Open Access

Analysis of changes in microRNA expression profiles in response to the troxerutin-mediated antioxidant effect in human dermal papilla cells

  • Authors:
    • Kyung Mi Lim
    • Sungkwan An
    • Ok‑Kyu Lee
    • Myung Joo Lee
    • Jeong Pyo Lee
    • Kwang Sik Lee
    • Ghang Tai Lee
    • Kun Kook Lee
    • Seunghee Bae
  • View Affiliations

  • Published online on: May 4, 2015     https://doi.org/10.3892/mmr.2015.3717
  • Pages: 2650-2660
  • Copyright: © Lim et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Dermal papilla (DP) cells function as important regulators of the hair growth cycle. The loss of these cells is a primary cause of diseases characterized by hair loss, including alopecia, and evidence has revealed significantly increased levels of reactive oxygen species (ROS) in hair tissue and DP cells in the balding population. In the present study, troxerutin, a flavonoid derivative of rutin, was demonstrated to have a protective effect against H2O2‑mediated cellular damage in human DP (HDP) cells. Biochemical assays revealed that pretreatment with troxerutin exerted a protective effect against H2O2‑induced loss of cell viability and H2O2‑induced cell death. Further experiments confirmed that troxerutin inhibited the H2O2‑induced production of ROS and upregulation of senescence‑associated β‑galactosidase activity. Using microRNA (miRNA) microarrays, the present study identified 24 miRNAs, which were differentially expressed in the troxerutin‑pretreated, H2O2‑treated HDP cells. Subsequent prediction using bioinformatics analysis revealed that the altered miRNAs were functionally involved in several cell signaling pathways, including the mitogen‑activated protein kinase and WNT pathways. Overall, these results indicated that ROS‑mediated cellular damage was inhibited by troxerutin and suggested that the use of troxerutin may be an effective approach in the treatment of alopecia.

Introduction

Trihydroxyethylrutoside (troxerutin) is one of the flavonoid rutoside derivatives. It exhibits non-mutagenic properties and has a functional role in the treatment of chronic venous insufficiency (CVI) (1,2). A number of studies have demonstrated other beneficial effects of troxerutin, in vitro and in vivo, and it may be effective in reducing different cytotoxicities. In particular, troxerutin has been observed to exhibit an inhibitory effect on the neurotoxicity induced by high cholesterol mediated cognitive deficits, kainic acid-triggered excitotoxic damage and β-amyloid oligomerization (35). In addition, troxerutin has a photoprotective effect against ultraviolet B (UVB) radiation in human skin cells, including dermal fibroblasts and keratinocytes (6,7). Troxerutin also exerts a protective effect against γ-radiation in mice (8,9). Although the precise cellular mechanisms underlying the effects of troxerutin remain to be fully elucidated, these reports can be summarized as a single meaningful finding, that troxerutin inhibits the production of reactive oxygen species (ROS). In vivo investigations have demonstrated that CVI-bearing patients have increased levels of ROS, and troxerutin has a protective effect against oxygen-derived free radical scavengers on the endothelium in these patients (10,11). In addition, the aforementioned neurotoxicities are inhibited following troxerutin application by reducing the production of ROS (3,4,12). UVB and γ-radiation are known ROS stimulators (13,14), and a previous study demonstrated that troxerutin protects against radiation-induced lipid peroxidation (9). These studies suggest that this toxerutin may offer a novel therapeutic strategy for ROS-induced diseases.

Dermal papilla (DP) cells are located at the base of hair follicles and are important in the induction of growth and maintenance of epithelial cells, which are the predominant components of hair follicles (15). In response to hormonal changes, DP cells direct the follicular epithelial cells to enter the hair growth cycle, which involves anagen, an active growing phase; catagen, a short transitionary regressive phase; and telogen, a dormant resting phase (15). An increasing body of evidence has demonstrated excessive loss of viability and death of DP cells in balding regions of the scalp, compared with non balding regions, due to increased levels of 5α-reductase (16), a converting enzyme for androgenic hormones and intracellular ROS (17). In addition, previous reports have indicated that oxidative stress is generated by the exposure of androgen sensitive prostate cancer cells to high levels of androgens (18), and that lipid peroxides increase the levels of ROS and apoptosis of the hair follicle cells (19). Furthermore, DP cells in the balding scalp grow more slowly in vitro, compared with cells from the non balding scalp. The reduced proliferative activity of balding DP cells is associated with changes in the expression levels of senescence-associated (SA) β-galactosidase, oxidative stress markers, superoxide dismutase and catalase (20). These findings indicate that oxidative stress is important in the loss of DP cells and in hair production.

In the present study, the hypothesis that troxerutin inhibits ROS-mediated cellular dysfunction in human DP (HDP) cells was investigated. In addition, using micro (mi)RNA microarrays and bioinformatics analysis, the role of troxerutin in the regulation of the expression and mechanisms of specific miRNAs was evaluated. The present study aimed to examine troxerutin as a potential novel chemical agent for the preven tion and/or treatment of alopecia.

Materials and methods

Cell culture and viability

The HDP cells were purchased from Innoprot (Biscay, Spain) and cultured in Dulbecco’s modified Eagle’s medium, containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin streptomycin (Gibco Life Technologies, Grand Island, NY, USA) at 37°C and 5% CO2. The cells were plated at a density of 4×103/well in a 96-well plate. At 70–80% confluence, the cells were treated with troxerutin (Sigma-Aldrich, St. Louis, MO, USA) at concentrations ranging between 0 and 60 μM for 24 h at 37°C. Subsequently, 10 μl water soluble tetrazolium salt assay solution (EZ-Cytox Cell Viability Assay kit; Itsbio, Seoul, Korea) was added to each well and, following incubation for 30 min at 37°C, the optical density was measured at 490 nm using an iMark microplate reader (Bio Rad Laboratories, Inc., Hercules, CA, USA). To examine troxerutin mediated ROS protection, the cells were pretreated with troxerutin at the following concentrations: 0, 5, 10 and 15 μM for 8 h. Subsequently, 750 μM H2O2 was added to each well. Following incubation for 24 h at 37°C, cell viability was evaluated using an EZ-Cytox Cell Viability Assay kit. The level of cell viability (%) was normalized to that of 0.1% dimethyl-sulfoxide (DMSO; Sigma-Aldrich)-treated cells. Each experiment was repeated at least three times. The P-value was determined using Student’s t-test and P<0.05 was considered to indicate a statistically significant difference.

