Eur J Dermatol 2013; 23(6): 807-11
Investigative report
Copyright © 2017 John Libbey Eurotext. Téléchargé par BIBLIOTHEQUE DIVISION DES ACQUISITIONS UNIVERSITE LAVAL le 07/06/2017.
Hiroki HIRAO Masatoshi JINNIN Asako ICHIHARA Akihiko FUJISAWA Katsunari MAKINO Ikko KAJIHARA Keisuke SAKAI Satoshi FUKUSHIMA Yuji INOUE Hironobu IHN Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan
Reprints: M. Jinnin
Article accepted on 8/20/2013
doi:10.1684/ejd.2013.2190
P
Detection of hair root miR-19a as a novel diagnostic marker for psoriasis Background: Objective biomarkers that reflect the diagnosis and disease activity have not been in clinical use for psoriasis. Objectives: We investigated the hair root miR-19a levels, regulatory microRNA of TNF-␣, and evaluated the possibility that miR-19a can be a biomarker of psoriasis. Materials and methods: microRNAs were extracted from hair roots of patients with psoriasis (n = 18) and healthy controls (n = 22). Samples from 10 atopic dermatitis patients and 4 dermatomyositis patients were also included as the disease controls. miR-19a levels were determined by quantitative real-time PCR. Results: Hair root levels of miR-19a were significantly up-regulated only in psoriasis compared with normal controls. In characteristics (ROC) curve analysis for hair root miR19a, to distinguish psoriasis patients from normal subjects, the areas under curve (AUC) was 0.87. Relative miR-19a levels were inversely and significantly correlated with duration between symptom onset and the first visit to the hospital in psoriasis patients. Conclusions: Our results indicated hair root miR-19a levels are effective as a disease marker. Key words: microRNA, psoriasis, hair root
soriasis is one of the chronic inflammatory skin diseases characterized by erythemas or papules with silver-white scales. Abnormal proliferation and differentiation of keratinocytes as well as infiltration of lymphocytes and neutrophils are responsible for the skin lesion, histopathologically. Cytokines, including tumor necrosis factor (TNF)-␣ or interleukin (IL)-17, have been proved to contribute to the inflammation of psoriasis: expression of these cytokines is known to be increased in lesional psoriatic skin [1-7] and monoclonal antibodies against these cytokines (e.g. infliximab, etanercept, or ustekinumab) improve the disease dramatically [8]. However, their serum levels do not reflect the disease activity and disease markers for psoriasis have not been used clinically. We focused on the possibility that miRNAs in serum are useful as diagnosis markers for various diseases. miRNAs, non-coding short ribonucleic acid molecules, an average of 22 nucleotides long, are post-transcriptional regulators that bind to complementary sequences in the 3’ UTRs of target mRNAs, leading to the inhibition of translation [9]. They play important roles in diverse cellular processes, including cell development or proliferation, and are thought to be involved in the pathogenesis of various human diseases. miRNAs are also present in human serum in a remarkably stable form, which is protected from endogenous RNase activity by microvesicles, such as exosomes or shedding vesicles [10]. Previously, we determined the serum levels of miR-19a, the regulatory miRNA of TNF-␣ [11], in psoriasis patients and control subjects but we could not find a statistically significant difference [12].
EJD, vol. 23, n◦ 6, November-December 2013
Recently, we confirmed the presence of miRNAs in hair roots [13] and showed that hair miRNA levels as well as serum miRNA levels can be novel and independent biomarkers. Thus, in this report, we determined the possibility that we can use hair root miR-19a levels as disease markers of psoriasis.
Materials and methods Clinical assessment and patient material A single hair root was obtained from the occiput (nonlesional skin) of each individual of 18 psoriasis patients (age range; 22-84 years) by gently plucking hairs. Control hair root samples were collected from 22 normal subjects. Samples from 10 atopic dermatitis patients and 4 dermatomyositis patients were also included as the disease controls. All hair root samples were stored at –80 ◦ C prior to use. Institutional review board approval and written informed consent were obtained before patients and healthy volunteers were entered into this study, according to the Declaration of Helsinki. Clinical data reported in this study were obtained at the time of sampling hair roots. Patients were evaluated for disease duration between the onset of disease and the first visit to the hospital, Psoriasis Area Severity Index (PASI) score, body surface area (BSA) of involved skin, the presence of arthritis, and nail change (table 1).
807
To cite this article: Hirao H, Jinnin M, Ichihara A, Fujisawa A, Makino K, Kajihara I, Sakai K, Fukushima S, Inoue Y, Ihn H. Detection of hair root miR-19a as a novel diagnostic marker for psoriasis. Eur J Dermatol 2013; 23(6): 807-11 doi:10.1684/ejd.2013.2190
Table 1. Summary of the information on each psoriasis patient.
