Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone

  • Authors:
    • Myung Joo Lee
    • Hwa Jun Cha
    • Kyung Mi Lim
    • Ok‑Kyu Lee
    • Seunghee Bae
    • Chun‑Ho Kim
    • Kee‑Ho Lee
    • Yu Na Lee
    • Kyu Joong Ahn
    • Sungkwan An
  • View Affiliations

  • Published online on: March 12, 2015     https://doi.org/10.3892/mmr.2015.3478
  • Pages: 1205-1212
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Abstract

Clinical evidence has demonstrated that the accumulation of 5α‑dihydrotestosterone (DHT) in dermal papilla cells (DPCs) is implicated in androgenetic alopecia. Whether this accumulation in DHT may have direct cellular effects leading to androgenetic alopecia remains to be elucidated. The present study aimed to determine whether DHT affects cell growth, cell cycle arrest, cell death, senescence and the induction of reactive oxygen species (ROS), and whether these effects are mediated by microRNA (miRNA)‑dependent mechanisms. The cell viability and cell cycle were determined, levels of ROS were examined and senescence‑associated β‑galactosidase assays were performed in normal human DPCs (nHDPCs). Furthermore, miRNA expression profiling was performed using an miRNA microarray to determine whether changes in the expression levels of miRNA were associated with the cellular effects of DHT. The results revealed that DHT decreased cell growth by inducing cell death and G2 cell cycle arrest, and by increasing the production of ROS and senescence in the nHDPCs. In addition, 55 miRNAs were upregulated and 6 miRNAs were downregulated inthe DHT‑treated nHDPCs. Bioinformatic analysis demonstrated that the putative target genes of these upregulated and downregulated miRNAs were involved in cell growth, cell cycle arrest, cell death, senescence and the production of ROS. Specifically, the target genes of five highly upregulated and downregulated miRNAs were identified and were associated with the aforementioned effects of DHT. These results demonstrated that the expression of miRNA was altered in the DHT‑treated nHDPCs and suggest the potential mechanisms of DHT‑induced cell growth repression, cell cycle arrest, cell death, senescence and induction of ROS.

Introduction

The 5α-dihydrotestosterone (DHT) androgen is produced primarily by 5α-reductase in the testes (1). DHT regulates male reproductive development, testes formation, growth of skeletal muscle and hair growth, through activation of the androgen receptor (2). The affinity of DHT is 10-fold greater than that of teststerone for the androgen receptor, and leads to its hyperactivation, which induces shortening of the anagen phase of hair follicle growth (35).

The hair growth cycle is modulated predominantly by dermal papilla cells (DPCs), which are mesenchymal cells located at the base of hair follicles, regulating formation of the hair follicle and hair growth cycle through secretion of growth factors and cytokines (611). Previous studies have demonstrated that DHT inhibits protein kinase C, regulates of the expression of B-cell lymphoma 2 (bcl-2)/blc-2-associated x protein (bax), and upregulates the expression of dickkopf 1 in the DPCs, leading to cell apoptosis, shortening of the hair cycle, a reduction in hair growth, and hair loss (1214).

MicroRNAs (miRNAs) are a class of small (~22 nt) noncoding RNAs, which bind to mRNAs in a sequence-specific manner to regulate the translation of target genes (15,16). miRNAs are important in development, apoptosis and cell growth (17). Various studies have been performed to investigate the role of miRNAs in dermal papilla cells from the balding and non balding scalp (14). In addition, investigations using mice, in which Dicer, a key enzyme of miRNA metabolism, has been knocked out, have revealed that miRNAs are essential for the morphogenesis and maintenance of hair follicles (18).

However, although DHT is well known as a key regulator of balding and hair follicle morphogenesis, DHT-dependent alterations of the miRNA expression profile and putative mechanisms remain to be elucidated. The present study investigated the cellular effects of DHT and the miRNA expression prolife in normal human DPCs (nHDPCs).

Materials and methods

Cells and culture conditions

The nHDPCs were purchased from Innoprot (Biscay, Spain) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2.