Analysis of cell cycle

The HDP cells (2×106), which had been treated with troxerutin and/or H2O2 were trypsinized using 0.25% trypsin-EDTA (Gibco Life Technologies), washed once with phosphate-buffered saline (PBS), and used for analysis. The cell cycle distribution was measured using propidium iodide (PI; Sigma-Aldrich) staining solution, containing 50 μg/ml PI, 0.5% Triton X-100 (Sigma-Aldrich) and 100 μg/ml RNase (Qiagen, Hilden, Germany). The cells were fixed in 70% cold ethanol (Merck Millipore, Darmstadt, Germany) and incubated for 1 h at −20°C. Subsequently, PI staining solution was added to the fixed cells, followed by incubation for 1 h in the dark at 37°C. The PI fluorescence intensity was detected using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The mean PI fluorescence intensity was calculated based on the measurements of 10,000 cells using the FL2-H channel.

Analysis of intracellular levels of ROS

The HDP cells (2×106), which had been treated with troxerutin and/or H2O2 were washed with PBS and trypsinized. Intracellular ROS levels were measured using 2′7′-dichlorofluorescein diacetate fluorescent dye (DCF-DA; Sigma-Aldrich), as described previously (21). The cells were resuspended in 10 μM DCF-DA and further incubated at room temperature for 1 h in the dark. The intensity of the resulting fluorescence was measured using a BD FACSCalibur flow cytometer (BD Biosciences). The mean DCF fluorescence intensity was calculated based on measurements of 10,000 cells using the FL1-H channel. The M1 range was calculated as the percentage of each subpopulation of cells exhibiting increased DCF-DA fluorescence.

Analysis of cellular senescence

The HDP cells (2×106), which had been treated with troxerutin and/or H2O2 were washed in PBS and fixed for 5 min at room temperature in 2% formaldehyde/0.2% glutaraldehyde (Sigma-Aldrich). The cells were washed with PBS and incubated for 15 h at 37°C with SA β-galactosidase staining solution (BioVision, Milpitas, CA, USA). The stained cells (blue) were observed using a bright-field microscope (CKX41; Olympus Corporation, Tokyo, Japan; magnification, ×200), counted in three different fields and the percentage of stained cells was determined.

Analysis of miRNA expression profiles

The HDP, which had been cells treated with troxerutin and/or H2O2 were washed in cold PBS and trypsinized for RNA purification. The total RNA was extracted and purified from the cells using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The integrity (RNA integrity number >8.0) and purity (A260/280 and A260/230 values >1.8) were confirmed using an Agilent 2100 Bioanalyzer® (Agilent Technologies, Inc., Santa Clara, CA, USA) and a MaestroNano® microvolume spectrophotometer (Maestrogen, Las Vegas, NV, USA), respectively. Samples (100 ng) of RNA meeting these criteria were first dephosphorylated by incubation with calf intestinal alkaline phosphatase (Agilent Technologies, Inc.) at 37°C for 30 min. Subsequently, cyanine 3-pCp labeling solution (Agilent Technologies, Inc.) and T4 RNA ligase (Agilent Technologies, Inc.) were added to the dephosphorylated RNA samples and incubated at 16°C for 2 h. Following the labeling reaction, the samples were dried and treated with GE Blocking Agent (Agilent Technologies, Inc.). The samples were hybridized to the SurePrint G3 Human v16 miRNA 8×60 K (Agilent Technologies, Inc.) microarray at 55°C, with constant rotation at 20 rpm in an Agilent Microarray Hybridization Chamber(Agilent Technologies, Inc.) for 20 h. The array was then washed and scanned using an Agilent SureScan Microarray scanner and the images captured were quantified using Agilent Feature Extraction software (version 10.7; Agilent Technologies, Inc.). The data were analyzed with the assistance of GeneSpring GX software version 7.3 (Agilent Technologies). In addition, fold-change analysis was performed to select those with ≥2.0-fold between the H2O2-treated control cells and those treated with troxerutin and H2O2.

Bioinformatic analysis of altered miRNAs

Analysis of the biological significance of the altered miRNAs in the present study was performed, as previously described (21). First, the putative target genes of the altered miRNAs were predicted using MicroCosm Targets Version 5 (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/). Subsequently, the target genes were grouped into four categories: Aging, skin development, apoptosis and cell proliferation, based on the AmiGo 2 Gene Ontology (GO) analysis tool (amigo.geneontology.org/cgi-bin/amigo/browse.cgi). The putative target genes of each miRNA were further analyzed for biologic function using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway within the Database for Annotation, Visualization and Integrated Discovery, (DAVID; http://david.abcc.ncifcrf.gov/home.jsp) bioinformatics resources (version 6.7), according to the standard procedures (22). The ‘KEGG_pathway’ category was processed by setting the threshold of the EASE score, a modified Fisher’s exact P-value, to 0.1. The KEGG pathways, identified as having a percentage of involved target genes / total target genes in each pathway >1% were selected.

Results

H2O2-induced cell damage is inhibited by troxerutin in HDP cells

HDP cells are a major component of skin and direct hair growth, and loss or senescence of these cells is a key cause of hair loss (15). Previous reports have demonstrated that cisplatin-and androgen overexposure-mediated cellular dysfunction, including apoptosis, can induce alopecia and occur predominantly by stimulating the production ROS in HDP cells (16,23,24). In our previous study, pretreatment of human dermal fibroblasts and HaCaT keratinocytes with troxerutin was observed to protect against UVB-mediated cell death (6,7). UVB light is an important inducer of ROS production in several types of cell (13); therefore, the present study examined the possible role of troxerutin in protecting against ROS-induced cell stress and damage in HDP cells. Initially, troxerutin-mediated cytotoxicity in HDP cells was screened for. No significant changes in cell viability were detected following treatment with between 0 and 60 μM troxerutin for 24 h (Fig. 1A), indicating that troxerutin was non toxic in the HDP cells. Subsequently, the protective role of trox erutin against ROS-induced cell damage was determined using H2O2, a ROS inducer. The HDP cells were pretreated with several concentrations (0–60 μM) of troxerutin for 6 h, followed by the addition of 750 μM H2O2 and incubation for an additional 24 h. The results revealed that the maximum protective effect against ROS induced cell damage in the HDP cells occured folowing pretreatment with 10 μM troxerutin (Fig. 1B). Treatment with H2O2 alone decreased cell viability to 77.33±2.44%; however, pretreatment with 10 μM troxerutin maintained cell viability at 90.88±2.24% following H2O2 exposure (P<0.05; Fig. 1B). Concentrations of troxerutin >15 μM did not significantly enhance the protective effect of 10 μM troxerutin (Fig. 1B and data not shown). These results suggested that pretreatment with troxerutin inducedresistance against H2O2-mediated cytotoxicity in the HDP cells.

