Implication of microRNA regulation in para-phenylenediamine-induced cell death and senescence in normal human hair dermal papilla cells

Para-phenylenediamine (PPD) is a major component of hair coloring and black henna products. Although it has been largely demonstrated that PPD induces allergic reactions and increases the risk of tumors in the kidney, liver, thyroid gland and urinary bladder, the effect on dermal papilla cells remains to be elucidated. Therefore, the current study evaluated the effects of PPD on growth, cell death and senescence using cell-based assays and microRNA (miRNA) microarray in normal human hair dermal papilla cells (nHHDPCs). Cell viability and cell cycle analyses demonstrated that PPD exhibited a significant cytotoxic effect on nHHDPCs through inducing cell death and G2 phase cell cycle arrest in a dose-dependent manner. It was additionally observed that treatment of nHHDPCs with PPD induced cellular senescence by promoting cellular oxidative stress. In addition, the results of the current study indicated that these PPD-mediated effects were involved in the alteration of miRNA expression profiles. Treatment of nHHDPCs with PPD altered the expression levels of 74 miRNAs by ≥2-fold (16 upregulated and 58 downregulated miRNAs). Further bioinformatics analysis determined that these identified miRNA target genes were likely to be involved in cell growth, cell cycle arrest, cell death, senescence and the induction of oxidative stress. In conclusion, the observations of the current study suggested that PPD was able to induce several cytotoxic effects through alteration of miRNA expression levels in nHHDPCs.


Introduction
Para-phenylenediamine (PPD), also known as 1,4-diaminobenzene, is a key primary precursor of the oxidative dyes used in hair coloring and tattoos (1,2). However, accumulating evidence has suggested that this compound sensitizes skin to allergic reactions (3,4). Further studies have identified that these allergic reactions predominantly occur due to PPD-mediated activation of dendritic cells (5). Additionally, PPD is a potential carcinogen, which was reported to increase the risk of tumorigenesis in the kidney, liver, thyroid gland and urinary bladder in mice and rats (6,7). In addition, in vitro studies have demonstrated that PPD induced reactive oxygen species (ROS)-mediated DNA damage in uroepithelial cells and activated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in Chang liver cells (8,9). A case report demonstrated that PPD induced severe acute hair loss in females within six days of application (10). However, it remains to be elucidated whether PPD contributes to hair loss by inducing damage to normal human hair dermal papilla cells (nHHDPCs).
MicroRNAs (miRNAs) are small (18-24 nucleotides) noncoding RNAs that repress the translation of target genes through imperfect base pairing to the 3'-untranslated region of their target mRNAs (11,12). miRNAs have been reported to be key regulators of apoptosis, proliferation and differentiation (13). Regarding the role of miRNA in hair, it has been reported that the expression levels of miRNA-31 (miR-31) were upregulated in the anagen phase of the hair growth cycle and controlled the expression levels of Krt16, Krt17, Dlx3 and Fgf10 (14). In addition, miR-24 was reported to regulate the development of hair follicles by targeting the hair keratinocyte stemness regulator Tcf-3 (15). Furthermore, a previous study demonstrated that Dicer, an miRNA-processing enzyme, was essential for the morphogenesis of hair follicles (16).
The aim of the current study was to investigate the effects of PPD on cell growth, death and senescence in nHHDPCs.
Propidium iodide (PI)-based cell cycle analysis. The cell cycle was analyzed using PI (Sigma-Aldrich) staining of DNA. nHHDPCs were plated and treated with various concentrations of PPD (0, 200, 400 and 600 µM) for 24 h. Cells were then trypsinized (using 0.25% trypsin-EDTA; Gibco Life Technologies), centrifuged (3,500 x g, 2 min), washed with phosphate-buffered saline (PBS; Gibco Life Technologies) and fixed in 70% ethanol (Merck Millipore, Darmstadt, Germany) at 4˚C for 3 h. The fixed cells were incubated with staining solution [50 µg/ml PI, 0.1 µg/ml RNase (Life Technologies, Grand Island, NY, USA) and 0.05% Triton X-100 (Sigma-Aldrich) in PBS] at 37˚C for 1 h and then analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The mean PI fluorescence intensity was determined based on analysis of 10,000 cells using the FLH-2 detection channel (585±42 nm).
