Exosomal miR-365b-5p derived from keratinocyte promotes melanogenesis by directly targeting GLI2

Exosomal miR-365b-5p derived from keratinocyte promotes melanogenesis by directly targeting GLI2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Exosomal miR-365b-5p derived from keratinocyte promotes melanogenesis by directly targeting GLI2 HaiRu Zhao, ChanSong Jo, JaeSung Hwang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5384239/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract In previous studies, we analyzed that exosomal microRNA (miRNA) secreted by keratinocytes exposed to Ultraviolet B(UVB) light regulate melanogenesis in melanocytes. Through functional experiments, it was determined that a subgroup of exosomal miRNAs had distinct impacts on melanogenesis. In the current study, we focused on hsa-miR-365b-5p which founded upregulated in UVB-irradiated keratinocyte exosomes and confirmed to exert enhancing effects on melanogenesis in human melanocyte. Hsa-miR-365b-5p is a specific, mature microRNA derived from the precursor hsa-miR-365. We demonstrated that the overexpression of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM) resulted in an approximate 50% increase in melanin content relative to the control group. Furthermore, treatment with an inhibitor of hsa-miR-365b-5p substantiated its specific regulatory role in melanogenesis, as inhibition resulted in a nearly 90% reduction in melanin production. Notably, hsa-miR-365b-5p upregulates the expression of genes associated with melanogenesis, including MITF, TYR, TRP1, and TRP2. Additionally, we established that GLI Family Zinc Finger 2 (GLI2) functions as a repressor of MITF, with its inhibition via siRNA leading to increased melanogenesis. Moreover, we constructed a luciferase reporter vector containing the 3' UTR of GLI2, confirming that hsa-miR-365b-5p specifically targets GLI2, a known repressor of MITF. These findings elucidate the regulatory pathways governing melanogenesis and underscore the significant role of hsa-miR-365b-5p in this biological process. melanogenesis microRNA hsa-miR-365b-5p GLI2 melanocyte Figures Figure 1 Figure 2 Introduction Melanocytes possess numerous dendritic extensions that interface with keratinocytes[ 1 ]. The production of melanin, a process termed melanogenesis, involves a series of sequential enzymatic reactions, culminating in the translocation of melanin into adjacent keratinocytes via melanosomes[ 2 ]. During the initial stages of melanin synthesis, several key enzymes, including tyrosinase (TYR), tyrosine hydroxylase I (THI), and phenylalanine hydroxylase (PAH), play critical roles in melanin production. In the later stages, tyrosinase-associated protein 1 (TYRP-1) and tyrosinase-associated protein 2 (TYRP-2), also known as Dopachrome tautomerase (DCT), are involved in the enzymatic pathways[ 3 ]. The melanocyte-specific MITF-M promoter contains binding motifs associated with several positive regulatory factors that facilitate the expression of the intricately regulated MITF-M transcription. One of regulator of MITF-M expression is the transcription factor GLI2[ 4 ], an effector of Hedgehog signaling that also mediates the actions of the TGF-β/SMAD pathway[ 5 ]. TGF-β has the capacity to enhance GLI2 mRNA expression while concurrently inhibiting MITF expression through various mechanisms[ 6 ]. Nevertheless, GLI2 plays a significant role in melanoma by promoting cellular invasion and resistance to BRAF inhibitors, which aligns with its function in the downregulation of MITF[ 7 ]. In melanoma cell lines, a negative correlation exists between GLI2 expression and MITF-M expression, with GLI2 and MITF-M mutually suppressing each other's expression[ 5 ]. Hsa-miR-365b-5p is a specific, mature microRNA (miRNA) derived from the precursor hsa-miR-365. This miRNA has been identified as a critical regulator in various cellular processes. Recent studies have also shown that hsa-miR-365b-3p, another mature form of the miRNA, exerts tumor-suppressive effects by inhibiting cell proliferation and promoting apoptosis in non-small cell lung cancer (NSCLC) cells[ 8 ]. Additionally, miR-365b-3p has been found to regulate cell cycle progression in human retinoblastoma cells, indicating its broader role in controlling tumor growth and cell division across different cancer types[ 9 ]. In