Copyright © 2017 John Libbey Eurotext. Téléchargé par BIBLIOTHEQUE DIVISION DES ACQUISITIONS UNIVERSITE LAVAL le 07/06/2017.
Patient
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Age
Duration (month)
PASI score (point)
BSA (%)
Arthritis
Nail change
miR-19a
45 66 67 22 40 84 45 29 41 57 43 28 41 56 58 44 56 40
3 300 84 1 192 12 14 144 180 60 12 216 240 12 84 240 300 240
0.6 25.9 12.0 0.6 2.0 4.9 2.0 3.1 13.2 ND 3.4 ND 9.9 5.6 3.8 30.0 6.5 5.8
3 80 55 2 10 25 15 20 15 50 20 30 30 30 5 50 20 10
+ + + + + + -
+ ND + ND + ND + ND ND ND -
5330.3 608.9 245.6 10297.5 235.6 407.3 955.4 326.3 160.9 1269.5 24833.5 11.9 213.8 10513.8 448.8 119.4 18.8 56.1
PASI score; psoriasis area and severity index score, BSA; body surface area of involved skin, ND; not determined. Relative miR-19a levels corrected by U6 levels in the same samples are shown, when the minimum value in normal subjects was set at 1 (see figure 2).
RNA extraction from a hair root A single hair root, washed in water and ethanol, was incubated overnight with 0.8ml Isogen® (Nippon Gene, Toyama, Japan) at 4 ◦ C. After chloroform was added, samples were mixed well by vortexing and were incubated for 5 min. Aqueous and organic phases were separated by centrifuge and RNA was precipitated from the aqueous phase by isopropanol [13].
Quantitative real-time PCR For cDNA synthesis from total RNA, including small RNA derived from hair roots, we used mir-X miRNA First Strand Synthesis and SYBR qRT-PCR Kit® (Takara Bio Inc) [14]. The cDNA and primers were mixed with SYBR Premix for quantitative real-time PCR with Takara Thermal Cycler Dice TP800. The U6 primer pair and universal 3’ primer were included in the kit. The sequences of 5’ primer of hsa-miR-19a were designed based on miRBase (http://www.mirbase.org). DNA amplification was performed by 40∼50 cycles of denaturation for 5 sec at 95 ◦ C and annealing for 20 sec at 60 ◦ C. The levels of miR19a in hair roots were normalized by U6 levels in the same samples.
Statistical analysis Differences between 2 groups were compared using MannWhitney’s U test. Correlations were assessed by Pearson’s correlation coefficient. p
Investigative report
Copyright © 2017 John Libbey Eurotext. Téléchargé par BIBLIOTHEQUE DIVISION DES ACQUISITIONS UNIVERSITE LAVAL le 07/06/2017.
Hiroki HIRAO Masatoshi JINNIN Asako ICHIHARA Akihiko FUJISAWA Katsunari MAKINO Ikko KAJIHARA Keisuke SAKAI Satoshi FUKUSHIMA Yuji INOUE Hironobu IHN Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan
Reprints: M. Jinnin
Article accepted on 8/20/2013
doi:10.1684/ejd.2013.2190
P
Detection of hair root miR-19a as a novel diagnostic marker for psoriasis Background: Objective biomarkers that reflect the diagnosis and disease activity have not been in clinical use for psoriasis. Objectives: We investigated the hair root miR-19a levels, regulatory microRNA of TNF-␣, and evaluated the possibility that miR-19a can be a biomarker of psoriasis. Materials and methods: microRNAs were extracted from hair roots of patients with psoriasis (n = 18) and healthy controls (n = 22). Samples from 10 atopic dermatitis patients and 4 dermatomyositis patients were also included as the disease controls. miR-19a levels were determined by quantitative real-time PCR. Results: Hair root levels of miR-19a were significantly up-regulated only in psoriasis compared with normal controls. In characteristics (ROC) curve analysis for hair root miR19a, to distinguish psoriasis patients from normal subjects, the areas under curve (AUC) was 0.87. Relative miR-19a levels were inversely and significantly correlated with duration between symptom onset and the first visit to the hospital in psoriasis patients. Conclusions: Our results indicated hair root miR-19a levels are effective as a disease marker. Key words: microRNA, psoriasis, hair root
soriasis is one of the chronic inflammatory skin diseases characterized by erythemas or papules with silver-white scales. Abnormal proliferation and differentiation of keratinocytes as well as infiltration of lymphocytes and neutrophils are responsible for the skin lesion, histopathologically. Cytokines, including tumor necrosis factor (TNF)-␣ or interleukin (IL)-17, have been proved to contribute to the inflammation of psoriasis: expression of these cytokines is known to be increased in lesional psoriatic skin [1-7] and monoclonal antibodies against these cytokines (e.g. infliximab, etanercept, or ustekinumab) improve the disease dramatically [8]. However, their serum levels do not reflect the disease activity and disease markers for psoriasis have not been used clinically. We focused on the possibility that miRNAs in serum are useful as diagnosis markers for various diseases. miRNAs, non-coding short ribonucleic acid molecules, an average of 22 nucleotides long, are post-transcriptional regulators that bind to complementary sequences in the 3’ UTRs of target mRNAs, leading to the inhibition of translation [9]. They play important roles in diverse cellular processes, including cell development or proliferation, and are thought to be involved in the pathogenesis of various human diseases. miRNAs are also present in human serum in a remarkably stable form, which is protected from endogenous RNase activity by microvesicles, such as exosomes or shedding vesicles [10]. Previously, we determined the serum levels of miR-19a, the regulatory miRNA of TNF-␣ [11], in psoriasis patients and control subjects but we could not find a statistically significant difference [12].
