Identification of MicroRNA Targeting Mlph and Affecting Melanosome Transport

Melanosomes undergo a complex maturation process and migrate into keratinocytes. Melanophilin (Mlph), a protein complex involving myosin Va (MyoVa) and Rab27a, enables the movement of melanosomes in melanocytes. In this study, we found six miRNAs targeting Mlph in mouse using two programs (http://targetscan.org and DianaTools). When melan-a melanocytes were treated with six synthesized microRNAs, miR-342-5p, miR-1839-5p, and miR-3082-5p inhibited melanosome transport and induced melanosome aggregation around the nucleus. The other microRNAs, miR-5110, miR-3090-3p, and miR-186-5p, did not inhibit melanosome transport. Further, miR-342-5p, miR-1839-5p, and miR-3082-5p decreased Mlph expression. The effect of miR-342-5p was the strongest among the six synthesized miRNAs. It inhibited melanosome transport in melan-a melanocytes and reduced Mlph expression in mRNA and protein levels in a dose-dependent manner; however, it did not affect Rab27a and MyoVa expressions, which are associated with melanosome transport. To examine miR-342-5p specificity, we performed luciferase assays in a mouse melanocyte-transfected reporter vector including Mlph at the 3′-UTR (untranslated region). When treated with miR-342-5p, luciferase activity that had been reduced by approximately 50% was restored after inhibitor treatment. Therefore, we identified a novel miRNA affecting Mlph and melanosome transport, and these results can be used for understanding Mlph expression and skin pigmentation regulation.


Introduction
Melanocytes, a type of skin cell, produce melanin and are located in the basal layer of the epidermis. Melanin is a biological pigment comprising the reddish-yellow type called pheomelanin and the brownish-black type eumelanin [1,2]. The differences in skin color among individuals from different races result from the type of melanin produced and not from the number of melanocytes [3].
Melanosomes are lysosome-related organelles that produce, store, and transport melanin, and result in the pigmentation in melanocytes [4]. The transport of mature melanosomes from the perinuclear area to the dendrite tips of melanocytes is facilitated by microtubule-and actin-dependent motor proteins [5]. Actin-dependent melanosome transport is associated with the tripartite complex proteins, Rab27a, melanophilin (Mlph), and myosin Va (MyoVa). Rab27a is a GTPase and a member of the Ras oncogene family, and its effector molecule is Slac2-a/Mlph, which recruits the actin-dependent motor protein MyoVa [5][6][7]. Defects in melanosome transport cause a condition known as Griscelli syndrome (GS). Griscelli and colleagues found two girls with skin hypopigmentation and silver-gray hair [8]. There are three types of GS, GS types 1, 2, and 3, caused by mutations in MyoVa, Rab27a, and Mlph, respectively [9]. All three types cause skin and hair hypopigmentation; however, GS types 1 and 2 cause neurological impairment and immunological impairment, respectively, as side effects. However, with Mlph mutation, only depigmentation is reportedly apparent and no other side effects are observed [10][11][12]. Therefore, Mlph could be an important target gene for depigmentation studies.
The roles of many uncharacterized miRNAs in the skin have been studied. For example, miR-31 enhances cell proliferation and migration in keratinocytes, and aids in skin wound healing [13]. A previous study showed that miR-218 decreased melanogenesis by directly inhibiting microphthalmia-associated transcription factor (MITF) in melan-a mouse melanocytes [14]. Recent studies targeted the 3´-UTR of genes associated with melanosome transport. For example, miR-203 significantly suppressed the growth of human and canine melanoma cells and inhibited melanosome transport through the suppression of MITF and Rab27a expressions [15]. In addition, miR-145 reportedly decreases MyoVa and FSCN1 expressions in human melanoma cells [16]. However, miRNAs that only target Mlph among the motor proteins associated with melanosome transport in mouse melanocytes have not yet been reported. In this study, we attempted to find miRNAs that regulate Mlph expression.

Identification of miRNAs Targeting Mlph in Mice
To identify miRNA targeting Mlph in mice, we used two programs. One was TargetScan, and the other was DIANA-microT v3.0. These two online bioinformatics programs predicted miRNAs that could directly target the 3 -UTR of Mlph. In all, 265 miRNAs were predicted by TargetScan and 17 by DIANA-microT v3.0. We synthesized six miRNAs and inhibitors (Inh) identified by both programs (Figure 1). The microRNA sequences are shown in Table 1.