Cell viability assay

The viability of the nHDPCs was measured using a water-soluble tetrazolium salt (WST-1) assay (EZ-Cytox Cell Viability Assay kit; Itsbio, Seoul, Korea). For the cell viability assay, the nHDPCs were plated at a density of 5×103 cells/well in 96-well plates. After 24 h, the cells were treated with doses of DHT between 0 and 1 mM at 37°C for 24, 48, or 72 h. The cells were then incubated with WST-1 reagent at 37°C for 30 min, and the optical density was determined at 450 nm using a microplate reader (iMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell cycle assay

A propidium iodide (PI) staining based cell cycle assay was performed using standard procedures, as described previously (10). The nHDPCs (2×106) were plated in 60 mm culture dishes and treated with DHT for 24 h. The cells were then trypsinized with 0.25% Trypsin-EDTA (Gibco Life Technologies) at 37°C, pelleted, washed with phosphate-buffered saline (PBS), and fixed with 70% ethanol at 4°C for 3 h. The DNA in the fixed cells was stained using staining solution containing 50 μg/ml PI (Sigma-Aldrich), 0.5% Triton X-100 (Bioshop, Burlington, ON, Canada), and 100 μg/ml RNase (Bioshop) at 37°C for 1 h. Following staining, the cells were analyzed using a FL2 channel with an excitation wavelength of 488 nm and an emission wavelength of 578 nm, on a FACSCaliber flow cytometer (BD Biosciences, San Jose, CA, USA).

Reactive oxygen species (ROS) measurement

The measurement of ROS was performed, as previously reported, using 2′,7′-dichlorofluorescein diacetate (DCF-DA) (19). The nHDPCs (2×106) were plated in 60 mm culture dishes and treated with DHT at 37°C for 24 h. 2′, 7′-Dichlorodihydrofluorescin diacetate (DCF-DA; 20 μM) was added to the culture medium, and the cells were incubated at 37°C for 1 h. The cells were then trypsinized with 0.25% Trypsin-EDTA at 37°C, pelleted, washed with PBS, and analyzed using a FL1 channel with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACSCaliber flow cytometer (BD Biosciences).

Senescence-associated β-galactosidase (SA-β-gal) assay

For the detection of senescent cells, an SA-β-gal assay was performed, as previously described (20). Briefly, the nHDPCs (2×106) were plated in 60 mm culture dishes and treated with DHT at 37°C for 24 h. The cells were then fixed with Fixative Solution (Senescence Detection kit; Biovision, Milpitas, CA, USA) and stained using a Staining Solution mix (Senescence Detection kit) supplemented with X-gal at 37°C for 24 h. Images of the SA-β-gal stained cells were captured using a camera mounted to a light microscope (CKX41; Olympus Corporation, Tokyo, Japan), and the number of stained cells were counted in five randomly selected microscopic fields from each condition.

miRNA microarray

The RNA in the cells was isolated using TRIzol reagent (Gibco Life Technologies), according to the manufacturer’s instructions. The RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the RNA quality was evaluated using spectrophotometry at the 260/280 nm ratio (Ultrospec 2100 Pro UV-Vis; Amersham Biosciences, GE Healthcare Life Sciences, Piscataway, NJ, USA). Samples with an RNA integrity score >7.8 and an RNA quality score >2.0 were used for the microarray. A total of 100 ng RNA was labeled with cyanine dye (Cy3) using an Agilent miRNA labeling kit (Agilent Technologies). The labeled RNAs were purified using Micro Bio-Spin P-6 columns (Bio-Rad Laboratories, Inc.) and hybridized using a SurePrint G3 Human v16 miRNA Microarray kit (8×60 K; Release 16.0; Agilent Technologies) at 65°C for 20 h. The microarray was scanned using an Agilent microarray scanner (Agilent Technologies), and the images were analyzed using Agilent Feature Extraction version 10.7 software (Agilent Technologies). The digitized data were analyzed and the fold change was determined using GeneSpring GX version 11.5 software (Agilent Technologies).

miRNA target gene prediction and biological function analysis

The putative target genes of significant miRNAs were identified using the probability of interaction by target accessibility (PITA; http://genie.weizmann.ac.il), microRNAorg (http://www.microrna.org) and TargetScan (http://www.targetscan.org) target prediction systems. The Gene Ontologies (GOs) of the putative target genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resource 6.7 (http://david.abcc.ncifcrf.gov).

Statistical analysis

The data are presented as the mean ± standard deviation. Statistical significance was calculated using Student’s two-tailed t-test. Statistical analyses were conducted using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). P<0.01 was considered to indicate a statistically significant difference, unless otherwise indicated.