H2O2-induced cell death is inhibited by troxerutin in HDP cells

Our previous study demonstrated that troxerutin effects the level of cell death induced by UVB irradiation (6,7) and Fig. 1 shows the protective role of troxerutin against ROS-induced cell damage, observed in the present study. Therefore, to examine whether troxerutin is involved in the response to ROS stress, which is known to inhibit cell-cycle progression and induce cell death, the present study investigated changes in the cell cycle and in cell death in the HDP cells pretreated with different concentrations of troxerutin prior to H2O2 exposure. The HDP cells were pretreated with 0, 5 or 10 μM troxerutin for 6 h, followed by treat ment of the cells with 750 μM H2O2 for an additional 24 h. Subsequently, the cells were analyzed by flow cytometry. At concentrations of 5 and 10 μM, pretreatment with troxerutin caused a decrease in the number of cells in the sub G1 phase, indicative of cell death (Fig. 2A). H2O2 increased the percentage of the non-pretreated cells in the sub-G1 phase to 10.63±0.43%; however, this value increased to only 9.38±0.11 and 4.53±0.53% in the cells pretreated with 5 and 10 μM troxerutin, respectively (P<0.05; Fig. 2A and B). Therefore, these results suggested that troxerutin overcame the effect of H2O2-mediated cell death, resulting in a diminution of cells in the sub-G1 phase.

Hydrogen peroxide-induced ROS production is inhibited by troxerutin in HDP cells

Although the above results indicated that troxerutin had a protective effect against H2O2-mediated cell death, it remained to be elucidated whether troxerutin also regulates the level of intracellular H2O2-induced ROS in HDP cells. To examine this, the presents study performed a fluorescent DCF-DA staining assay following treatment with troxerutin and H2O2 in the HDP cells. The DCF-positive cells were then analyzed using flow cytometry to determine and compare the levels of intracellular ROS in the control cells, non-troxerutin pretreated cells, troxeruti only treated cells and troxerutin pretreated/H2O2 treated cells. As shown in Fig. 3, in the control and troxerutin-only-treated cells, 3.58±0.15 and 0.89±0.11% were DCF-positive (P<0.05; Fig. 3B), suggestive of ROS respectively, whereas treatment with H2O2 alone increased the level of ROS to 46.36±2.33%. The cells pretreated with troxerutin were 19.92±1.95% DCF-positive following H2O2 treatment, indicating that troxerutin reduced the H2O2-induced production of ROS in the HDP cells.

Hydrogen peroxide-induced senescence is inhibited by troxerutin in HDP cells

Increased ROS are one of the key mediators of cellular senescence (25). A previous report demonstrated that the premature senescence of balding DP cells is associated with changes in the expression of SA β-galactosidase and also suggested that oxidative stress may be involved in the premature senescence of these cells (20). The present study assayed for the presence of SA β-galactosidase activity to investigate whether troxerutin affects H2O2-induced senescence, thereby contributing to its protective effect against ROS-mediated cell damage. H2O2 treatment increased the number of SA β-galactosidase-positive cells to 32.11±3.32% compared with the control; however, only 18.22±5.21% of the cells pretreated with troxerutin were SA β-galactosidase-positive following treatment with H2O2 (Fig. 4). These data indicated that troxerutin has the potential to inhibit cellular senescence in HDP cells.

Troxerutin-mediated protective effects against ROS are involved in changes in miRNA expression in HDP cells

The present study also analyzed the miRNA expression profiles of non-pretreated and troxerutin-pretreated HDP cells treated with H2O2, as miRNAs can be involved in cell death, ROS scavenging and senescence (21,2628). In the microarray, a total of 24 miRNAs were detected with a ≥2.0-fold change in expression levels between the two groups. Among these, 10 miRNAs were upregulated and 14 were downregulated in the H2O2-treated cell, which had been pretreated with troxerutin (Table I). To investigate the biological value of the microarray data, several bioinformatic analyses were performed to predict the putative target genes of the altered miRNAs, and the GO and signaling pathways of the target genes. The putative target genes of each miRNA were deterfmined using MicroCosm Targets Version 5, following which GO analysis was performed for the target genes. Subsequently, the target genes of each miRNA were categorized into four biological functions: Aging, skin development, apoptosis and cell proliferation. Several target genes of each miRNA were found to be involved in these four biological functions at different levels (Tables II and III). For example, has-miR-602, which was the most highly upregulated miRNA (6.91-fold) based on the microarray data, potentially targets 34 genes, six of which were involved in aging, and one of the remaining 28 target genes was involved in skin development (Table II). Similarly, the target genes of the downregulated miRNAs were also differentially involved in the four functions (Table III), indicating that the altered miRNAs identified by the microarray analysis had distinct biological roles associated with the protective effect of troxerutin in H2O2-treated HDP cells. Therefore, the present study further analyzed the signaling pathways associated with the upregulated and downregulated miRNAs using KEGG pathway analysis and the DAVID bioinformatics tool (22), the results of which are presented in Tables IV and V, respectively. The results demonstrated that the altered miRNAs are functionally involved in shared and unique pathways among the miRNAs. For example, hsa-miR-602 was identified to be functionally involved in MAPK, insulin, and calcium signaling pathways, whereas has-miR-205 3p was found to be involved in cancer, MAPK, Wnt and cell adhesion signaling pathways. Overall, these results indicated that the miRNA expression patterns of non-pretreated and troxerutin-pretreated H2O2-treated HDP cells can be distinguished, and those which are significant changed may be involved in troxerutin-mediated protection against H2O2-induced cellular stress through the regulation of multiple signaling pathways.