a previous study, we demonstrated that exosomal microRNAs (miRNAs) derived from keratinocytes exposed to UVB radiation may regulate melanogenesis in melanocytes[ 10 ]. The present study aims to investigate the effects of hsa-miR-365b-5p on melanogenesis in melanocytes and its associated target genes. Result Keratinocyte-derived exosomal hsa-miR-365b-5p promotes melanogenesis of human melanocyte To evaluate the effects of hsa-miR-365b-5p on melanogenesis in NHEM were transfected with either miR mimics or inhibitors. The results demonstrated that hsa-miR-365b-5p significantly increased melanin contents in NHEM by approximately 50% compared to the control group (Fig. 1 A). In contrast, when NHEM were co-transfected with both hsa-miR-365b-5p mimics and inhibitors, melanin content decreased by 90% relative to the hsa-miR-365b-5p mimic group (Fig. 1 A). Additionally, the mRNA levels (Fig. 1 B) and protein expression (Fig. 1 C, D) of melanogenesis-related genes, including MITF, TYR, TRP1, and TRP2, were significantly elevated following transfection with miR-365b-5p mimics and reduced upon co-transfection with hsa-miR-365b-5p inhibitors. These findings indicate that hsa-miR-365b-5p promotes melanogenesis in human melanocytes. MITF repressor GLI2 is a direct target gene of hsa-miR-365b-5p To identify target genes of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM), we utilized TargetScan ( https://www.targetscan.org ), which predicted that the GLI2 gene, a known repressor of MITF, is a target of hsa-miR-365b-5p (Fig. 2 A). Previous studies have demonstrated that GLI2 represses MITF transcription in both human melanoma cells[ 5 ] and mouse melanocytes[ 11 ]. We confirmed that the silencing of GLI2 resulted in a 35% increase in melanin contents (Fig. S1 A) and 1.5-fold increase in MITF mRNA levels compared to the control group (Fig. S1 B). Additionally, the mRNA levels(Fig. S1 B) and protein expression(Fig. S1 C) of melanogenesis-related genes, including TYR, TRP1, and TRP2, were significantly upregulated following GLI2 silencing. To investigate the binding of hsa-miR-365b-5p to GLI2, we constructed a luciferase vector incorporating the 3'-UTR of human GLI2 within the pmirGLO luciferase reporter vector. The luciferase reporter assay demonstrated that the hsa-miR-365b-5p mimic resulted in a decrease in luciferase activity (Fig. 2 B). When co-transfected with an inhibitor, luciferase activity in HEK-293T cells transfected with the pmirGLO-GLI2 3'-UTR vector increased compared to the hsa-miR-365b-5p mimic group (Fig. 2 B). qPCR and Western blot analyses revealed that the hsa-miR-365b-5p mimic significantly reduced GLI2 mRNA levels (Fig. 2 C) and protein expression (Fig. 2 D, E) in human melanocytes. These results confirm that hsa-miR-365b-5p directly targets the MITF repressor gene GLI2, thereby promoting melanogenesis in human melanocytes. Materials and Methods In this study, hsa-miR-365b-5p mimic and inhibitor were chemically synthesized by BIONEER (Daejeon, Korea). Normal Human epidermal melanocyte (NHEM) was purchased from PromoCell (Heidelberg, Germany) and culture in Medium 254 (Invitrogen, Carlsbad, CA, USA) containing 1% human melanocytes growth supplement (HMGS, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Welgene, Daegu, Korea). Cells were maintained at 37°C in a 5% CO2 atmosphere. HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene, Daegu, Korea). Cells were maintained at 37°C in a 5% CO2 atmosphere. The 3′-UTR fragment of GLI2 containing the binding site of hsa-miR-365b-5p was cloned into the pmirGLO vector (Promega,USA) and purchased from Cosmogenetech (Seoul, Korea) in order to identify the target sequences recognized by the miRNAs. Discussion MicroRNAs have been identified as potential prognostic, diagnostic, and predictive biomarkers for several years. In our previous study, we investigated the role of exosomal miRNAs secreted by keratinocytes exposed to UVB radiation in regulating melanogenesis in melanocytes[ 10 ]. In this study, we focused specifically on hsa-miR-365b-5p and confirmed its significant role in promoting melanogenesis. Overexpression of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM) resulted in approximately a 50% increase in melanin content compared to the control group. Moreover, treatment with an inhibitor of hsa-miR-365b-5p further