Due to the key role of MITF as an essential regulator of the melanogenic process, several studies have investigated miRNAs with potential effects on MITF expression and consequent regulation of mRNA levels in melanogenic enzymes [3,30,31,53] (Table 1). Some preliminary studies concerning melanogenesis regulation by miRNAs have been performed in fiber-producing animals, such as alpaca, due to the contribution of melanin synthesis to coat color and the specific interest of alpaca breeders in animal color coat modulation. Differences in miRNA profiles from alpaca skins with different colored coats were identified and most differentially expressed miRNAs showed predicted targets involved in pigmentation [54,55]. These results led to further investigation concerning the functional role of selected miRNAs. Data obtained by Zhu Z et al. (2010) demonstrated the functional role of miR-25 in reducing MITF mRNA and protein, TYR, and TYRP-1 expression in cultured melanocytes. In addition, an inverse relationship was observed between miR-25 level and coat color. Similarly, in alpaca melanocytes, miR508-3p also directly targets MITF, binding to the 3′-UTR of the gene. miR508-3p overexpression downregulated MITF expression, resulting in a decrease in TYR, TYRP-2, and melanin production [56]. Interestingly, in 2012, Dong C. et al. investigated the role of miR-137, another miR-targeting MITF, in a transgenic mice model [57]. Initially investigating melanoma cells lines, where its overexpression downregulated MITF [58], it was found that miR-137 also decreased the expression of the MITF protein and its downstream genes in transgenic mice. Notably, miR-137 had an impact on coat color, demonstrating that modulating a specific miR may significantly regulate melanogenesis, at least in animal models [57].

Other studies have identified miRNAs regulating skin pigmentation in human melanocytes. Particular interest has been shown in miR-675, another miR which targets MITF and was to be found expressed at low levels in the hyperpigmented skin of melasma patients. In melanocytes or keratinocytes derived from melasma patients, miR-675 upregulation decreased TYRP-1 and TYRP-2 expression, whereas its knockdown increased their expression. Interestingly, miR-675 was also identified in exosomes released from keratinocytes into the extracellular environment [5,59,60]. Another miR involved in melanogenesis by binding the 3′-UTR in MITF is miR-218, which downregulated TYR, TYRP-1, and DCT mRNA and protein levels, reducing melanin content in immortalized melan-a murine melanocytes. In agreement with these data, miR-218 also suppressed melanogenesis in human pigmented skin organotypic culture (OTC) through MITF repression. Anti-miR-218 was also found to promote melanogenesis in human primary melanocytes [61].

Recently, other miRNAs, including miRNA-183 cluster, miR-340, miR-141-3p, and miR-200a-3p, have been investigated for their role in regulating MITF expression and melanogenesis [62]. Results of this research show that the miRNA-183 cluster targets the 3′-UTR of MITF in B16 mouse melanoma cells. miRNA-183 cluster overexpression decreased MITF, TYR, TYRP-1, and DCT expression and melanin production. Conversely, knockdown of the miRNA-183 cluster increased MITF, TYR, TYRP-1, and DCT expression and, consequently, melanin levels. The miR-183 cluster was also involved in regulating the MEK/ERK signaling pathway implicated in cell proliferation and migration, by modulating mitogen-activated protein kinase 1 (MEK1), extracellular regulated protein kinases1/2 (ERK1/2), and CREB expression [62]. miR-340, which was first identified in melanoma cell lines for its ability to bind specifically to the 3′-UTR of MITF [64], has been also investigated in immortalized human epidermal melanocytes (Pig-1), where it downregulates MITF expression and melanin synthesis [63]. Recently, miR-141-3p and miR-200a-3p have been identified as MITF regulators [52]. In this study, comparing miRNA expression profiles in B16-4A5 mouse melanoma cells which were treated or untreated with α-MSH led to 13 miRNAs being identified as differentially expressed through miRNA array analysis. miR-141-3p, miR-200a-3p, and miR-148a-3p, which target MITF, were downregulated in α-MSH-stimulated cells when compared to untreated cells. Furthermore, miR-141-3p and miR-200a-3p overexpression suppressed MITF expression and TYR activity in B16-4A5 cells. Notably, the inhibitory effect on melanin synthesis was confirmed in a three-dimensional tissue culture model of the human epidermis (3D-MHE model) [52].

Besides the abovementioned studied group of miRNAs, with significant regulatory roles in MITF, other miRNAs are involved in melanogenesis by regulating the expression of other molecular targets, including melanogenic enzymes, transcription factors, or components of the signaling pathways which regulate melanogenesis (Table 2).

Several miRNAs, including miR-450b-5p, miR-1208, miR-326, miR-434-5p, miR330-5p, miR-125, miR-145, and miR-203, have been predicted as targeting TYR and most also target other genes, including MITF [65,66].