Table I

MicroRNAs with ≥2-fold change in expression in troxerutin pretreated H2O2-treated human dermal papilla cells.

Table I

MicroRNAs with ≥2-fold change in expression in troxerutin pretreated H2O2-treated human dermal papilla cells.

microRNAChange relative to controlDirection of regulationChromosome
hsa-miR-150-3p4.13Up19
hsa-miR-181a-2-3p2.31Up9
hsa-miR-205-3p4.28Up1
hsa-miR-21-3p2.92Up17
hsa-miR-29b-1-5p3.72Up7
hsa-miR-3127-5p2.19Up2
hsa-miR-371a-5p2.30Up19
hsa-miR-3663-3p2.53Up10
hsa-miR-42982.01Up11
hsa-miR-6026.91Up9
hsa-miR-1181−3.14Down19
hsa-miR-12022.78Down6
hsa-miR-1224-5p−4.66Down3
hsa-miR-12902.15Down1
hsa-miR-135a-3p5.61Down3
hsa-miR-28-5p2.95Down3
hsa-miR-378a-5p2.01Down5
hsa-miR-4271−2.16Down3
hsa-miR-452-5p−2.51DownX
hsa-miR-572−4.26Down4
hsa-miR-575−8.01Down4
hsa-miR-629-3p3.12Down15
hsa-miR-9392.22Down8
hsa-miR-940−2.30Down16

[i] miR, microRNA.

Table II

Predicted targets of microRNAs upregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

Table II

Predicted targets of microRNAs upregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

microRNAAgingSkin developmentApoptosisCell proliferation
hsa-miR-150-3pBCL3, INHBA, ARHGEF2, RHOA, ATG7, MAP3K5, MECOM, PLAGL1BCL3, INHBA, MECOM, ARHGEF2, RHOA, NDN, PROX1, BTRC, TGFBI
hsa-miR-181a-2-3pLMNA, SRFSRFLMNA, MED1, BDNF, TIAL1, SRPK2, CITED2, AGAP2, PSMD7, MAPK8SRF, MED1, BDNF, TIAL1, SRPK2, CITED2, SOX11, FBXW7
hsa-miR-205-3pTBX3, CDK6, MNT, IL1B, TECP2L1, PNPT1, ATRAPC, DBIBRCA1, HDAC2, SOS2, SIX4, GRAM, MDM4, CUL5, NBN, MNT, IL1B, TBX3, WNT5A, RAD21, MAP3K5, RASSF6, CREB1, GLO1, API5, SOS1, APC, MSX2, FGF2, SOX2, DUSP1, GSK3B, PSMA5,MITF, HiPK2, HOXA13, PARK7, NAIP, BCLAF1MITF, GRAM, MDM4, CUL5, NBN, CDK13, CASK, PURA, MNT, IL1B, TBX3, HDAC2, CDK6, MSX2, FGF2, SOX2, HiPK2, BRCA1, WNT5A, EVI5, TOB1, NUMB
hsa-miR-21-3pCDK6MAP2K4, MAP3K1, BCL2L11, SMAD3, CUL3, SOX4, BAG4, RNF41, AMIGO2, SLC11A2, KDM28, DAB2IP, FOXO3, CCAR1, ROBO2, TRIM32, DSG1CUL3, SOX4, NR6A1, FTO, TRIM32, FOXO3, SMAD3, CDK6, KDM28, DAB2IP, CD274, PBRM1
hsa-miR-29b-1-5pNR3C1, SIRT1NR3C1, SIRT1, REST, PTK2, SOS2, NUAK2, PSMD7NR3C1, SIRT1, REST, PTK2, FGF18, INSR, PBRM1
hsa-miR-3127
hsa-miR-371a-5pLEF1, ATP7A, COL8A1LEF1, SOX2, CITED2, STK4, RB1CC1, BARD1, GSK3B, PSMF1, NR4A2, DYRK2, RPS6KA1, ITSN1, MAP3K1LEF1, SOX2, CITED2, STK4, COL8A1, RNF10, MAPRE1, BTG3, CCR2, FRS2, PRMT5
hsa-miR-3663-3pFAS, CASP2, CDKN1A, PTH1RADAMTS2, BCL11B, COL3A1, COL1A1FAS, CASP2, BCL11B, USP28, TGFB2, DDX5, COMP, PIGT, CDKN1A, TIAL1, PPP2R1B, PSMA2, MEF2DFAS, TIAL1, TGFB2, USP28, CDKN1A, BCL11B, VSIG
hsa-miR-4298HMGA1, AMFRMED1, FGF2, TRAF5, CCAR1HMGA1, MED1, WT1, FGF2
hsa-miR-602EDN1, VDR, SOD2, HTT, SLC34A2, CHEK1APCNOG, ERBB4, PSMD2, PIM1, EDN1, VDR, SOD2, DYRK2, ALDH1A2, CLI2, SEMA3A, HTT, APC, H1F0, PPARG, BCL2L15, JMY, TP53BP2, MYO18A, SHFNOG, ERBB4, PIM1, PPARG, CLI2, CDC27, CDK13, LIFR, EDN1, VDR, SOD2, STAT3, APC, ALDH1A2, ACSL6, PPP1R8, EMX2, CDK9, RTKN2, ID4, ZEB1

[i] miR, microRNA.