Detection of cellular senescence. For the assessment of cellular senescence, nHHDPCs (2x10 6 ) were seeded into 60-mm cell culture dishes and treated with 0 or 400 µM PPD. Following 48 h of treatment, cells were fixed using Fixative solution (included in Senescence Detection kit; BioVision, Inc., Milpitas, CA, USA) and senescence-associated-β-galactosidase (SA-β-gal) activity was measured using the Staining Solution Mix, including Staining Solution, Staining Supplements and X-gal substrate for (SA-β-gal) within the Senescence Detection kit, according to the manufacturer's instructions. Cells stained for SA-β-gal were counted under a light microscope (CKX41; Olympus Corporation, Tokyo, USA) (magnification, x200) and the percentage of SA-β-gal positive cells were calculated.
Detection of intracellular ROS. Staining for ROS in cultured cells was conducted using a 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Sigma-Aldrich) assay. Briefly, 2x10 6 nHHDPCs were plated in 60-mm culture dishes and treated with PPD. Following treatment for 24 h, the cells were stained by adding DCF-DA to the culture medium to a final concentration of 20 µM and then incubating for 1 h. Distribution of the stained cell population was determined using a FACSCalibur flow cytometer. miRNA expression profiling. In order to analyze the miRNA expression profile, nHHDPCs (2x10 6 ) were seeded into 60-mm culture dishes and treated with 400 µM PPD. Following 24 h of treatment, total RNA was purified using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Total RNA was dephosphorylated and labeled with pCp-Cy3 using an Agilent miRNA Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Labeled RNAs were hybridized using a SurePrint G3 Human v16 miRNA 8x60K microarray (Agilent Technologies, Inc.) at 65˚C for 20 h. The miRNA expression profile was digitized using Feature Extraction version 10.7 software (Agilent Technologies). Fold changes in miRNA expression levels were determined using GeneSpring GX software, version 11.5 (Agilent Technologies).
Prediction of potential target genes of PPD-regulated miRNAs and gene ontology (GO) analysis. Potential target genes of PPD-regulated miRNAs were predicted using the DNA Intelligent Analysis (DIANA) microT-CDS version 5.0 bioinformatics tool (http://diana.cslab.ece.ntua. gr/). GO of each putative target gene was identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource, version 6.7 (http://david.abcc.ncifcrf.gov). Target genes were categorized into four GO terms: Aging, skin development, apoptosis and cell proliferation. Furthermore, target gene-associated signaling pathways were determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway algorithm (http://david.abcc.ncifcrf.gov/summary.jsp) within the DAVID resource.
Statistical analyses. Values are expressed as the mean ± standard error of the mean of three independent experiments. Statistical significance was determined by Student's t-test and P<0.05 was considered to indicate a statistically significant difference between values.

PPD treatment reduces the proliferation rate of nHHDPCs.
Previous studies reported that exposure to >250 µM and >60 µM PPD resulted in considerable cytotoxicity in dendritic cells (17) and keratinocytes (18), respectively. Therefore, the current study aimed to determine the cytotoxic effects of PPD on nHHDPCs at concentrations of 0, 100, 200, 300, 400, 500 and 600 µM using a WST-1-based cell viability assay. As shown in Fig. 1, the viability of nHHDPCs was significantly reduced following exposure to PPD for 24 h. Maximal toxicity was observed at 600 µM, at which concentration cell viability was reduced to 58.33±2.39% of the control value (n=3; P<0.05). The IC 25 (a 25% reduction in viability) of PPD was 400 µM, at which concentration cell viability was reduced to 74.42±6.08% of control value (P<0.05) (Fig. 1). Figure 1. Effect of PPD on viability of nHHDPCs. nHHDPCs (5x10 3 ) were seeded into 96-well plates and treated with various concentrations of PPD (0, 100, 200, 300, 400, 500 and 600 µM) for 24 h. Cell viability was measured using the water-soluble tetrazolium salt assay. Values are presented as the mean ± standard error of the mean of the percentage of control optical density of experiments performed in triplicate. * P<0.05 vs. 0 µM PPD. PPD, para-phenylenediamine; nHHDPCs, normal human hair dermal papilla cells.