Effect of miRNAs on Mlph Expression in Melan-a Melanocytes
To investigate whether the six synthesized miRNAs affect melanosome transport, melan-a mouse melanocytes were treated with 10 nM of the miRNAs. After 72 h transfection, we found that melanosome aggregation was induced. Three miRNAs, miR-342-5p, miR-3082-5p, and miR-1839-5p, induced melanosome aggregation in melan-a melanocytes. However, the other three did not induce melanosome aggregation (Figure 2a). Among the six miRNAs, miR-342-5p had the strongest inhibitory effect on the Mlph protein expression level compared to the negative control ( Figure 2b). (A) After 24 h, melan-a melanocytes were treated with miRNAs. We examined cells transfected with six representative microRNAs. After 72 h, the cells were observed in bright field using a digital microscope (iRiS TM , Logosbio, Anyang, Korea). Melanosome aggregation was estimated by counting cells with perinuclear melanosome aggregation in three random microscopic fields per well at 200× magnification. (B) Melan-a melanocytes treated with 10 nM miRNAs for 72 h. Six miRNAs showed effects on protein expression level from Mlph. Values are presented as the mean ± SD from three wells.

Effect of miR-342-5p on Mlph Expression in Melan-a Melanocytes
To investigate whether miR-342-5p affected melanosome transport in a dose-dependent manner, miR-342-5p was treated with 2.5 nM, 5 nM, and 10 nM miR-342-5p for 72 h (Figure 3a). Treatment with 2.5 nM, 5 nM, and 10 nM miR-342-5p increased melanosome aggregation in a dose-dependent manner compared with control. miR-342-5p decreased the mRNA and protein levels of Mlph in a dose-dependent manner (Figure 3b,c). The mRNA Mlph expression level was measured using quantitative real-time PCR. The aggregation of melanosomes around the nucleus was imaged using HMB45 antibody immunofluorescence ( Figure 3d).

Effect of mir-342-5p on the Rab27a and MyoVa Expressions
To determine whether miR-342-5p affects other key melanosome transport protein expressions, we examined Rab27a and MyoVa expressions together. miR-342-5p decreased the level of Mlph but did not affect the levels of MyoVa and Rab27a. To check the specificity of the effect of miR-342-5p on Mlph expression, the rescue effect was examined using an inhibitor of miR-342-5p (Figure 4a). The inhibitor of miR-342-5p increased the levels of Mlph. miR-342-5p or scrambled mimic (Scr mimic) were co-transfected with a luciferase-UTR construct into melan-a melanocytes, and luciferase activity was determined.

miR-342-5p Directly Targets Mlph in Melan-a Melanocytes
We identified the binding site of mmu-miR-342-5p in the 3´-UTR of Mlph (Figure 4b). To identify the specificity of miR-342-5p, we constructed a sensor vector by joining the 3 -UTR region of mouse Mlph to a luciferase reporter pGLuc-basic vector. To determine the target sequence recognized by the miRNA, miR-342-5p was treated with an inhibitor, while the control was not. Treatment with miR-342-5p reduced luciferase activity, and treatment of miR-342-5p with inhibitor restored the luciferase activity (Figure 4c).