Table III

Predicted targets of microRNAs downregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

Table III

Predicted targets of microRNAs downregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

microRNAAgingSkin developmentApoptosisCell proliferation
hsa-miR-1181
hsa-miR-1202CLNB, PNPT1, SLC18A2CLN8, RRN3, PIK3CG, ETS1, DRAM1, DNAJC10, STEAP3, IKBKG, SOS1, NOD1RRN3, PIK3CG, ETS1, CDC6, BCAT1, NRP1, ERG, SESN1, FZD6, CD276, GAS8, RPS15A
hsa-miR-1224-5pHMGA2, AQP2, SLC1A2APCHMGA2, AQP2, APC, FGFR1, ADORA1, SATB1, STAT5BRBBP7, APC, CD160, RC3H1, HMGA2, FGFR1, ADORA1, SATB1, STAT5B, NOLC1
hsa-miR-1290HMGA2, NUAK1, TERF2, SLC1A2, FADS1, DDCAPC, COL8A1HMGA2, APC, RRN3, ITGAV, CSE1L, NOTCH1, GAS, BMI1, FOXC1, ROBO1, USP28HMGA2, BMI1, NUAK1, APC, MLL2, RRN3, ITGAV, CSE1L, NOTCH1, GAS, HES1, NPR3, CDC27, COL8A1, CDKN2B, FOXC1, ROBO1, USP28, FIGF, NRAS
hsa-miR-135a-3pTFAP2ATFAP2A, POU3F3, RRP8, PEG3, DYRK2,TFAP2A, POU3F3, DERL2, RERG, COL8A1, CEP120
hsa-miR-28-5pMST4 CNTFR, STK4, BAG1, SON, NR4A3, PAK2CNTFR, STK4, HTR4, FTSJ2, SESN1, TNS3, RAP18, DERL2
hsa-miR-378a-5pPMLDFFA, ITSN1, CTSB, ROBO2, DEPTOR, RAG1, RFFL, IL24, PML, VHL, FRZB, STK4, BAG1, ITGB2FZD3, RAC2, CCND2, FER, PML, VHL, FRZB, STK4, NUDC, PDAP1, ITGAL, PELI1, HNF4A, CD33
hsa-miR-4271HMGA1, AMFR, SLC6A3ALDH1A2, SPN, EIF2AK3, FOXO3, WNT7B, MAPK1, CYLD, MAPT, MEF2D, DAPL1, EP300COL4A3BP, FOXO4, PDGFB, WNT7B, MAPK1, ALDH1A2, CDK2, SPN, MXD1, FOXO3, TGFBR3, CNOT8, MBD2, CD209, CDON, HOXD13,
hsa-miR-452-5pTIMP3SPRY2, PAX3, SOX7, LRP6, SNAI2, CSNK2A2, FGD4, PKN2, ITGA6, PDCD6IPSPRY2, PAX3, SOX7, LRP6, SNAI2, RPA1, EPS8, NFIB, MAPRE1, ODZ1, CDCA7L, CD47, E2F3, PURA,
hsa-miR-572HIP1, CASP10, E2F2, MAP3K1RUNX1, CDC27, ROS1
hsa-miR-575ZBTB16, HIP1, PDPK1, BRAF, CASP10, E2F2, MAP3K1, DNM1LZBTB16, NR3C2, NDEL1, ROS1, BRFOX2, KIF15
hsa-miR-629-3pSOD2, VDR, EDN1, CHEK1, SLC34A2THOC1, MYO18A, TP53BP2, APC, PPARG, PIM1, PSMD2, SOD2, VDR, EDN1, ERBB4, PERP, BCL2L15DLG3, RTKN2, CDK9, STAT3, EPHB1, ACSL6, LIFR, EREG, APC, PPARG, PIM1, STAT6, PDGFC, ZEB1, NOLC1, ID4, SOD2, VDR, EDN1, ERBB4, CDK13, CDC27
hsa-miR-939TIMP1, ATM, CDKN1A, NEK6, SCL34A2, PRELP, SLC1A2NGFR, COL1A1TNF, BCL6, BTC, NRG1, IHH, TIMP1, ATM, WNK3, CLIP3, NEK6, NGFR, MT3, TRAIP, CDKN1A, NACC1, IP6K2, PAX7, CAMK1D, CASP10, USP7, CSNK2A2, THRA, INHBB, BCL2L2BCL6, BTC, NRG1, IHH, GRN, TRAIP, CDKN1A, TNF, E2F8, RXRB, RARA, DRD2, CSF1, TIMP1, ATM, NGFR, MT3, NOS2, AGGF1, ELN
hsa-miR-940

[i] miR, microRNA.

Table IV

Functional annotation chart for miRNAs upregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

Table IV

Functional annotation chart for miRNAs upregulated in response to troxerutin pretreatment in H2O2-exposed human dermal papilla cells.

microRNAPutative target genes (n)KEGG pathwayGenes involved in the term (n)Involved genes/total genes (%)P-value
miR-150-3p184Wnt signaling pathway52.76.00E-02
Neurotrophin signaling pathway42.21.20E-01
Ubiquitin mediated proteolysis42.21.50E-01
Adherens junction31.61.70E-01
miR-181a-2-3p189Endocytosis63.22.90E-02
Chemokine signaling pathway63.23.00E-02
Ubiquitin mediated proteolysis52.63.90E-02
Pancreatic cancer42.13.00E-02
Adherens junction42.13.50E-02
Nucleotide excision repair31.66.40E-02
miR-205-3p944Pathways in cancer192.02.50E-01
MAPK signaling pathway171.81.70E-01
Wnt signaling pathway151.69.20E-03
miR-21-3p210Cell adhesion molecules73.34.70E-03
Ubiquitin mediated proteolysis62.92.30E-02
Long-term potentiation52.48.60E-03
Oocyte meiosis52.44.20E-02
miR-29b-1-5p265Insulin signaling pathway51.98.50E-02
Cell cycle41.52.00E-01
Wnt signaling pathway41.52.90E-01
Jak-STAT signaling pathway41.53.00E-01
mir-3127-5p205
miR-371a-5p351Spliceosome82.34.20E-03
Wnt signaling pathway72.03.60E-02
mir-3663-3p305MAPK signaling pathway123.95.90E-03
Pathways in cancer113.65.50E-02
Neurotrophin signaling pathway72.32.00E-02
Pancreatic cancer51.63.50E-02
Chronic myeloid leukemia51.64.00E-02
mir-4298185Oocyte meiosis52.78.70E-03
Neuroactive ligand receptor interaction52.71.20E-01
Calcium signaling pathway42.21.40E-01
Phosphatidylinositol signaling system31.61.10E-01
miR-602302MAPK signaling pathway72.32.20E-01
Insulin signaling pathway62.05.30E-02
Alzheimer’s disease62.01.00E-01
Calcium signaling pathway62.01.30E-01

[i] miR, microRNA; MAPK, mitogen activated protein kinase; JAK, Janus kinase; STAT, signal transducers and activators of transcription.

