MicroRNA-141-3p and microRNA-200a-3p regulate α-melanocyte stimulating hormone-stimulated melanogenesis by directly targeting microphthalmia-associated transcription factor

In recent years, it has been reported that non-coding RNAs, especially microRNAs (miRNAs) and long non-coding RNAs, act as melanogenesis-regulating molecules in melanocytes. We found that the expression levels of miR-141-3p and miR-200a-3p were decreased significantly by α-melanocyte-stimulating hormone (α-MSH) stimulation in mouse melanocyte B16-4A5 cells, as demonstrated by a miRNA array. Overexpression of miR-141-3p and miR-200a-3p in B16-4A5 cells suppressed melanogenesis and tyrosinase activity. Moreover, both miR-141-3p and miR-200a-3p showed direct targeting of microphthalmia-associated transcription factor using a luciferase reporter assay. Furthermore, topical transfection of miR-141-3p and miR-200a-3p to three-dimensional reconstructed human skin tissue inhibited α-MSH-stimulated melanin biosynthesis. Taken together, our findings indicate that downregulation of miR-141-3p and miR-200a-3p during the α-MSH-stimulated melanogenesis process acts as an important intrinsic signal. This result is expected to lead to the development of miRNA-based whitening therapeutics.


Results
Expression profiles of miR-141 and miR-200a in α-MSH-stimulated B16-4A5 murine melanoma cells. To investigate the expression profiles of miRNAs in α-MSH-stimulated B16-4A5 cells, we subjected total RNA extracted from cells treated with or without α-MSH to 3D-Gene ® mouse miRNA Oligo chips. The expression levels of 13 miRNAs (log2 ratios >2 or <−2) were distinct between α-MSH-stimulated and non-treated B16-4A5 cells (Table 1). We then focused on the downregulation of miR-141-3p and miR-200a-3p because their nucleotide sequences were identical except for two nucleotides (Fig. 1A) and the target genes of both miRNAs were predicted to be common from TargetScan Mouse 7.1.
To confirm the results from the miRNA array analysis, we examined the expression levels of miR-141-3p and miR-200a-3p in α-MSH-stimulated and non-treated B16-4A5 cells by the quantitative real-time polymerase chain reaction (qRT-PCR). As expected, the expression levels of both miRNAs in α-MSH-stimulated cells were significantly downregulated as compared with control non-treated cells. Additionally, we confirmed the downregulation of miR-148a-3p, which targets Mitf similar to results reported previously ( Fig. 1B and Supplementary  Fig. 1).

Overexpression of miR-141-3p and miR-200a-3p in B16-4A5 cells inhibits α-MSH-stimulated
melanogenesis by suppressing Mitf expression. The melanin content, a marker of melanogenesis, increased with time after α-MSH stimulation. To disclose the role of miR-141-3p or miR-200a-3p in this response, we first measured melanin contents and tyrosinase activities in B16-4A5 cells transfected with miR-141-3p or miR-200a-3p, and with and without α-MSH treatment. As shown in Fig. 2A,B, the melanin contents and tyrosinase activities in transfected cells were suppressed approximately 30%. These were the same inhibition levels achieved following treatment with 200 µM arbutin. In contrast, the melanin content and tyrosinase activity in cells transfected with non-specific control miRNA were not changed compared with untransfected α-MSH-stimulated B16-4A5 cells. Additionally, the melanin contents and tyrosinase activities in cells transfected with non-specific control miRNA, miRNA-141-3p, or miR-200a-3p were also not change compared with  We constructed pmir GLO-Mitf/miR-141-3p and miR-200a-3p mutated sensor-B and -E to confirm these observations (Fig. 5C). Luciferase activities in cells transfected with pmir GLO-Mitf/miR-141-3p, and miR-200a-3p sensor-B and E (wild-type), were significantly reduced, although in cells transfected with mutants of the seed region the decrease was absent (Fig. 5D). These results strongly suggest that the target region of Mitf is the common binding site for miR-141-3p and miR-200a-3p.