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PPD treatment increases cell death and cell cycle arrest in nHHDPCs. The present study investigated whether PPD-induced loss of cell viability occurred due to cell cycle arrest and cell death. Cells were treated with PPD (0, 200, 400 and 600 µM) for 24 h and the distribution of cells in the different cell cycle phases was analyzed using flow cytometry. As shown in Fig. 2, treatment with 200, 400 and 600 µM PPD led to significant accumulation in the sub-G 1 phase, compared with that of control DMSO-treated cells (P<0.05). In addition, the proportion of cells in G 1 /G 2 was significantly reduced by PPD (P<0.05), indicating that PPD increased the G 2 population. These data therefore demonstrated that PPD induced cell death and G 2 arrest in nHHDPCs.
PPD treatment leads to the accumulation of intracellular ROS and senescence-like growth. Chye et al (8) observed that PPD increased intracellular ROS and induced apoptosis in Chang normal human liver cells. Therefore, the current study investigated the effects of PPD on the regulation of intracellular ROS production in nHHDPCs. Intracellular ROS levels were determined using an DCF-DA probe, which is oxidized to fluorescent DCF in the presence of ROS. As presented in Fig. 3A, exposure to PPD resulted in a marked increase in  Table II. Predicted targets of microRNAs upregulated in response to para-phenylenediamine treatment in normal human hair dermal papilla cells. fluorescent DCF-positive cells compared with that of the control cells, indicating that PPD increased intracellular ROS production in nHHDPCs. As ROS have been implicated in cellular senescence (19), the present study investigated whether the PPD-mediated increase in ROS was associated with increased senescence by analyzing the activity of SA-β-gal, a marker of cellular senescence. Consistent with the observed ROS increase, PPD was identified to promote an increase in SA-β-gal activity (Fig. 3B), indicating that PPD induces cellular senescence in nHHDPCs.

Biological processes and target genes
Identification of differentially expressed miRNAs in PPD-treated nHHDPCs. Fig. 2 and 3 demonstrated that cell cycle arrest in G 2 phase and PPD-mediated cell death were characterized by increased ROS production. Therefore, in order to determine whether ROS-mediated cell cycle arrest and cell death are associated with miRNA expression, miRNA microarray analysis was conducted using the SurePrint G3 Human v16 miRNA 8x60K microarray, which contained 2,006 human miRNA probes. Significant miRNAs exhibiting a ≥2.0-fold increase or reduction in expression were selected using GeneSpring GX software. As presented in Table I, PPD differentially regulated the expression levels of 74 miRNAs. Notably, 16 of 74 miRNAs were significantly upregulated and 58 miRNAs were significantly downregulated. In particular, miR-425-3p exhibited the greatest increase in expression (230.60-fold) and miR-3656 the greatest reduction (112.15-fold), compared with the corresponding miRNAs in control cells. These results suggested that the PPD-mediated cellular effects were associated with alterations in expression of specific miRNAs.
Bioinformatic analysis of PPD-modulated miRNAs. As miRNAs perform their biological functions through regulation of target mRNA translation (11), the present study aimed to predict the target genes of the miRNAs deregulated in response to PPD. The biological functions of the upregulated and downregulated genes were then determined following categorization into the four groups: Aging, apoptosis, cell proliferation and skin development, using DAVID (Tables II  and III, respectively). In addition, in order to identify the specific signaling pathways of the deregulated miRNAs, the correlation between KEGG pathway-associated genes and the target genes of each miRNA were analyzed. The meaningful KEGG pathways with a value >1% (percentage of target genes/total genes involved in each pathway) were selected. The analysis identified a wide distribution of cellular functions, which are presented in Tables IV and V. The results indicated that the upregulated miRNAs were implicated in signaling pathways in cancer, ubiquitin-mediated proteolysis, melanogenesis, cell cycle, Wnt signaling, MAPK signaling, neurotrophin signaling, cell adhesion molecules (CAMs), long-term potentiation, natural killer cell-mediated cytotoxicity, calcium signaling, neuroactive ligand-receptor interactions, glycosphingolipid biosynthesis, arrhythmogenic right ventricular cardiomyopathy, axon guidance, ErbB signaling, gonadotropin-releasing hormone signaling, tight junctions and viral myocarditis (Table IV). In addition, PPD-induced downregulated miRNAs were implicated in signaling pathways in cancer, regulation of actin cytoskeleton, Wnt Table II. Continued.