Discussion
Melanosomes are fully matured during transport from the melanocyte nucleus to the melanocyte dendrite tip. Then, melanosomes are transferred to the keratinocytes [17,18]. During melanosome transport, three proteins, Mlph, Rab27a, and MyoVa, form a tripartite complex. These are the key regulators of melanosome transport [19,20].
In this study we identified miRNAs that affected Mlph expression, and attempted to understand their effect. When the melan-a melanocytes were treated with the selected miRNAs, the miRNAs were observed to suppress melanosome transport (Figure 2a). Significant aggregation of melanosomes in the perinuclear area was evident in the transfection of miR-342-5p in melan-a melanocytes (Figure 3a). miR-342-5p decreased the protein levels of Mlph in a dose-dependent manner (Figure 3b). Previous studies have showed that miR-5110 suppresses pigmentation in alpaca melanocytes by co-targeting MLPH and WNT family member 1 [21]. However, miR-5110 has no effect on melanosome transport in melan-a melanocytes. miR-342-5p decreased the levels of Mlph, but did not affect Rab27a and MyoVa expressions (Figure 4a). Until now there has been no report on the miRNA targeting Mlph in mice or humans. From this study, we found miRNA that affects the expression of Mlph in mice, and we can expect miRNAs with similar function in humans, which is an area of research to be explored in future studies. According to several previous studies, Mlph expression is very important for melanosome transport. Mutations in Mlph have been shown to cause defects in melanosome transport in melanocytes [7]. Treatment with miR-342-5p reduced the mRNA level of Mlph (Figure 3c) by targeting the 3´-UTR of Mlph.
miR-3082-5p and miR-1839-5p downregulated Mlph and Rab27a expression levels (data not shown). There are reports that one miRNA can target two genes simultaneously. For example, a study published in Japan in 2013 showed that one miRNA can target two genes simultaneously in a gastric cancer cell line [22]. miR-224 targets DPYSL2 (dihydropyri-midinase-like-2) and KLAS, which is a proto-oncogene [22]. DPYSL2 encodes the collapsin response mediator protein 2 (CRMP2), a cytosolic protein [23]. This protein is necessary for the growth and promotion of neurons. It is usually tethered to the cell membrane, and is essential for tissue signaling. Therefore, miR-3082-5p and miR-1839-5p can be considered to target two genes simultaneously, similar to miR-224. miR-3082-5p is reportedly controlled in mouse melanoma cells by curcumin and could be a potential anti-cancer miRNA [24]. However, miR-1839-5p has not yet been investigated.
It has been reported that miR-342-5p is upregulated in Alzheimer's disease (AD) transgenic mouse models, including APP/PS1, PS1DE9, and PS1-M146V lines [25]. miR-342-5p binds directly to the 3'-UTR of AnkG mRNA and decreases AnkG levels through translation repression in AD transgenic mouse neurons. However, there have been no reports on the effects of miR 342-5p on Mlph expression. We found a novel miRNA that affects Mlph and melanosome transport and could be important for understanding Mlph expression and skin pigmentation regulation.

Transfection
One day before transfection, melan-a melanocytes were seeded in six-well plates at 60% confluency, and miRNAs, inhibitors, and 20 nM siRNA mixed RNAi MAX (Invitrogen) were applied according to the manufacturer's protocol.

Immuno-Fluorescence (IF)
One day before transfection, melan-a melanocytes were seeded in one chamber at 60% confluency and miRNA plus 20 nM siRNA mixed RNAi MAX (Invitrogen) were applied according to the manufacturer's protocol. Melan-a melanocytes transfected with miRNA and siRNA after 72 h were washed with DPBS (Dulbecco's Phosphate-Buffered Salin) and then were fixed in 2 mL formalin for 15 minutes at room temperature. Chambers were washed three times with PBS and were then permeabilized with 1 mL of 0.1% Tripton for 15 minutes and then blocked using 1 mL of 2% BSA(Bovine Serum Albumin). After thorough washing, sections were incubated with first antibody (HMB45) diluted with 2% BSA for two to three days at 4 • C with shaking. FITC-conjugated antibody was diluted 2% BSA or PBS treated for one hour at 4 • C with shaking followed by washing three times with PBS. The cells were stained with 4 -6-diamidino-2-phenylindole DAPI) for 15 minutes at room temperature.
Values are presented as mean ± SD from three wells.

Detection and Quantification of Melanosome Aggregation
Melan-a melanocytes were seeded in 24-well plates and cultured for 24 h. The cells were then rinsed in DPBS and treated with samples of RPMI-1640 containing 2% FBS for three days. The cells were observed under bright field using a digital microscope (iRiS TM , Logosbio, Korea). Evaluation of melanosome aggregation was performed by counting cells with perinuclear melanosome aggregates in three random microscopic fields per well at 200× magnification.
Values are presented as mean ± SD from three wells.

Luciferase Assay
We constructed a sensor vector by joining the regions with a possible binding site from the 3 -UTR of mouse Mlph to a luciferase reporter pGLuc-basic vector purchased from Cosmogenetech (Seoul, Korea) in order to identify the target sequences recognized by the miRNAs. For amplification of these mRNAs, melan-a melanocytes were seeded in six-well plates at a concentration of 3.0 × 10 5 /well the day before the transfection. The vector, at a concentration of 0.5 µg/well, and 10 nM miRNAs or nonspecific control miRNA was used for the co-transfection of the cells using the cationic liposomes