Results

DHT induced cytotoxicity in nHDPCs

To determine whether DHT was associated with cell viability in nHDPCs, the present study analyzed the viability of DHT-treated nHDPCs after 24, 48, and 72 h using a WST-1 assay. Low concentrations of DHT (<0.1 mM) demonstrated no significant toxicity in the nHDPCs at any of the time-points assessed. However, as shown in Fig. 1, cytotoxicity was significantly increased by 1 mM DHT in the nHDPCs at every time-point assessed. Thus, it was determined that 1 mM DHT-induced cytotoxicity in the nHDPCs following exposure for ≥24 h, which led to an exposure duration of 24 h being selected for use in the subsequent experiments.

DHT induces cell death and cell cycle arrest in nHDPCs

Previous experiments established that high levels of DHT induce apoptosis (14,21). In agreement with the previous experiments (Fig. 2), the present study demonstrated that 1 mM DHT increased cell death between 3.36 and 15.62% in the nHDPCs. In addition, the G1/G2 ratio was significantly increased by concentrations of DHT >10−6 M, in a dose-dependent manner. The DHT-induced increment in G1/G2 ratio indicated that DHT-induced G2 cell cycle arrest. Therefore, high-doses of DHT reduced cell viability through induction of cell death and G2 cell cycle arrest in the nHDPCs.

DHT increases ROS levels in nHDPCs

DHT can induce ROS in prostate cancer cell lines, which express the androgen receptor at a high level (14,2224). Additionally, ROS are a key inducer of retinoblastoma-mediated senescence (25). As nHPDCs also express androgens at a high level (26), the present study investigated whether 1 mM DHT-induced ROS in these cells. The levels of ROS were determined using DCF-DA staining in untreated nHDPCs and in 1 mM DHT-treated nHDPCs. As shown Fig. 3A, DHT significantly increased the level of ROS in the nHDPCs. In addition, the cellular effect underlying the effect of 1 mM DHT in enhancing ROS levels in the nHDPCs was investigated. As shown in previous experiments in a prostate cell line (23), accumulated ROS induced senescence in the nHDPCs, as assessed by SA-β-gal activity (Fig. 3B).

As DHT induced growth arrest, cell death, cell cycle arrest, ROS production and senescence, comparative microarray analysis of miRNAs was performed to identify the miRNA signatures in the DHT-treated nHDPCs. Total RNA was extracted from the untreated nHDPCs and nHDPCs treated with 1 mM DHT for 24 h. The total RNA was labeled with Cy3 and hybridized to microarray-containing probes for 1,205 annotated miRNAs. The untreated cells were then compared with the 1 mM DHT-treated nHDPCs, in which 55 miRNAs that were upregulated and 6 were downregulated, by more than two-fold (Table I). Among the five miRNAs significantly upregulated in the DHT-treated nHDPCs, the level of miR-3663-3p increased by 219.04-fold, miR-485-3p by 200.81-fold, miR-7 by 173.64-fold, miR-125a-3p by 154.55-fold, and miR-4271-by 108-fold. In addition, in the five miRNAs, which were significantly downregulated in the DHT-treated nHDPCs, the level of miR-450a decreased by 95.69-fold, miR-1181 by 93.76-fold, miR-3656 by 2.84-fold, miR-4286 by 2.29-fold and miR-370 by 2.24-fold.

Table I

miRNAs exhibiting a ≥2-fold change in expression following treatment of the nHDPCs with DHT.

Table I

miRNAs exhibiting a ≥2-fold change in expression following treatment of the nHDPCs with DHT.

