16-F10 cells transfected with miRNA-24 mimics/inhibitors/NC were detected by RT-qPCR, Western Blot, and Immunofluorescence. To investigate the pathway that KLF6 influenced, KLF6 was silenced by RNA interference (RNAi), and the melanogenesis pathway's gene expression was examined. The expression and phosphorylation of STAT3 protein in the pathway were inhibited. RNAi and Stattic were utilized to downregulate its protein expression and phosphorylation for detecting tyrosine expression. BALB/c mice were subcutaneously injected with antagomiRNA-24, a miRNA-24 inhibitor. The influence of miRNA-24 on the downregulation of KLF6 in relation to melanogenesis was examined. Results: MiRNA-24 is related to coat color. KLF6 was chosen as the target gene, and the targeted interaction was validated. The expression trends of miRNA-24 and KLF6 in the skin inversely correlated. The mRNA and protein expression of KLF6 were altered in B16- F10 cells after transfected. These suggested that miRNA-24 downregulates KLF6. Silencing of KLF6 resulted in the downregulation of genes linked to melanogenesis, including WNT1, WNT2, PRKACA, MAPK1, TYR, and STAT3. The suppression of protein and phosphorylation levels of STAT3 decreaseed the mRNA level of TYR, suggesting that KLF6 influences TYR expression by modulation of STAT3 expression and phosphorylation. BALB/c mice injected with antagomiRNA-24 showed increased melanin content and decreased KLF6 expression. Conclusion: MiRNA-24 regulates melanogenesis in the Cashmere goat coat by downregulation of KLF6, thus mediating STAT3 expression and phosphorylation.

Keywords

Acknowledgement

This study was funded by the National Natural Science Foundation of China (NSFC, No. 32172806), the Natural Science Foundation of Jilin Province (20240101262JC) and Scientific research project of Education Department of Jilin Province (JJKH20220399KJ).