Table V

Functional annotation chart for miRNAs downregulated in response to troxerutin in H2O2-exposed HDP cells.

Table V

Functional annotation chart for miRNAs downregulated in response to troxerutin in H2O2-exposed HDP cells.

microRNAPutative target genes (n)KEGG pathwayGenes involved in the term (n)Involved genes/total genes (%)P-value
miR-11812
miR-1202241Pathways in cancer83.31.50E-01
Insulin signaling pathway52.11.10E-01
Phosphatidylinositol signaling system41.77.60E-02
ABC transporters31.21.20E-01
mTOR signaling pathway31.21.50E-01
Inositol phosphate metabolism31.21.60E-01
miR-1224-5p213Axon guidance41.91.00E-01
miR-1290593Pathways in cancer172.94.00E-02
Insulin signaling pathway132.27.60E-04
Regulation of actin cytoskeleton122.06.30E-02
MAPK signaling pathway122.01.90E-01
ErbB signaling pathway111.92.80E-04
miR-135a-3p140
miR-28-5p157MAPK signaling pathway74.51.20E-02
Axon guidance42.56.60E-02
miR-378a-5p366Wnt signaling pathway71.93.60E-02
TGF-β signaling pathway41.11.70E-01
miR-4271361Jak-STAT signaling pathway71.97.80E-02
Lysine degradation41.15.20E-02
miR-452-5p327Oocyte meiosis82.31.30E-03
Wnt signaling pathway72.02.60E-02
ECM-receptor interaction51.43.80E-02
Small cell lung cancer51.43.80E-02
miR-5726
miR-575241MAPK signaling pathway83.37.70E-02
Prostate cancer62.57.70E-03
Melanoma52.11.70E-02
Cell cycle52.19.60E-02
Aldosterone-regulated sodium reabsorption41.71.90E-02
mTOR signaling pathway41.73.50E-02
Androgen and estrogen metabolism31.29.40E-02
miR-629-3p441PPAR signaling pathway61.41.20E-02
miR-939365Calcium signaling pathway102.41.30E-02
Regulation of actin cytoskeleton92.18.90E-02
ErbB signaling pathway51.21.20E-01
p53 signaling pathway40.91.80E-01
Wnt signaling pathway61.42.20E-01
miR-940

[i] miR, microRNA; mTOR, mammalian targets of rapamycin; MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; Jak, Janus kinase; STAT, signal transducers and activators of transcription; TGF, transforming growth factor; PPAR, peroxisome proliferator-activated-receptor.

Discussion

In the present study, the protective effect of troxerutin against H2O2-induced oxidative stress in HDP cells was confirmed using biochemical assays. Notably, pretreatment with troxerutin decreased the cell death, ROS production and cellular senescence, which was mediated by exposure to H2O2. Although the specific signaling pathways involved in the protective effect were not demonstrated, the findings of the present study are important in that they identify troxerutin as a candidate agent for use in the prevention and/or treatment of alopecia. A growing body of evidence suggests the role of oxidative stress in alopecia, and that the prevention of oxidative stress may offer novel strategies for the intervention and reversal of alopecia and even graying of hair (17). A previous case study confirmed increased oxidative stress in alopecia areata patients compared with healthy individuals (29). In addition, a study using a mouse model demonstrated that hair dye-induced hair loss is caused predominantly by H2O2-induced oxidative stress (30). Oxidative stress stimulates the production of a known inhibitor of hair follicles, tumor growth factor-β, in DPC cells, which induces the onset of androgenic alopecia (24). Our previous studies demonstrated that troxerutin has a photoprotective effect against UV radiation on dermal fibroblasts and keratinocytes (6,7), and several clinical and theoretical reports have revealed that UV radiation has negative effects on hair growth through the induction of oxidative stress, acute telogen effluvium and follicular micro-inflammation in follicular stem cells (3133). Therefore, countering oxidative stress can be considered an important strategy to overcome stress-or androgen-dependent alopecia, and the results of the present study confirmed that troxerutin inhibited oxidative stress-induced cellular damage in the DPC cells. In addition, our previous studies and the present studies demonstrated low levels of cytotoxicity of troxerutin on dermal fibroblasts, keratinocytes and DP cells (6,7). Therefore, further investigation of the clinical effect of topical application of troxerutin to the scalp is required.

Using miRNA microarray analysis, the present study identified 24 miRNAs in the HDP cells treated with troxerutin and H2O2, which were differentially expressed compared with the cells treated with H2O2 only. Of these, has-miR-602 was the most markedly upregulated by troxerutin in the H2O2-treated HDP cells (6.91-fold), and has been reported to downregulate the expression of the RASSF1A and TP73 tumor suppressor genes (34). Several reports have revealed that H2O2 induces the expression of TP73 (35) and that the anticancer drug cisplatin, which has been reported to induce alopecia in patients, stimulates ROS-induced apoptosis and functionally upregulates the expression of p73 (3638). Although the cellular functions of miR-602, RASSF1A and TP73 have not been investigated in DP cells, the data of the present study suggested that the interaction between miR-602 and the two genes may be functionally involved in ROS-induced cellular stress and even alopecia-associated mechanisms. The biological functions of has-miR-575, which was the most downregulated miRNA in the results of the present study, have not been reported previously; however, it may regulate H2O2-mediated cellular stress. PDPK1, also termed PDK1, is a putative target of miR-575 (Table III) and a well-known kinase, which phosphorylates and activates Akt1 kinase, induces cell proliferation and protects against H2O2-induced apoptosis (39,40). The present study also classified the biological functions of differentially expressed miRNAs by troxerutin in the H2O2-treated HDP cells. KEGG pathway analysis of the target genes of the upregulated and downregulated miRNAs revealed 18 and 23 pathways, respectively, were statistically enriched. Among these, the WNT and MAPK signaling pathways, which were the most markedly enriched pathways associated with the target genes of the upregulated and downregulated miRNAs, are involved in the regulation of H2O2-mediated cellular stress, including apoptosis and antioxidative mechanisms (4145), suggesting that the miRNAs altered by troxerutin may be involved in protective mechanisms against H2O2-induced damage through the regulation of these pathways.