validated its specific regulatory effect on melanogenesis, as inhibition led to a decrease in melanin production. Numerous studies have investigated miRNAs that may influence MITF expression and, consequently, the regulation of mRNA levels in melanogenic enzymes, due to MITF's essential role as a key regulator of the melanogenic process[ 12 – 15 ]. These findings have stimulated further research into the functional significance of specific miRNAs. Our results identified GLI2 as a repressor of MITF by demonstrating that the transfection of GLI2-specific small interfering RNA resulted in the de-repression of MITF transcription. This indicates that GLI2 inhibits melanogenesis by suppressing MITF expression at the transcriptional level. Furthermore, we constructed a luciferase reporter vector containing the 3' UTR of GLI2, confirming that hsa-miR-365b-5p directly targets GLI2, which is known to repress MITF. The protein GLI2 is known to be reduced in melanoma cells, where elevated levels of GLI2 significantly impede both basal and TGF-β-induced cellular migration and invasion[ 7 ]. Consequently, further investigations are warranted to ascertain the role of hsa-miR-365b-5p in regulating melanoma cell functionality and its implications for melanoma progression. There are three exosomal microRNAs have been identified as up-regulators of melanogenesis, despite their significant downregulation in exosomes derived from UVB-irradiated keratinocytes[ 10 ]. However, we have not yet characterized hsa-miR-365b-5p as the most influential regulators in this context. Thus, it is essential to explore the effects of other identified microRNAs on melanogenesis and their potential target genes in human melanocytes. Our findings contribute critical insights into the regulatory mechanisms underlying melanogenesis, emphasizing the pivotal role of hsa-miR-365b-5p in this biological process. These results underscore the necessity for additional research to fully elucidate the intricate network of regulatory interactions that govern melanocyte biology and melanoma progression. Declarations Author Contribution Author Contributions StatementFirst author H.R.Z and corresponding author J.S.H. decided on the subject of the study. H.R.Z wrote the manuscript, and revised the manuscript through discussion with C.S.J. It helped with the research. Acknowledgement Acknowledgements This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP23C0001). References Slominski A, Tobin DJ, Shibahara S, Wortsman J (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155–1228 JJ Nordlund (2007) The melanocyte and the epidermal melanin unit: an expanded concept. Dermatol Clin 25:271–281 SA D’Mello GJ, Finlay BC, Baguley ME, Askarian-Amiri (2016) Signaling pathways in melanogenesis. Int J Mol Sci 17:1144 Vachtenheim J, Ondrušová. L (2013) A critical transcription factor in melanoma transcriptional regulatory network, Recent Advances in the Biology, Therapy and Management of Melanoma. 4:71–82 Javelaud D, Alexaki V, Pierrat M, Hoek KS, Dennler S, Van Kempen L et al (2011) GLI2 and M-MITF transcription factors control exclusive gene expression programs and inversely regulate invasion in human melanoma cells. Pigment cell melanoma Res 24:932–943 Dennler S, André J, Verrecchia F, Mauviel A (2009) Cloning of the human GLI2 promoter. J Biol Chem 284:31523–31531 Alexaki V, Javelaud D, Van Kempen LC, Mohammad KS, Dennler S, Luciani F et al (2010) GLI2-mediated melanoma invasion and metastasis. J Natl Cancer Inst 102:1148–1159 Wang J, Wang X, Wu G, Hou D, Hu Q (2013) MiR-365b-3p, down-regulated in retinoblastoma, regulates cell cycle progression and apoptosis of human retinoblastoma cells by targeting PAX6. FEBS Lett 587:1779–1786 Zhang X, Wang J, Pan Y, Zhao J, Pan Y, Yan Y et al (2021) MicroRNA-365b-3p represses the proliferation and promotes the apoptosis of nonsmall cell lung cancer cells by targeting PPP5C Retraction in/10.3892/ol. 2023.14010, Oncology Letters. 