miRNAFold changeDirection of changeChromosome
Has-let-7a*28.48Up9
hsa-miR-1181−93.76Down19
hsa-miR-1207-5p5.23Up8
hsa-miR-1225-5p3.08Up16
hsa-miR-12462.49Up2
hsa-miR-124935.73Up22
hsa-miR-125a-3p154.55Up19
hsa-miR-12682.38Up15
hsa-miR-12835.55Up2
hsa-miR-12902.06Up1
hsa-miR-13240.88Up17
hsa-miR-13490.23Up14
hsa-miR-135a*45.73Up3
hsa-miR-138 2*52.92Up16
hsa-miR-146a−2.01Down5
hsa-miR-148b50.96Up12
hsa-miR-150*98.34Up19
hsa-miR-153940.93Up18
hsa-miR-154*−2.24Down14
hsa-miR-17*60.48Up13
hsa-miR-19153.07Up10
hsa-miR-19784.06Up1
hsa-miR-19732.20Up4
hsa-miR-20235.08Up10
hsa-miR-28 5p36.51Up3
hsa-miR-324-5p36.40Up17
hsa-miR-3613-3p78.20Up13
hsa-miR-364650.21Up20
hsa-miR-36512.98Up9
hsa-miR-3656−2.84Down11
hsa-miR-3663-3p219.04Up10
hsa-miR-369-3p36.36Up14
hsa-miR-37040.16Up14
hsa-miR-371-5p78.83Up19
hsa-miR-37848.73Up5
hsa-miR-409-5p44.22Up14
hsa-miR-423-5p49.28Up17
hsa-miR-427036.98Up3
hsa-miR-4271108.00Up3
hsa-miR-42812.81Up5
hsa-miR-4286−2.29Down8
hsa-miR-429153.23Up9
hsa-miR-42992.14Up11
hsa-miR-43135.70Up14
hsa-miR-431*25.62Up14
hsa-miR-431738.79Up18
hsa-miR-432734.79Up21
hsa-miR-450a−95.69DownX
hsa-miR-483-5p39.06Up11
hsa-miR-485-3p200.81Up14
hsa-miR-500a2.30UpX
hsa-miR-513a-5p2.68UpX
hsa-miR-513b47.72UpX
hsa-miR-550a18.95Up7
hsa-miR-57239.29Up4
hsa-miR-6303.53Up15
hsa-miR-642b105.36Up19
hsa-miR-7173.64Up9
hsa-miR-7625.26Up16
hsa-miR-770-5p56.99Up14
hsa-miR-87462.43Up5

[i] The direction of change is relative to the control. miRNA/miR, microRNA; Up, upregulated; Down, downregulated.

Subsequently, the putative target genes of DHT-regulated miRNAs were identified using the PITA, microRNAorg and Targetscan target prediction systems (Table II). A total of 587 putative target genes of the upregulated miRNAs and 140 putative target genes of the downregulated miRNAs were identified in PITA. Using microRNAorg, 488 putative target genes of upregulated miRNAs and 312 putative target genes of downregulated miRNAs were found, and 691 putative target genes of upregulated miRNAs and 219 putative target genes of down regulated miRNAs were identified using Targetscan. Of these, 339 were overlapping target genes of upregulated miRNAs and 111 were overlapping target genes of downregulated miRNAs in all three target prediction systems.

Table II

Number of significant miRNA targets using three prediction databases.

Table II

Number of significant miRNA targets using three prediction databases.

DatabaseTarget miRNAs (n)Overlapping miRNAs in all three databases (n)
Upregulated target miRNAs339
 Targetscan691
 PITA587
 microRNAorg488
Downregulated target miRNAs111
 Targetscan219
 PITA140
 microRNAorg312

[i] miRNA, microRNA; PITA, probability of interaction by target accessibility.

To investigate a association between the aforementioned effects of DHT and the putative miRNA target genes, GO analysis of each putative target gene was performed using DAVID. The genes were classified according to GO terms associated with the five effects of DHT and the number of putative target genes associated with each GO term were counted. As shown in Table III, the putative target genes of the uppregulated and downregulated miRNAs were associated with five antioxidant-associated GO terms, 17 apoptosis and cell death-associated terms, 11 proliferation and cell growth-associated terms, 1 age associated term and 14 cell cycle-associated GO terms. The miRNAs and their putative target genes are shown in Table IV. Overall, these results demonstrated that DHT exerted negative effects, which were associated with an alteration in cellular miRNA expression profiles.

Table III

Genes grouped according to the GO terms, associated with the effects of 5α-dihydrotestosterone.

Table III

Genes grouped according to the GO terms, associated with the effects of 5α-dihydrotestosterone.