In conclusion, the present study revealed a novel role of troxerutin as a putative antioxidant agent in HDP cells. In addition, the results revealed 24 differentially expressed miRNAs and determined the putative involvement of 18 signaling pathways associated with upregulated miRNAs and 23 signaling pathways associated with downregulated miRNAs in the troxerutin mediated protective effect against H2O2-induced cell damage. Although further experiments are required to confirm the differentially expressed miRNAs and their target genes, the results of the present study may assist in elucidating the mechanism underlying the troxerutin-mediated protection and miRNA-associated signaling pathways in HDP cells.

Acknowledgments

This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (grant no. HN13C0075). Dr Seunghee Bae was supported by the KU Research Professor Program of Konkuk University.

References

1 

Marzin D, Phi HV, Olivier P and Sauzieres J: Study of mutagenic activity of troxerutin, a flavonoid derivative. Toxicol Lett. 35:297–305. 1987. View Article : Google Scholar : PubMed/NCBI

2 

Boisseau MR, Taccoen A, Garreau C, Vergnes C, Roudaut MF and Garreau-Gomez B: Fibrinolysis and hemorheology in chronic venous insufficiency: a double blind study of troxerutin efficiency. J Cardiovasc Surg (Torino). 36:369–374. 1995.

3 

Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B and Zhang ZF: Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain. 134:783–797. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Lu J, Wu DM, Zheng YL, et al: Troxerutin counteracts domoic acid induced memory deficits in mice by inhibiting CCAAT/enhancer binding protein beta-mediated inflammatory response and oxidative stress. J Immunol. 190:3466–3479. 2013. View Article : Google Scholar : PubMed/NCBI

5 

Babri S, Amani M, Mohaddes G, Alihemmati A and Ebrahimi H: Protective effects of troxerutin on beta-amyloid (1–42)-induced impairments of spatial learning and memory in rats. Neurophysiology. 44:387–393. 2012. View Article : Google Scholar

6 

Lee KS, Cha HJ, Lee GT, et al: Troxerutin induces protective effects against ultraviolet B radiation through the alteration of microRNA expression in human HaCaT keratinocyte cells. Int J Mol Med. 33:934–942. 2014.PubMed/NCBI

7 

Cha HJ, Lee KS, Lee GT, et al: Altered miRNA expression profiles are involved in the protective effects of troxerutin against ultraviolet B radiation in normal human dermal fibroblasts. Int J Mol Med. 33:957–963. 2014.PubMed/NCBI

8 

Ping X, Junqing J, Junfeng J and Enjin J: Radioprotective effects of troxerutin against gamma irradiation in V79 cells and mice. Asian Pac J Cancer Prev. 12:2593–2596. 2011.

9 

Maurya DK, Salvi VP and Krishnan Nair CK: Radioprotection of normal tissues in tumor bearing mice by troxerutin. J Radiat Res. 45:221–228. 2004. View Article : Google Scholar : PubMed/NCBI

10 

Flore R, Gerardino L, Santoliquido A, et al: Enhanced oxidative stress in workers with a standing occupation. Occup Environ Med. 61:548–550. 2004. View Article : Google Scholar : PubMed/NCBI

11 

Hladovec J: Protective effect of oxygen-derived free radical scavengers on the endothelium in vivo. Physiol Bohemoslov. 35:97–103. 1986.PubMed/NCBI

12 

Lu J, Wu DM, Hu B, Zheng YL, Zhang ZF and Wang YJ: NGF-Dependent activation of TrkA pathway: A mechanism for the neuroprotective effect of troxerutin in D-galactose-treated mice. Brain Pathol. 20:952–965. 2010.PubMed/NCBI

13 

Heck DE, Vetrano AM, Mariano TM and Laskin JD: UVB light stimulates production of reactive oxygen species: unexpected role for catalase. J Biol Chem. 278:22432–22436. 2003. View Article : Google Scholar : PubMed/NCBI

14 

Leach JK, Van Tuyle G, Lin PS, Schmidt-Ullrich R and Mikkelsen RB: Ionizing radiation induced, mitochondria-dependent generation of reactive oxygen/nitrogen. Cancer Res. 61:3894–3901. 2001.PubMed/NCBI

15 

Driskell RR, Clavel C, Rendl M and Watt FM: Hair follicle dermal papilla cells at a glance. J Cell Sci. 124:1179–1182. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Hibberts NA, Howell AE and Randall VA: Balding hair follicle dermal papilla cells contain higher levels of androgen receptors than those from non balding scalp. J Endocrinol. 156:59–65. 1998. View Article : Google Scholar : PubMed/NCBI

17 

Trüeb RM: Oxidative stress in ageing of hair. Int J Trichology. 1:6–14. 2009. View Article : Google Scholar : PubMed/NCBI

18 

Ripple MO, Henry WF, Rago RP and Wilding G: Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells. J Natl Cancer Inst. 89:40–48. 1997. View Article : Google Scholar : PubMed/NCBI

19 

Naito A, Midorikawa T, Yoshino T and Ohdera M: Lipid peroxides induce early onset of catagen phase in murine hair cycles. Int J Mol Med. 22:725–729. 2008.PubMed/NCBI

20 

Bahta AW, Farjo N, Farjo B and Philpott MP: Premature senescence of balding dermal papilla cells in vitro is associated with p16 (INK4a) expression. J Invest Dermatol. 128:1088–1094. 2008. View Article : Google Scholar

21 

Lee EJ, Cha HJ, Ahn KJ, An IS, An S and Bae S: Oridonin exerts protective effects against hydrogen peroxideinduced damage by altering microRNA expression profiles in human dermal fibroblasts. Int J Mol Med. 32:1345–1354. 2013.PubMed/NCBI

22 

Huang DW, Sherman BT, Tan Q, et al: The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol. 8:R1832007. View Article : Google Scholar : PubMed/NCBI

23 

Luanpitpong S, Nimmannit U, Chanvorachote P, et al: Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism. Apoptosis. 16:769–782. 2011. View Article : Google Scholar : PubMed/NCBI

24 

Shin H, Yoo HG, Inui S, et al: Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells. BMB Rep. 46:460–464. 2013. View Article : Google Scholar : PubMed/NCBI

25 

Colavitti R and Finkel T: Reactive oxygen species as mediators of cellular senescence. IUBMB Life. 57:277–281. 2005. View Article : Google Scholar : PubMed/NCBI