21:1–10 Yoon J, Jo C, Hwang J (2024) Comprehensive Analysis of Exosomal MicroRNAs Derived from UVB-Irradiated Keratinocytes as Potential Melanogenesis Regulators. Int J Mol Sci 25:3095 Choi H, Shin JH, Kim ES, Park SJ, Bae I, Jo YK et al (2016) Primary cilia negatively regulate melanogenesis in melanocytes and pigmentation in a human skin model. PLoS ONE 11:e0168025 Vachtenheim J (2010) Transcription physiology of pigment formation in melanocytes: central role of MITF. Exp Dermatol 19:617–627 Levy C, Khaled M, Fisher DE (2006) MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 12:406–414 Nguyen NT, Fisher DE (2019) MITF and UV responses in skin: From pigmentation to addiction. Pigment cell melanoma Res 32:224–236 Chen T, Zhao B, Liu Y, Wang R, Yang Y, Yang L et al (2018) MITF-M regulates melanogenesis in mouse melanocytes. J Dermatol Sci 90:253–262 Additional Declarations No competing interests reported. Supplementary Files figureS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Dec, 2024 Reviews received at journal 15 Dec, 2024 Reviews received at journal 12 Dec, 2024 Reviewers agreed at journal 05 Dec, 2024 Reviewers agreed at journal 03 Dec, 2024 Reviewers invited by journal 13 Nov, 2024 Editor assigned by journal 05 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 03 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5384239","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":377650237,"identity":"930215d3-ef31-4615-b186-e7fa4b087d7e","order_by":0,"name":"HaiRu Zhao","email":"","orcid":"","institution":"Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"HaiRu","middleName":"","lastName":"Zhao","suffix":""},{"id":377650238,"identity":"a219d8bd-cd97-439c-8622-80c95ec8db5f","order_by":1,"name":"ChanSong Jo","email":"","orcid":"","institution":"Kyung Hee University","correspondingAuthor":false,"prefix":"","firstName":"ChanSong","middleName":"","lastName":"Jo","suffix":""},{"id":377650239,"identity":"80315a6f-62ec-4d5b-ad62-a0428db1500c","order_by":2,"name":"JaeSung Hwang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACxgYeMC0nIQERMCBaizHxWhgYIFoSZxCthbn/7DGJnztq02fO7jFg+FHDYGzeQMhhM/LSJHvPHM+dLXPGgLHnGIOZzAGCWnjMbvC2HcudJ5FjwMDbwGAjQchhjP1nzG7+bTuWLgfUwviXKC0NOWa3edtqEqSBWpiBtpgR1jIjx/y3bNsBw5kz0goOyxyTMCaoxbD/jLHh27Y6eYkbyRsfvqmxMZxBUEsDmDoMJg8wMBC0g4FBHkLVEVY5CkbBKBgFIxcAAGyVO16Kymi4AAAAAElFTkSuQmCC","orcid":"","institution":"Kyung Hee University","correspondingAuthor":true,"prefix":"","firstName":"JaeSung","middleName":"","lastName":"Hwang","suffix":""}],"badges":[],"createdAt":"2024-11-04 02:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5384239/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5384239/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69327996,"identity":"0810dcad-88aa-46e8-91de-1abd5aeb55ed","added_by":"auto","created_at":"2024-11-19 08:25:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-365b-5p promotes melanogenesis in human melanocyte.\u003c/strong\u003e (A). Melanin contents in NHEM changed by miR-365b-5p mimic and inhibitor. (B). NHEM melanin pellet after lysis by NaOH. (C). miR-365b-5p mimic effects on melanogenesis related gene mRNA level determined by qPCR (D). melanogenesis related gene protein expression changes by miR-365b-5p mimic and detected by Western Blot. (E). Blot analysis by image J. (Values are presented as the mean ± SD from n4. Data were analyzed using Student’s un-paired t-test. **, p\u0026lt;0.01; ***, ###, p\u0026lt;0.001.)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5384239/v1/222e89faab59e358f6e25762.jpeg"},{"id":69327997,"identity":"8cc439db-2433-47fd-81b8-8b1d1909ec6a","added_by":"auto","created_at":"2024-11-19 08:25:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-365b-5p directly target to GLI2 in human melanocyte. \u003c/strong\u003e(A). Predicted target gene binding site of miR-365b-5p by TargetScan (https://www.targetscan.org). (B). miR-365b-5p or/and inhibitor co-transfected with luciferase-UTR construct into NHEM and luciferase activity was determined. (C). miR-365b-5p or/and inhibitor transfected into NHEM and GLI2 mRNA level determined by qPCR. (D). GLI2 protein expression detected by western blot. (E). Blot analysis by image J. (Values are presented as the mean ± SD from n4. Data were analyzed using Student’s un-paired t-test. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, ###, p\u0026lt;0.001.)