Discussion
The purpose of this study was to show that miR-141-3p and miR-200a-3p regulate α-MSH-stimulated melanogenesis. We demonstrated that overexpression of both miR-141-3p and miR-200a-3p significantly decreased melanin content and tyrosinase activity. Furthermore, we also identified Mitf as a common target for miR-141-3p and miR-200a-3p during α-MSH-stimulated melanogenesis by using a luciferase reporter assay. The binding of miR-141-3p and miR-200a-3p to the Mitf 3′-UTR was prevented by including a point mutation in each individual binding region of the Mitf 3′-UTR. These results suggest that changes of endogenous miR-141-3p and miR-200a-3p levels are closely related to α-MSH-stimulated melanogenesis (Fig. 7). MITF plays a central role in melanogenesis signalling in melanocytes. It is a member of the MYC family of basic helix-loop-helix leucine zipper transcription factors and is most closely related to transcription factor E3, transcription factor EC, and transcription factor proteins 33 . MITF expression is regulated by at least four different transcriptional molecules (paired box family of transcription factor 3, sex determining region Y-box 10, Wnt/β-catenin pathway effector lymphoid enhancer-binding factor 1, and cAMP pathway effector cAMP response element binding protein) 34 . Nine isoforms have been reported in humans, each with a different 5′ specificity (MITF-A, -B, -C, -D, -E, -H, -J, -M, and -MC) 35 . Tachibana et al., and other researchers, demonstrated that melanocyte-specific MITF (MITF-M) consists of 419 amino acid residues and its mRNA expression is detected exclusively in melanocytes and pigmented melanoma cells 36,37 . MITF-M transactivates several genes involved in melanogenesis, such as melanogenic enzymes (tyrosinase, TRP-1 and DCT or TRP-2, respectively), melanosome biogenesis-related proteins (pre-melanosome protein 17, G protein-coupled receptor 143, solute carrier family 24 member 5, and melanosome transport proteins, which are members of the RAS oncogene family (RAB7A, RAB27A)), melanosome delivery proteins (adaptor-related protein complex 1 subunits (AP1B1, AP1G1, AP1S1, AP1S2)), subunit 1 BLOC3 complex (BLOC3S1), subunit 1 BLOC2 complex (BLOC2S1), BLOC3 complex subunits 1, 2, and 3, and membrane proteins (melanocortin 1 receptor, endothelin receptor type B, c-Kit receptor, and melastatin) 38 . From these findings, MITF has become a potential therapeutic target for skin pigmentation disorders or whitening.
The miRNA-200 family is highly conserved among vertebrate species and consists of five members that form two clusters located in two different chromosomes 39 41 . The difference between the seed sequences in both groups is only one nucleotide. Therefore, it can be predicted that the miRNA-200 family targets many common genes to increase the efficiency of genetic regulation.
It is widely understood that miRNA-200 family members serve as crucial molecules for tumorigenesis, cell growth, angiogenesis, and the metastasis processes in each type of cancer cell [42][43][44][45] . In addition, several  56 . The results of the current study reveal the mechanisms by which miR-141-3p and miR-200a-3p transcriptionally regulate melanogenesis; i.e. direct targeting of Mitf. This adds miR-141-3p and miR-200a-3p to the previously reported group of miRNAs that control melanogenesis. From these reports, it is conceivable that controlling miR-141-3p and/or miR-200-3p is a promising approach for treating photodamage.
Although MITF small interfering RNA creams have been used safely for melasma in the clinical setting, the effects of these agents are extremely low owing to the difficulty of RNAi-materials penetrating through the stratum corneum barrier of the skin 57 . Hence, several studies are ongoing to develop revolutionary RNAi-based therapeutics that eliminate the drawbacks of conventional methods. For example, ultra-deformable, elastic, and   (Toray Industries). For efficient hybridization, this microarray adopts a columnar structure to stabilize spot morphology and enable micro-beads agitation. Total RNA was labelled with Cy5 using the Amino Allyl MessageAMP II aRNA Amplification Kit (Applied Biosystems). The Cy5-labeled a RNA pools were mixed with hybridization buffer, and hybridized for 16 h. Hybridization was performed according to the supplier's protocols (www.3d-gene.com). Hybridization signals were obtained by using a 3D-Gene Scanner, and processed by 3D-Gene Extraction software (Toray Industries). Detected signals for each gene were normalized by the global normalization method; the median of the detected signal intensity was adjusted to 25.
Biological replicates were not prepared for microarray analysis. Technical and biological replicates were prepared for the qRT-PCR validation experiments.