Wu et al. used a miR-434-5p homologue to target TYR in cells cultured in vitro as well as in an animal model and showed efficient melanin synthesis reduction. Similarly, miR-330-5p downregulated TYR in melanoma cells and normal melanocytes, inducing depigmentation without affecting cell proliferation [66]. This miR has been also identified in exosomes derived from keratinocytes [56]. In this study, exosomes carrying miR-330-5p caused a decrease in melanin production and TYR expression in melanocytes. Similarly, miR-330-5p overexpression in melanocytes confirmed its inhibitory activity on melanogenesis. Several other miRNAs, such as miR-203, which targets Kinesin Superfamily Protein 5b, involved in melanosome transfer, and miR-3196, with unknown target genes, have been identified in human-derived exosomes from keratinocytes and have shown an ability to increase melanin content in melanocytes [67]. Indeed, exosomes released by Black keratinocytes, as well as Caucasian UV-irradiated keratinocytes, were able to induce increased TYR activity and melanogenic gene expression in melanocytes, at least partly as a result of miR content.

As reported above, several miRNAs which directly target MITF are involved in melanogenesis. Conversely, MITF expression may be subject to more complex regulation. For example, it has been shown that miR-21a-5p overexpression downregulated the SOX5 target, as well as β-catenin and CDK2 protein expression, in normal human melanocytes. In agreement with SOX5 involvement in melanogenesis, its downregulation induced an increase in MITF and melanogenic enzymes’ expression, with consequent stimulation of melanogenesis [68]. Several other miRNAs have been identified as regulators of SOX transcription factors [56,69,74]. miR-145, which was initially identified as a miR regulated by UV treatment and forskolin in murine melan-a melanocytes, significantly modulates several genes involved in pigmentation. miR-145 overexpression or downregulation reduced or increased the expression of several genes involved in melanin biosynthesis, such as SOX9, MITF, TYR, and TYRP-1 [74,75], and in melanosome transfer, such as Myosin Va (Myo5a), Rab27a, and fascin1 (Fscn1), respectively [76]. Notably, miR-145 targeted the 3′-UTR binding site of Myo5a, a molecular motor involved in the intracellular trafficking of vesicles and organelles. A further miR that binds an SOX family component is miR-380-3p, which targets SOX6 by binding to the 3′-UTR region. In alpaca melanocytes, miR-380-3p overexpression downregulated SOX6 and increased β-catenin. Differently from studies indicating that β-catenin induces MITF transcription, in this study, miR-380-3p overexpression caused decreased mRNA levels of melanin-related genes including MITF, TYR, TYRP-1, and DCT, suggesting other SOX6 activities in MITF regulation [56].

Recently, miR-200c has been identified as a SOX1 regulator. Specifically, miR-200c induced SOX1 downregulation by direct binding to the SOX1 3′-UTR, leading to intranuclear β-catenin upregulation and increased expression of MITF-dependent gene expression as well as melanogenesis. These effects were induced in normal human epidermal melanocytes (NHEM) treated with exosomes containing miR-200c, produced by normal human epidermal keratinocytes (NHEK). Notably, miR-200c was found at low levels in exosomes derived from keratinocytes in vitiligo lesions compared to exosomes from NHEK, with a consequent inhibition in melanin production [69]. Some miRNAs have also been involved in modulating the extracellular ligands which regulate melanogenic signaling pathways. An example is miR-27a-3p, which directly targets Wnt3a, a component of the Wnt signaling pathway. miR-27a-3p was found to be expressed at higher levels in alpaca white skin compared to brown skin [54] and was similarly related to skin color in mice [70]. Furthermore, its functional role in downregulating Wnt3a, and thus inhibiting β-catenin and melanogenesis, was confirmed in mouse melanocytes transfected with miR-27a-3p [70]. In addition, miR-379 was shown to modulate insulin-like growth factor receptor I (IGFR1) in alpaca melanocytes. Insulin-like growth factor 1 (IGF1), mainly produced in dermal cells, has been shown to improve melanogenesis through the cAMP pathway. Likewise, miR-379 overexpression reduced melanogenesis by inhibiting the cAMP response element (CRE)-binding protein (CREB)/(MITF) pathway in alpaca melanocytes [71].