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5384239/v1/a47ea5e7fd0f08d7f28ef5f6.jpeg"},{"id":69328009,"identity":"02f63206-39ca-44ac-8cad-f5379e89e7e0","added_by":"auto","created_at":"2024-11-19 08:25:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":823462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5384239/v1/2cc3eba6-6229-40cb-a959-292d2f9ac775.pdf"},{"id":69327998,"identity":"e5da21d2-ffc7-46a1-8d34-745a4a330a3e","added_by":"auto","created_at":"2024-11-19 08:25:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":201910,"visible":true,"origin":"","legend":"","description":"","filename":"figureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5384239/v1/42f1bce3915291abd8521a6b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal miR-365b-5p derived from keratinocyte promotes melanogenesis by directly targeting GLI2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMelanocytes possess numerous dendritic extensions that interface with keratinocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The production of melanin, a process termed melanogenesis, involves a series of sequential enzymatic reactions, culminating in the translocation of melanin into adjacent keratinocytes via melanosomes[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During the initial stages of melanin synthesis, several key enzymes, including tyrosinase (TYR), tyrosine hydroxylase I (THI), and phenylalanine hydroxylase (PAH), play critical roles in melanin production. In the later stages, tyrosinase-associated protein 1 (TYRP-1) and tyrosinase-associated protein 2 (TYRP-2), also known as Dopachrome tautomerase (DCT), are involved in the enzymatic pathways[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe melanocyte-specific MITF-M promoter contains binding motifs associated with several positive regulatory factors that facilitate the expression of the intricately regulated MITF-M transcription. One of regulator of MITF-M expression is the transcription factor GLI2[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], an effector of Hedgehog signaling that also mediates the actions of the TGF-β/SMAD pathway[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TGF-β has the capacity to enhance GLI2 mRNA expression while concurrently inhibiting MITF expression through various mechanisms[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nevertheless, GLI2 plays a significant role in melanoma by promoting cellular invasion and resistance to BRAF inhibitors, which aligns with its function in the downregulation of MITF[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In melanoma cell lines, a negative correlation exists between GLI2 expression and MITF-M expression, with GLI2 and MITF-M mutually suppressing each other's expression[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHsa-miR-365b-5p is a specific, mature microRNA (miRNA) derived from the precursor hsa-miR-365. This miRNA has been identified as a critical regulator in various cellular processes. Recent studies have also shown that hsa-miR-365b-3p, another mature form of the miRNA, exerts tumor-suppressive effects by inhibiting cell proliferation and promoting apoptosis in non-small cell lung cancer (NSCLC) cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, miR-365b-3p has been found to regulate cell cycle progression in human retinoblastoma cells, indicating its broader role in controlling tumor growth and cell division across different cancer types[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In a previous study, we demonstrated that exosomal microRNAs (miRNAs) derived from keratinocytes exposed to UVB radiation may regulate melanogenesis in melanocytes[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The present study aims to investigate the effects of hsa-miR-365b-5p on melanogenesis in melanocytes and its associated target genes.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eKeratinocyte-derived exosomal hsa-miR-365b-5p promotes melanogenesis of human melanocyte\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of hsa-miR-365b-5p on melanogenesis in NHEM were transfected with either miR mimics or inhibitors. The results demonstrated that hsa-miR-365b-5p significantly increased melanin contents in NHEM by approximately 50% compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, when NHEM were co-transfected with both hsa-miR-365b-5p mimics and inhibitors, melanin content decreased by 90% relative to the hsa-miR-365b-5p mimic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, the mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D) of melanogenesis-related genes, including MITF, TYR, TRP1, and TRP2, were