A, Antioxidant-associated genes

Accession No.GO termUpregulated (n)Downregulated (n)
GO:0006733Oxidoreduction coenzyme metabolic process30
GO:0006979Response to oxidative stress52
GO:0042542Response to hydrogen peroxide20
GO:0015980Energy derivation by oxidation of organic compounds03
GO:0055114Oxidation reduction66
B, Apoptosis and cell death-associated genes

Accession No.GO termUpregulated (n)Downregulated (n)
GO:0006916Anti-apoptosis74
GO:0008624Induction of apoptosis by extracellular signals40
GO:0042981Regulation of apoptosis177
GO:0043066Negative regulation of apoptosis84
GO:0043065Positive regulation of apoptosis82
GO:0006917Induction of apoptosis60
GO:0006915Apoptosis95
GO:0043067Regulation of programmed cell death184
GO:0010941Regulation of cell death184
GO:0043069Negative regulation of programmed cell death90
GO:0060548Negative regulation of cell death90
GO:0043068Positive regulation of programmed cell death82
GO:0010942Positive regulation of cell death82
GO:0012502Induction of programmed cell death60
GO:0008219Cell death116
GO:0016265Death116
GO:0012501Programmed cell death95
C, Proliferation and cell growth-associated genes

Accession No.GO termUpregulated (n)Downregulated (n)
GO:0008283Cell proliferation93
GO:0008284Positive regulation of cell proliferation85
GO:0042127Regulation of cell proliferation127
GO:0008285Negative regulation of cell proliferation43
GO:0030308Negative regulation of cell growth30
GO:0040008Regulation of growth74
GO:0048638Regulation of developmental growth20
GO:0045926Negative regulation of growth30
GO:0001558Regulation of cell growth42
GO:0045927Positive regulation of growth2
GO:0040007Growth30
D, Aging-associated genes

Accession No.GO termUpregulated (n)Downregulated (n)
GO:0007568Aging30
E, Cell cycle-associated genes

Accession No.GO termUpregulated (n)Downregulated (n)
GO:0051726Regulation of cell cycle130
GO:0045786Negative regulation of cell cycle50
GO:0051327M phase of meiotic cell cycle40
GO:0051321Meiotic cell cycle42
GO:0045930Negative regulation of mitotic cell cycle20
GO:0010948Negative regulation of cell cycle process20
GO:0007346Regulation of mitotic cell cycle40
GO:0022403Cell cycle phase82
GO:0010564Regulation of cell cycle process30
GO:0007049Cell cycle144
GO:0022402Cell cycle process94
GO:0000278Mitotic cell cycle60
GO:0000075Cell cycle checkpoint20
GO:0000087M phase of mitotic cell cycle30

[i] GO, Gene Ontology.

Table IV

Target genes of significantly regulated miRNAs in DHT-treated nHDPCs.

Table IV

Target genes of significantly regulated miRNAs in DHT-treated nHDPCs.

A, Targets of up regulated miRNAs
miRNAAntioxidantApoptosis and cell deathProliferation and cell growthAgingCell cycle
a3663-3pGAPDHS, NDUFA8, GAPDH, DEGS2, DCXRCARD9, ADAFOXS1, ENO1
a485-3pAPOA4, PRDX1, NDUFAB1, NQO2GNRH1, PRDX1
a7CYP11A1, UCP2, NQO2 NEIL1, BCKDHA, FADS3, ALKBH2DAPL1, CASP12, DDX41, DAPK3, BCL2L12, CRYAA, CSTB, INHABMP10, LBX1, INHA, IL34, CKLF, SLC3A2, ENO3, BDKRB1, OGFR,RNF167, INHA, CDC37, CRYAA
a125-3pBCKDHA, NDUFS7, FTMT, PLOD3, TH, COX6B1, HGD, AKR1C1PYCARD, LGALS12, TGFB1, LRDD, GML, ADABDKRB1, SCGB3A1, NPPA, TGFB1, ENO1, E4F1, FTMT, AGER, ADA, FGF6, PRG4, GMLAGER, ADA, TGFB1TUBB2A, SPAG5, PKMYT1, CDC20, TGFB1, E4F1, GML, CDK5RAP3 PARD6A, GPS2
a4271BCKDHA, NDUFS7, NDUFB11, NDUFB10, HAO2, NDUFS8, FADS3, FDX1L, ALOX12B, IL4I1, NSDHLGZMM, DAPL1, LRDD, ATP2A1, MGC29506SSTR4, PRTN3, GHRH, ILK, PYY, PRSS2, BARHL2, OGFR, ENO1BGLAP, PKMYT1, ILK
B, Targets of down-regulated miRNAs
miRNAAntioxidantApoptosis and cell deathProliferation and cell growthAgingCell cycle
a450aUQCRH, ALKBH2
a1181
a3656CARD9, INS, TMEM102, SFN, ATP2A1INS, SFN, SCGB3A1, VGFINS, SFN
a428NDUFB11, NMRAL1, FDX1LAARS, MUC5AC, DAPK3, CDK5, TGFB1, PROC, MIF, LRDD, TBRG4FANCG, CDK5, SERTAD1, TGFB1, TBRG4, PARD6A
a154IFIH1, CASP12, PF4, PRDX1VTI1B, PRDX1, RARRES3, GNL3