26 

Baehrecke EH: miRNAs: micro managers of programmed cell death. Curr Biol. 13:R473–R475. 2003. View Article : Google Scholar : PubMed/NCBI

27 

Wang Z, Liu Y, Han N, et al: Profiles of oxidative stress-related microRNA and mRNA expression in auditory cells. Brain Res. 1346:14–25. 2010. View Article : Google Scholar : PubMed/NCBI

28 

Faraonio R, Salerno P, Passaro F, et al: A set of miRNAs participates in the cellular senescence program in human diploid fibroblasts. Cell Death Differ. 19:713–721. 2012. View Article : Google Scholar :

29 

Bakry OA, Elshazly RM, Shoeib MA and Gooda A: Oxidative stress in alopecia areata: a case-control study. Am J Clin Dermatol. 15:57–64. 2014. View Article : Google Scholar

30 

Seo JA, Bae IH, Jang WH, et al: Hydrogen peroxide and monoethanolamine are the key causative ingredients for hair dye-induced dermatitis and hair loss. J Dermatol Sci. 66:12–19. 2012. View Article : Google Scholar : PubMed/NCBI

31 

Trüeb RM: Is androgenetic alopecia a photoaggravated dermatosis? Dermatology. 207:343–348. 2003. View Article : Google Scholar : PubMed/NCBI

32 

Camacho F, Moreno JC and García-Hernández MJ: Telogen alopecia from UV rays. Arch Dermatol. 132:1398–1399. 1996. View Article : Google Scholar : PubMed/NCBI

33 

Johnsson A, Kjeldstad B and Melø TB: Fluorescence from pilosebaceous follicles. Arch Dermatol Res. 279:190–193. 1987. View Article : Google Scholar : PubMed/NCBI

34 

Yang L, Ma Z, Wang D, Zhao W, Chen L and Wang G: MicroRNA-602 regulating tumor suppressive gene RASSF1A is overexpressed in hepatitis B virus-infected liver and hepatocellular carcinoma. Cancer Biol Ther. 9:803–808. 2010. View Article : Google Scholar : PubMed/NCBI

35 

Singh M, Sharma H and Singh N: Hydrogen peroxide induces. apoptosis in HeLa cells through mitochondrial pathway. Mitochondrion. 7:367–373. 2007. View Article : Google Scholar : PubMed/NCBI

36 

Surendiran A, Balamurugan N, Gunaseelan K, Akhtar S, Reddy KS and Adithan C: Adverse drug reaction profile of cisplatin-based chemotherapy regimen in a tertiary care hospital in India: An evaluative study. Indian J Pharmacol. 42:40–43. 2010. View Article : Google Scholar : PubMed/NCBI

37 

Casares C, Ramírez-Camacho R, Trinidad A, Roldán A, Jorge E and García-Berrocal JR: Reactive oxygen species in apoptosis induced by cisplatin: review of physiopathological mechanisms in animal models. Eur Arch Otorhinolaryngol. 269:2455–2459. 2012. View Article : Google Scholar : PubMed/NCBI

38 

Gong JG, Costanzo A, Yang HQ, et al: The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature. 399:806–809. 1999. View Article : Google Scholar : PubMed/NCBI

39 

Wang Z, Cui M, Sun L, et al: Angiopoietin-1 protects H9c2 cells from H2O2-induced apoptosis through AKT signaling. Biochem Biophys Res Commun. 359:685–690. 2007. View Article : Google Scholar : PubMed/NCBI

40 

Mora A, Komander D, van Aalten DM and Alessi DR: PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol. 15:161–170. 2004. View Article : Google Scholar : PubMed/NCBI

41 

Finkel T: Signal transduction by reactive oxygen species. J Cell Biol. 194:7–15. 2011. View Article : Google Scholar : PubMed/NCBI

42 

Shin SY, Kim CG, Jho EH, et al: Hydrogen peroxide negatively modulates Wnt signaling through downregulation of beta-catenin. Cancer Lett. 212:225–231. 2004. View Article : Google Scholar : PubMed/NCBI

43 

Korswagen HC: Regulation of the Wnt/beta-catenin pathway by redox signaling. Dev Cell. 10:687–688. 2006. View Article : Google Scholar : PubMed/NCBI

44 

Song X, Xu A, Pan W, et al: Minocycline protects melanocytes against H2O2-induced cell death via JNK and p38 MAPK pathways. Int J Mol Med. 22:9–16. 2008.PubMed/NCBI

45 

Ismail NS, Pravda EA, Li D, Shih SC and Dallabrida SM: Angiopoietin-1 reduces H2O2-induced increases in reactive oxygen species and oxidative damage to skin cells. J Invest Dermatol. 130:1307–1317. 2010. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

August-2015
Volume 12 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Lim KM, An S, Lee OK, Lee MJ, Lee JP, Lee KS, Lee GT, Lee KK and Bae S: Analysis of changes in microRNA expression profiles in response to the troxerutin-mediated antioxidant effect in human dermal papilla cells. Mol Med Rep 12: 2650-2660, 2015
APA
Lim, K.M., An, S., Lee, O., Lee, M.J., Lee, J.P., Lee, K.S. ... Bae, S. (2015). Analysis of changes in microRNA expression profiles in response to the troxerutin-mediated antioxidant effect in human dermal papilla cells. Molecular Medicine Reports, 12, 2650-2660. https://doi.org/10.3892/mmr.2015.3717
MLA
Lim, K. M., An, S., Lee, O., Lee, M. J., Lee, J. P., Lee, K. S., Lee, G. T., Lee, K. K., Bae, S."Analysis of changes in microRNA expression profiles in response to the troxerutin-mediated antioxidant effect in human dermal papilla cells". Molecular Medicine Reports 12.2 (2015): 2650-2660.
Chicago
Lim, K. M., An, S., Lee, O., Lee, M. J., Lee, J. P., Lee, K. S., Lee, G. T., Lee, K. K., Bae, S."Analysis of changes in microRNA expression profiles in response to the troxerutin-mediated antioxidant effect in human dermal papilla cells". Molecular Medicine Reports 12, no. 2 (2015): 2650-2660. https://doi.org/10.3892/mmr.2015.3717