significantly elevated following transfection with miR-365b-5p mimics and reduced upon co-transfection with hsa-miR-365b-5p inhibitors. These findings indicate that hsa-miR-365b-5p promotes melanogenesis in human melanocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMITF repressor GLI2 is a direct target gene of hsa-miR-365b-5p\u003c/h3\u003e\n\u003cp\u003eTo identify target genes of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM), we utilized TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which predicted that the GLI2 gene, a known repressor of MITF, is a target of hsa-miR-365b-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Previous studies have demonstrated that GLI2 represses MITF transcription in both human melanoma cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and mouse melanocytes[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We confirmed that the silencing of GLI2 resulted in a 35% increase in melanin contents (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and 1.5-fold increase in MITF mRNA levels compared to the control group (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Additionally, the mRNA levels(Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) and protein expression(Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) of melanogenesis-related genes, including TYR, TRP1, and TRP2, were significantly upregulated following GLI2 silencing. To investigate the binding of hsa-miR-365b-5p to GLI2, we constructed a luciferase vector incorporating the 3'-UTR of human GLI2 within the pmirGLO luciferase reporter vector. The luciferase reporter assay demonstrated that the hsa-miR-365b-5p mimic resulted in a decrease in luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). When co-transfected with an inhibitor, luciferase activity in HEK-293T cells transfected with the pmirGLO-GLI2 3'-UTR vector increased compared to the hsa-miR-365b-5p mimic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). qPCR and Western blot analyses revealed that the hsa-miR-365b-5p mimic significantly reduced GLI2 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E) in human melanocytes. These results confirm that hsa-miR-365b-5p directly targets the MITF repressor gene GLI2, thereby promoting melanogenesis in human melanocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eIn this study, hsa-miR-365b-5p mimic and inhibitor were chemically synthesized by BIONEER (Daejeon, Korea). Normal Human epidermal melanocyte (NHEM) was purchased from PromoCell (Heidelberg, Germany) and culture in Medium 254 (Invitrogen, Carlsbad, CA, USA) containing 1% human melanocytes growth supplement (HMGS, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Welgene, Daegu, Korea). Cells were maintained at 37\u0026deg;C in a 5% CO2 atmosphere. HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene, Daegu, Korea). Cells were maintained at 37\u0026deg;C in a 5% CO2 atmosphere.\u003c/p\u003e \u003cp\u003eThe 3\u0026prime;-UTR fragment of GLI2 containing the binding site of hsa-miR-365b-5p was cloned into the pmirGLO vector (Promega,USA) and purchased from Cosmogenetech (Seoul, Korea) in order to identify the target sequences recognized by the miRNAs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroRNAs have been identified as potential prognostic, diagnostic, and predictive biomarkers for several years. In our previous study, we investigated the role of exosomal miRNAs secreted by keratinocytes exposed to UVB radiation in regulating melanogenesis in melanocytes[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, we focused specifically on hsa-miR-365b-5p and confirmed its significant role in promoting melanogenesis. Overexpression of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM) resulted in approximately a 50% increase in melanin content compared to the control group. Moreover, treatment with an inhibitor of hsa-miR-365b-5p further validated its specific regulatory effect on melanogenesis, as inhibition led to a decrease in melanin production.