Finally, miRNAs involved in the regulation of melanosome processing and transfer have been also identified. Among them, miR-143-5p targets Myo5a, which belongs to the complex Rab27a/MLPH/Myo5a that connects melanosomes to the actin cytoskeleton in human melanocytes [72]. In a more recent study, Qi S. et al. inhibited miR-143-5p expression using Short Tandem Target Mimics (STTMs) in order to evaluate the functional role of miR-143-5p. As a consequence, increased melanogenic gene expression, including MITF, TYR, and TYRP-1, MLPH, and Rab27, and melanin production were observed [77]. This result indicates the effective possibility of modulating melanin biosynthesis by a miRNA-regulating approach. In addition, miR-143-5p overexpression induces a decrease in TGF-β-activated kinase 1 (TAK1) expression with consequent effects on melanocyte migration and proliferation and MITF downregulation in alpaca melanocytes. Therefore, miR-143-5p may regulate melanogenesis by modulating different gene targets [73]. By means of a bioinformatic approach, followed by experimental validation, miR-125b has also been shown to directly target SRC homology 3 domain-binding protein 4 (SH3BP4), a gene regulated by MITF, which may act by controlling melanogenic enzymes’ distribution to melanosomes or the mTOR signaling pathway. Indeed, when miR-125b was overexpressed in WM266-4 and MNT1 human melanoma cells, decreased levels of the melanogenic enzymes TYR and DCT were observed, although these genes are not direct miR-125b targets [78].

UV rays can modify intracellular functions in different ways: by directly or indirectly damaging DNA through reactive oxygen species (ROS), inducing apoptosis, cell cycle arrest, and carcinogenesis. The expression profiles of the miRNAs involved in these events are modified by UV irradiation [79,80,81]. Among the UV-induced effects, it is well known that there is a correlation between UVB irradiation and melanogenesis stimulation [1]. miRNA profiling in irradiated mouse melanocytes has been helpful in identifying miRNAs involved in melanogenesis, such as miR-145 [74]. Furthermore, several miRNAs targeting different pigmentation genes have been involved in UV ray effects, including miR-145, miR-137, miR-148, and miR-25 [57,82,83].

miR-340 upregulation was identified in pigmented cells treated with UVB irradiation among miRNAs related to UV irradiation. Studies have shown that miR-340 significantly represses RhoA protein expression and stimulates melanosome transport [81]. Recently, the functional role of miR-340 has been further investigated in immortalized human epidermal melanocytes (Pig-1), confirming that its expression is modulated by UV and that it downregulates MITF expression and melanin synthesis. Notably, miR-340 mimics decreased melanin content in irradiated cells [63]. A further example of a miR potentially being involved in UV-induced pigmentation modulation is miR-21. The latter has been largely investigated in melanoma and shown to be increased by UV radiation in melanocytes, keratinocytes, and fibroblasts [84]. In mouse skin melanocytes, miR-21 has been shown to enhance MITF expression by targeting SOX5 [85]. Inversely, miR-21a-5p has been observed with different levels of activity in UV-irradiated A375.S2 human melanoma and B16F10 melanoma mouse cells. Indeed, in these UV-treated cells, increased melanin content was associated with an increase in α-MSH expression and reduced EGFR and Akt phosphorylation. miR-21 overexpression negatively modulated these UV-induced effects [86]. Interestingly, this miR has been also involved in communication between melanocytes and keratinocytes. Extracellular vesicles derived from UVA-exposed melanocytes modified keratinocyte behavior by inducing miR-21 upregulation and TGF-β and IL-6/STAT3 signaling pathway activation [87]. UVA and UVB rays also induce changes in miRNA produced by keratinocytes [88,89]. Accumulating evidence indicates that exosomes and exosomal miRNAs represent an effective means of communication for melanocytes and keratinocytes [56]. Notably, in Caucasian human keratinocytes, UV rays induce the release of miR, such as hsa-miR-3196, which may stimulate melanin synthesis in melanocytes [67]. Recently, miR-675 production by human keratinocytes has been shown and its role as a paracrine regulator of melanogenesis has been confirmed in vitro, indicating increased exosomal miR-675 in cells irradiated with a 585 nm light emitting diode (LED) and its ability to attenuate melanogenesis in melanocytes [90].

Several miRNAs regulating melanogenesis play an important role in melanoma [91]. For instance, miR-340, miR-218, and miR-137, which inhibit melanogenesis, have a tumor suppressor role during melanomagenesis by negatively regulating MITF expression [58,61,64,92]. Other miRNAs which regulate melanogenesis, including miR-379, -200c, -203, -200a-3p, -125b, -183, -508-3p, -675, -143-5p, -141-3p, -145, and -330-5p, can act as tumor suppressors in melanomagenesis, although the target genes may differ from those identified in melanogenesis. On the contrary, miR-21a-5p, -25, and -27a-3p have shown an oncogenic role, being upregulated in metastatic melanoma and during tumor progression [93,94].

Interestingly, some miRNAs involved in both melanogenesis and melanomagenesis, such as miR-21a-3p, -25, -3196, -145, -137, -148, and -675 are induced by UV rays, the main environmental risk factor for melanoma [79,80,81,95].