References

  1. Sun Q, Lee W, Hu H, et al. Dedifferentiation maintains melanocyte stem cells in a dynamic niche. Nature 2023;616:774-82. https://doi.org/10.1038/s41586-023-05960-6
  2. Pavan WJ, Sturm RA. The genetics of human skin and hair pigmentation. Annu Rev Genomics Hum Genet 2019;20:41-72. https://doi.org/10.1146/annurev-genom-083118-015230
  3. Ohbayashi N, Fukuda M. Recent advances in understanding the molecular basis of melanogenesis in melanocytes. F1000Research 2020;9:608. https://doi.org/10.12688/f1000research.24625.1
  4. Weiner L, Fu W, Chirico WJ, Brissette JL. Skin as a living coloring book: how epithelial cells create patterns of pigmentation. Pigment Cell Melanoma Res 2014;27:1014-31. https://doi.org/10.1111/pcmr.12301
  5. Dumbuya H, Hafez SY, Oancea E. Cross talk between calcium and ROS regulate the UVA-induced melanin response in human melanocytes. FASEB J 2020;34:11605-23. https://doi.org/10.1096/fj.201903024R 
  6. Adelmann CH, Traunbauer AK, Chen B, et al. MFSD12 mediates the import of cysteine into melanosomes and lysosomes. Nature 2020;588:699-704. https://doi.org/10.1038/s41586-020-2937-x
  7. Kim J, Hong SC, Lee EH, Lee JW, Yang SH, Kim JC. Preventive effect of M. Cochinchinensis on melanogenesis via tyrosinase activity inhibition and p-PKC signaling in melan-A cell. Nutrients 2021;13:3894. https://doi.org/10.3390/nu13113894
  8. O'Carroll D, Schaefer A. General principals of miRNA biogenesis and regulation in the brain. Neuropsychopharmacology 2013;38:39-54. https://doi.org/10.1038/npp.2012.87
  9. Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 2010;79:351-79. https://doi.org/10.1146/annurevbiochem-060308-103103
  10. Chen S, Nie H, Huo Z, Yan X. Comprehensive analysis of differentially expressed mRNA, lncRNA and miRNA, and their ceRNA networks in the regulation of shell color in the Manila clam (Ruditapes philippinarum). Int J Biol Macromol 2024;256:128404. https://doi.org/10.1016/j.ijbiomac.2023.128404
  11. Bertrand JU, Petit V, Aktary Z, et al. Loss of dicer in newborn melanocytes leads to premature hair graying and changes in integrin expression. J Invest Dermatol 2024;144:601-11. https://doi.org/10.1016/j.jid.2023.08.023
  12. Shin SY, Gil HN, Choi JH, Lim Y, Lee YH. Agerarin inhibits α-MSH–induced TYR gene transcription via STAT3 suppression independent of CREB-MITF pathway. J Dermatol Sci 2018;91:107-10. https://doi.org/10.1016/j.jdermsci.2018.03.014
  13. Wang XY, Guan XH, Yu ZP, et al. Human amniotic stem cellsderived exosmal miR-181a-5p and miR-199a inhibit melanogenesis and promote melanosome degradation in skin hyperpigmentation, respectively. Stem Cell Res Ther 2021;12:501. https://doi.org/10.1186/s13287-021-02570-9
  14. Li J, Ba X, Li J, et al. MicroRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in cashmere goats. Res Vet Sci 2022;147:68-73. https://doi.org/10.1016/j.rvsc.2022.03.020
  15. Yang G, Wu M, Liu X, et al. MiR-24-3p conservatively regulates muscle cell proliferation and apoptosis by targeting common gene CAMK2B in rat and cattle. Animals 2022;12:505. https://doi.org/10.3390/ani12040505
  16. Wang Q, Huang Z, Xue H, et al. MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4. Blood 2008;111:588-95. https://doi.org/10.1182/blood-2007-05-092718
  17. Liu F, Zhang X, Peng Y, et al. miR-24 controls the regenerative competence of hair follicle progenitors by targeting Plk3. Cell Rep 2021;35:109225. https://doi.org/10.1016/j.celrep.2021.109225
  18. Amelio I, Lena AM, Bonanno E, Melino G, Candi E. miR-24 affects hair follicle morphogenesis targeting Tcf-3. Cell Death Dis 2013;4:e922. https://doi.org/10.1038/cddis.2013.426
  19. Bian Q, Chen B, Weng B, et al. circBTBD7 promotes immature porcine sertoli cell growth through modulating miR-24-3p/MAPK7 axis to inactivate p38 MAPK signaling pathway. Int J Mol Sci 2021;22:9385. https://doi.org/10.3390/ijms22179385
  20. Lopez JP, Fiori LM, Cruceanu C, et al. MicroRNAs 146a/b-5 and 425-3p and 24-3p are markers of antidepressant response and regulate MAPK/Wnt-system genes. Nat Commun 2017;8:15497. https://doi.org/10.1038/ncomms15497
  21. Jin F, Yang R, Wei Y, et al. HIF-1α-induced miR-23a~27a~24 cluster promotes colorectal cancer progression via reprogramming metabolism. Cancer Lett 2019;440–441:211-22. https://doi.org/10.1016/j.canlet.2018.10.025
  22. Otmani K, Rouas R, Lagneaux L, et al. Acute myeloid leukemia-derived exosomes deliver miR-24-3p to hinder the T-cell immune response through DENN/MADD targeting in the NF-κB signaling pathways. Cell Commun Signal 2023;21:253. https://doi.org/10.1186/s12964-023-01259-1
  23. Bajpai VK, Swigut T, Mohammed J, et al. A genome-wide genetic screen uncovers determinants of human pigmentation. Science 2023;381:eade6289. https://doi.org/10.1126/science.ade6289
  24. Kim GD, Ng HP, Chan ER, Mahabaleshwar GH. Kruppel-like factor 6 promotes macrophage inflammatory and hypoxia response. FASEB J 2020;34:3209-23. https://doi.org/10.1096/fj.201902221R
  25. Tian S, Zhou X, Zhang M, et al. Mesenchymal stem cellderived exosomes protect against liver fibrosis via delivering miR-148a to target KLF6/STAT3 pathway in macrophages. Stem Cell Res Ther 2022;13:330. https://doi.org/10.1186/s13287-022-03010-y
  26. Yang Y, Lv Z, An Q, et al. Tricholoma matsutake polysaccharides suppress excessive melanogenesis via JNK-mediated pathway: investigation in 8-methoxypsoralen induced B16–F10 melanoma cells and clinical study. Heliyon 2024;10:e29363. https://doi.org/10.1016/j.heliyon.2024.e29363
  27. Lee SW, Kim JH, Song H, Seok JK, Hong SS, Boo YC. Luteolin 7-sulfate attenuates melanin synthesis through inhibition of CREB- and MITF-mediated tyrosinase expression. Antioxidants 2019;8:87. https://doi.org/10.3390/antiox8040087