\u003c/p\u003e \u003cp\u003eNumerous studies have investigated miRNAs that may influence MITF expression and, consequently, the regulation of mRNA levels in melanogenic enzymes, due to MITF's essential role as a key regulator of the melanogenic process[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings have stimulated further research into the functional significance of specific miRNAs. Our results identified GLI2 as a repressor of MITF by demonstrating that the transfection of GLI2-specific small interfering RNA resulted in the de-repression of MITF transcription. This indicates that GLI2 inhibits melanogenesis by suppressing MITF expression at the transcriptional level. Furthermore, we constructed a luciferase reporter vector containing the 3' UTR of GLI2, confirming that hsa-miR-365b-5p directly targets GLI2, which is known to repress MITF.\u003c/p\u003e \u003cp\u003eThe protein GLI2 is known to be reduced in melanoma cells, where elevated levels of GLI2 significantly impede both basal and TGF-β-induced cellular migration and invasion[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, further investigations are warranted to ascertain the role of hsa-miR-365b-5p in regulating melanoma cell functionality and its implications for melanoma progression. There are three exosomal microRNAs have been identified as up-regulators of melanogenesis, despite their significant downregulation in exosomes derived from UVB-irradiated keratinocytes[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, we have not yet characterized hsa-miR-365b-5p as the most influential regulators in this context. Thus, it is essential to explore the effects of other identified microRNAs on melanogenesis and their potential target genes in human melanocytes. Our findings contribute critical insights into the regulatory mechanisms underlying melanogenesis, emphasizing the pivotal role of hsa-miR-365b-5p in this biological process. These results underscore the necessity for additional research to fully elucidate the intricate network of regulatory interactions that govern melanocyte biology and melanoma progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions StatementFirst author H.R.Z and corresponding author J.S.H. decided on the subject of the study. H.R.Z wrote the manuscript, and revised the manuscript through discussion with C.S.J. It helped with the research.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgements This research was supported by a grant of the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (grant number: HP23C0001).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSlominski A, Tobin DJ, Shibahara S, Wortsman J (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155\u0026ndash;1228\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJJ Nordlund (2007) The melanocyte and the epidermal melanin unit: an expanded concept. Dermatol Clin 25:271\u0026ndash;281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSA D\u0026rsquo;Mello GJ, Finlay BC, Baguley ME, Askarian-Amiri (2016) Signaling pathways in melanogenesis. Int J Mol Sci 17:1144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVachtenheim J, Ondrušov\u0026aacute;. L (2013) A critical transcription factor in melanoma transcriptional regulatory network, Recent Advances in the Biology, Therapy and Management of Melanoma. 4:71\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJavelaud D, Alexaki V, Pierrat M, Hoek KS, Dennler S, Van Kempen L et al (2011) GLI2 and M-MITF transcription factors control exclusive gene expression programs and inversely regulate invasion in human melanoma cells. Pigment cell melanoma Res 24:932\u0026ndash;943\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDennler S, Andr\u0026eacute; J, Verrecchia F, Mauviel A (2009) Cloning of the human GLI2 promoter. J Biol Chem 284:31523\u0026ndash;31531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexaki V, Javelaud D, Van Kempen LC, Mohammad KS, Dennler S, Luciani F et al (2010) GLI2-mediated melanoma invasion and metastasis. J Natl Cancer Inst 102:1148\u0026ndash;1159\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Wang X, Wu G, Hou D, Hu Q (2013) MiR-365b-3p, down-regulated in retinoblastoma, regulates cell cycle progression and apoptosis of human retinoblastoma cells by targeting PAX6. FEBS Lett 587:1779\u0026ndash;1786\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Wang J, Pan Y, Zhao J, Pan Y, Yan Y et al (2021) MicroRNA-365b-3p represses the proliferation and promotes the apoptosis of nonsmall cell lung cancer cells by targeting PPP5C Retraction in/10.3892/ol. 2023.14010, Oncology Letters. 21:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoon J, Jo C, Hwang J (2024) Comprehensive Analysis of Exosomal MicroRNAs Derived from UVB-Irradiated Keratinocytes as Potential Melanogenesis Regulators. Int J Mol Sci 25:3095\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi H, Shin JH, Kim ES, Park SJ, Bae I, Jo YK et al (2016) Primary cilia negatively regulate melanogenesis in melanocytes and pigmentation in a human skin model. PLoS ONE 11:e0168025\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVachtenheim J (2010) Transcription physiology of pigment formation in melanocytes: central role of MITF. Exp Dermatol 19:617\u0026ndash;627\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevy C, Khaled M, Fisher DE (2006) MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 12:406\u0026ndash;414\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen NT, Fisher DE (2019) MITF and UV responses in skin: From pigmentation to addiction. Pigment cell melanoma Res 32:224\u0026ndash;236\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Zhao B, Liu Y, Wang R, Yang Y, Yang L et al (2018) MITF-M regulates melanogenesis in mouse melanocytes. J Dermatol Sci 90:253\u0026ndash;262\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"melanogenesis, microRNA, hsa-miR-365b-5p, GLI2, melanocyte","lastPublishedDoi":"10.21203/rs.3.rs-5384239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5384239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn previous studies, we analyzed that exosomal microRNA (miRNA) secreted by keratinocytes exposed to Ultraviolet B(UVB) light regulate melanogenesis in melanocytes. Through functional experiments, it was determined that a subgroup of exosomal miRNAs had distinct impacts on melanogenesis. In the current study, we focused on hsa-miR-365b-5p which founded upregulated in UVB-irradiated keratinocyte exosomes and confirmed to exert enhancing effects on melanogenesis in human melanocyte. Hsa-miR-365b-5p is a specific, mature microRNA derived from the precursor hsa-miR-365. We demonstrated that the overexpression of hsa-miR-365b-5p in normal human epidermal melanocytes (NHEM) resulted in an approximate 50% increase in melanin content relative to the control group. Furthermore, treatment with an inhibitor of hsa-miR-365b-5p substantiated its specific regulatory role in melanogenesis, as inhibition resulted in a nearly 90% reduction in melanin production. Notably, hsa-miR-365b-5p upregulates the expression of genes associated with melanogenesis, including MITF, TYR, TRP1, and TRP2. Additionally, we established that GLI Family Zinc Finger 2 (GLI2) functions as a repressor of MITF, with its inhibition via siRNA leading to increased melanogenesis. Moreover, we constructed a luciferase reporter vector containing the 3' UTR of GLI2, confirming that hsa-miR-365b-5p specifically targets GLI2, a known repressor of MITF. These findings elucidate the regulatory pathways governing melanogenesis and underscore the significant role of hsa-miR-365b-5p in this biological process.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-365b-5p derived from keratinocyte promotes melanogenesis by directly targeting GLI2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 08:25:33","doi":"10.21203/rs.3.rs-5384239/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-20T22:23:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-15T20:10:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-13T03:57:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155924717708449039889389346810605784152","date":"2024-12-05T13:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187184724083049066230464124560887077280","date":"2024-12-04T01:54:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-13T08:38:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-05T17:16:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-05T17:14:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2024-11-04T02:33:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b980bcb7-0abc-42a5-b768-7595452e2fdc","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-01-18T00:53:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-19 08:25:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5384239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5384239","identity":"rs-5384239","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}