QRT-PCR.
To confirm reproducibility of the miRNA expression profiles obtained by the miRNA array analysis, we used TaqMan ® miRNA reverse transcription and TaqMan ® miRNA assay kits (Applied Biosystems).
After α-MSH stimulation for 24 h, total RNA was extracted from the cells by TRIzol reagent containing phenol/ guanidine isothiocyanate (Invitrogen) with DNase I treatment. Complementary DNA (cDNA) was then synthesized using reverse transcriptase with 12.5 ng of total RNA. The products were subjected to qRT-PCR using an Applied Biosystems StepOne ™ RT-PCR system. Expression levels were normalized to U6 as the internal control and quantified by the comparative Ct (ΔΔCt) method. QRT-PCR consisted of 45 cycles of 95 °C for 10 sec, 60 °C for 40 sec, and 72 °C for 1 sec, after an initial denaturation step (95 °C for 10 min).
To determine the levels of Mitf and Tyrosinase mRNAs, we prepared cDNA from total RNA samples using a high capacity RNA-to-cDNA kit (Applied Biosystems). Subsequently, qRT-PCR was performed using the SYBR ™ Green PCR Master Mix Kit. Primers for Mitf, tyrosinase, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were purchased from Takara Bio (Kusatsu, Japan, primer sequences are shown in Supplementary Table 1). The expression level of each gene was determined using the ΔΔCt method and normalized to that of Gapdh, which was used as the internal control. The PCR reaction consisted of 45 cycles of 95 °C for 15 s and 60 °C for 60 s, after an initial denaturation step (95 °C for 10 min).  www.nature.com/scientificreports www.nature.com/scientificreports/ Determination of melanin content. After α-MSH stimulation for 72 h, B16-4A5 cells were harvested with the culture medium using a rubber tipped cell scraper. The cell pellet was collected by centrifugation (5,000 × g for 15 min at 4 °C). The supernatants was transferred to an ultracentrifugation tube and centrifuged at 100,000 × g for 30 min at 4 °C for purification of secreted melanosomes (OptimaTM TLX ultracentifuge (Rotor: TLA-110), Beckman Coulter, CA, USA). The cell pellet containing crude melanosomes was lysed in 1 N NaOH for 1 h at 60 °C. The melanin content was measured at 475 nm using a Varioskan-LUX multimode microplate reader (Thermo Fisher Scientific) and was calculated from a standard curve of synthetic melanin.
Western blotting. For preparation of cell lysates, B16-4A5 cells, stimulated by α-MSH for 72 h were washed twice with PBS, then harvested with a cell scraper. The cell pellet after centrifugation at 15,000 × g for 30 min at 4 °C was resuspended in radioimmunoprecipitation assay buffer containing protease (25 × Complete ® ) and phosphatase inhibitors (Sigma-Aldrich). The protein content was measured with a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Each whole cell lysate was resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) buffer containing 2% mercaptoethanol and boiled at 98 °C for 5 min. Protein samples were subjected to SDS-PAGE in a 12% polyacrylamide gel and subsequently electroblotted onto a polyvinylidene difluoride membrane (GE Healthcare, Pittsburgh, PA, USA). After blocking non-specific binding sites for 1 h with 5% non-fat milk in TBST (tris-buffered saline containing 0.1% Tween 20), the membrane was incubated overnight at 4 °C with the various primary antibodies. The membrane was then washed 3 times in TBST, incubated further with a horseradish peroxidase-conjugated secondary antibody at room temperature, then washed again 3 times in TBST. Protein bands were detected using an enhanced chemiluminescence kit (GE Healthcare) and chemiluminescence detector (Davinchi Chem, Fujifilm Wako Pure Chemical, Osaka, Japan).
B16-4A5 cells were inoculated into 12-well plates (1 × 10 5 cells/mL), transfected with the sensor vector or mutated sensor vector plasmid and 40 nM of miR-141-3p or miR-200a-3p using Lipofectamine TM RNAiMAX, then cultured for 24 h. Luciferase activity was measured using a luciferase assay system according to the manufacturer's protocol (Promega, Madison, WI, USA). Relative luciferase activity is expressed as the ratio of measured luciferase activity to the control (non-specific miRNA).
Reconstructed skin model. 3D-MHE from a black donor (MEL-300-B, lot no. 28492) was purchased from Kurabo Industries (Osaka, Japan). Briefly, human epidermal keratinocytes and normal human melanocytes derived from dark skin donors were co-cultured on a collagen-coated membrane. After delivery from Kurabo, the in vitro skin model was immediately precultured for 12 h with DMEM/high glucose medium supplemented with 10% FBS, after which the tissue culture was washed twice with PBS, and transferred to a 12-well microplate.
Mature type of miR-141-3p and miR-200a-3p (40 nM, Applied Biosystems), that were designed to bind to and inhibit the activity of endogenous specific miRNAs when introduced into cells, were transfected from both sides (apical side: 12 h; basolateral side: 36 h: 12 h × 3, exchanging transfection medium every 12 h) by using cationic liposomes (RNAiMAX) according to the manufacturer's lipofection protocol. Non-specific control miRNA was used for detecting non-specific effects. Forty-eight hours after transfection, the tissue was cultured