Discussion
Although PPD has been widely used in hair dyes and tattoos, previous studies have demonstrated that PPD may be an important etiological factor for allergic contact dermatitis (20,21). However, the side effects of PPD in hair follicle cells remain to be fully elucidated. The results of the current study provided evidence for senescence and cell death as key responses of nHHDPCs to PPD. The senescence-associated alterations observed included G 2 phase arrest and increases in ROS production as well as SA-β-gal activity. To the best of   our knowledge, the present study was to first to report these responses in dermal papilla cells, although PPD-mediated hair loss has been described in a clinical report (10). PPD was reported to promote apoptosis through oxidative stress-induced DNA damage in kidney and liver cells (22,23). The data of the current study confirmed the apoptotic effect of PPD in dermal papilla cells; however, maximal toxicity was obtained at 600 µM, which only increased the proportion of apoptotic cells 9.21%. The most notable observation in the present study was the implication of PPD in the induction of G 2 phase arrest, cellular ROS production and senescence in dermal papilla cells. The proportion of cells in G 1 /G 2 phase was significantly reduced at 400 µM, at which concentration ROS production was increased by 50.61% compared with the control group. Consistent with the data described, 400 µM PPD increased the number of senescent cells by 15.90%. It has been previously demonstrated that PPD increased intracellular ROS levels and induced apoptosis in Chang normal human liver cells (8). In the present study, marked alterations in the levels of cell death and senescence were observed in dermal papilla cells following PPD treatment. The data collected indicated that PPD induced G 2 arrest and ROS production, which in turn triggered cellular senescence leading to cell death in HHDPCs. Under identical experimental conditions, the present study identified 74 miRNAs that were differentially expressed by ≥2-fold following PPD treatment in nHHDPCs. Among these, 16 miRNAs were significantly upregulated and 58 miRNAs were significantly downregulated in PPD-treated nHHDPCs. Of note, the expression levels of miR-146b-5p were significantly downregulated by 30.17-fold following PPD treatment of the cells. miR-146b-5p has been previously reported to negatively regulate cellular senescence via targeting interleukin-1 receptor-associated kinase 1 in fibroblasts (24). In addition, miR-378, which was downregulated by 36.78-fold in the present study, has been previously reported to promote cell survival, tumor growth and angiogenesis through targeting suppressor of fused and fused in sarcoma-1 (25). Together, miR-146b-5p and miR-378 are known to be critical miRNAs involved in cell survival and anti-senescence; thus, regulation of their expression is a promising strategy for the treatment of PPD-mediated cellular senescence in dermal papilla cells. The biological functions of potential target genes of the altered miRNAs were further demonstrated using GO analysis and the web-based program DAVID. The target genes were categorized into four GO terms: Aging, skin development, apoptosis and cell proliferation. Additionally, KEGG pathway analysis identified that the target genes of the miRNAs upregulated by PPD treatment were predominantly implicated in the Wnt and MAPK signaling pathways. The Wnt signaling pathway has been demonstrated to maintain the balance between cell proliferation and differentiation (26). Notably, the Wnt signaling pathway has an important involvement in hair follicle morphogenesis via activation of β-catenin (27). In addition, in bald patients, activation of Wnt signaling was reported to induce reactivation of hair growth (28). Therefore, the results of the KEGG pathway analysis in the present study indicated that PPD regulated hair growth, morphogenesis and proliferation of dermal papilla cells via miRNA-mediated regulation of the Wnt signaling pathway. MAPKs are important intracellular signaling molecules which have pivotal roles in proliferation, differentiation, development, transformation and apoptosis (29). Therefore, the results of the present study suggested that PPD regulated MAPK signaling pathways through altering the expression of specific miRNAs, which in turn altered cell proliferation in dermal papilla cells.
In conclusion, to the best of our knowledge the present study was the first to use cell-based assays and miRNA microarray analysis to demonstrate that PPD significantly induced dermal papilla cell death and senescence through alteration of the expression levels of specific miRNAs. The results of the current study also suggested that the identified miRNAs may be potential candidates for the development of novel treatment strategies for PPD-induced cell dysfunction. hsa-miR-874 99 ----miR, microRNA; hsa, homo sapiens; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; Jak-STAT, Janus kinase-signal transducer and activator of transcription; mTOR, mammalian target of rapamycin; TGF-β, transforming growth factor-β.