[i] ahsa miR. miR/miRNA, microRNA.

Discussion

The results of the present study provided evidence that DHT-induced growth arrest, cell death, cell cycle arrest, ROS production and senescence in nHDPCs. In the hair follicle, DHT is produced by 5α-reductase and it accumulates, which induces androgenetic alopecia through DHT-mediated cell death and decreased growth rate (2729). As shown in Figs. 1 and 2, 1 mM DHT repressed cell growth by inducing cell cycle arrest and cell death. In a previous report, activation of the androgen receptor provoked ROS-mediated senescence (30,31). As shown in Fig. 3, measurement of ROS revealed that 1 mM DHT significantly elevated the levels of ROS in the nHDPCs. In the nHDPCs, which exhibited increased activity of the androgen receptor by DHT, 1 mM DHT significantly increased the percentage of senescent cells (Fig. 3B). Specifically, an association was observed between the effects of DHT and the regulation of miRNAs by DHT. Using miRNA microarray analysis, 61 miRNAs (55 upregulated and 6 downregulated) were identified, in which the miRNA levels were increased of decreased by more than two-fold by DHT in the nHDPCs (Table I). One of these, miRNA-125a-3p has been demonstrated as a repressor of cell proliferation and migration through targeting Fyn (32). In addition, miR-485-5p (39.06-fold increase) inhibits cell growth and migration in breast cancer cell lines (33), whereas miRNA-7 regulates the mammalian target of rapamycin and phosphoinositide 3-kinase/Akt pathways, and targets Bcl-2, X-linked inhibitor of apoptosis protein and ETS2 repressor factor, which affect cell growth and the repression of intrinsic apoptosis (3439). Furthermore, the present study predicted the target genes of DHT-regulated miRNAs and performed GO analysis of potential target genes using the DAVID bioinformatics resources. A correlation was found between DHT-induced alterations in miRNA expression profiles and DHT-induced cellular effects, by grouping the target genes, according to GO terms, with five biological processes, which impacted in DHT-treated cells (Tables II and III). The results revealed that the DHT-induced alteration of the miRNA profile was associated with the aforementioned cellular effects of DHT, of induced cell growth, cell cycle arrest, cell death, ROS induction and senescence.

In conclusion, the present study demonstrated that DHT-induced growth arrest, cell death, cell cycle arrest, ROS production and senescence by upregulating and downregulating the expression of DHT-specific miRNAs in nHDPCs. These findings support the hypothesis that miRNA regulation is involved in DHT-induced androgenetic alopecia.

Acknowledgments

This study was supported by Konkuk University in 2013.

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July 2015
Volume 12 Issue 1

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Online ISSN:1791-3004

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APA
Lee, M.J., Cha, H.J., Lim, K.M., Lee, O., Bae, S., Kim, C. ... An, S. (2015). Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone. Molecular Medicine Reports, 12, 1205-1212. https://doi.org/10.3892/mmr.2015.3478
MLA
Lee, M. J., Cha, H. J., Lim, K. M., Lee, O., Bae, S., Kim, C., Lee, K., Lee, Y. N., Ahn, K. J., An, S."Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone". Molecular Medicine Reports 12.1 (2015): 1205-1212.
Chicago
Lee, M. J., Cha, H. J., Lim, K. M., Lee, O., Bae, S., Kim, C., Lee, K., Lee, Y. N., Ahn, K. J., An, S."Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone". Molecular Medicine Reports 12, no. 1 (2015): 1205-1212. https://doi.org/10.3892/mmr.2015.3478