EJD, vol. 23, n◦ 6, November-December 2013
Recently, we confirmed the presence of miRNAs in hair roots [13] and showed that hair miRNA levels as well as serum miRNA levels can be novel and independent biomarkers. Thus, in this report, we determined the possibility that we can use hair root miR-19a levels as disease markers of psoriasis.
Materials and methods Clinical assessment and patient material A single hair root was obtained from the occiput (nonlesional skin) of each individual of 18 psoriasis patients (age range; 22-84 years) by gently plucking hairs. Control hair root samples were collected from 22 normal subjects. Samples from 10 atopic dermatitis patients and 4 dermatomyositis patients were also included as the disease controls. All hair root samples were stored at –80 ◦ C prior to use. Institutional review board approval and written informed consent were obtained before patients and healthy volunteers were entered into this study, according to the Declaration of Helsinki. Clinical data reported in this study were obtained at the time of sampling hair roots. Patients were evaluated for disease duration between the onset of disease and the first visit to the hospital, Psoriasis Area Severity Index (PASI) score, body surface area (BSA) of involved skin, the presence of arthritis, and nail change (table 1).
807
To cite this article: Hirao H, Jinnin M, Ichihara A, Fujisawa A, Makino K, Kajihara I, Sakai K, Fukushima S, Inoue Y, Ihn H. Detection of hair root miR-19a as a novel diagnostic marker for psoriasis. Eur J Dermatol 2013; 23(6): 807-11 doi:10.1684/ejd.2013.2190
Table 1. Summary of the information on each psoriasis patient.
Copyright © 2017 John Libbey Eurotext. Téléchargé par BIBLIOTHEQUE DIVISION DES ACQUISITIONS UNIVERSITE LAVAL le 07/06/2017.
Patient
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Age
Duration (month)
PASI score (point)
BSA (%)
Arthritis
Nail change
miR-19a
45 66 67 22 40 84 45 29 41 57 43 28 41 56 58 44 56 40
3 300 84 1 192 12 14 144 180 60 12 216 240 12 84 240 300 240
0.6 25.9 12.0 0.6 2.0 4.9 2.0 3.1 13.2 ND 3.4 ND 9.9 5.6 3.8 30.0 6.5 5.8
3 80 55 2 10 25 15 20 15 50 20 30 30 30 5 50 20 10
+ + + + + + -
+ ND + ND + ND + ND ND ND -
5330.3 608.9 245.6 10297.5 235.6 407.3 955.4 326.3 160.9 1269.5 24833.5 11.9 213.8 10513.8 448.8 119.4 18.8 56.1
PASI score; psoriasis area and severity index score, BSA; body surface area of involved skin, ND; not determined. Relative miR-19a levels corrected by U6 levels in the same samples are shown, when the minimum value in normal subjects was set at 1 (see figure 2).
RNA extraction from a hair root A single hair root, washed in water and ethanol, was incubated overnight with 0.8ml Isogen® (Nippon Gene, Toyama, Japan) at 4 ◦ C. After chloroform was added, samples were mixed well by vortexing and were incubated for 5 min. Aqueous and organic phases were separated by centrifuge and RNA was precipitated from the aqueous phase by isopropanol [13].
Quantitative real-time PCR For cDNA synthesis from total RNA, including small RNA derived from hair roots, we used mir-X miRNA First Strand Synthesis and SYBR qRT-PCR Kit® (Takara Bio Inc) [14]. The cDNA and primers were mixed with SYBR Premix for quantitative real-time PCR with Takara Thermal Cycler Dice TP800. The U6 primer pair and universal 3’ primer were included in the kit. The sequences of 5’ primer of hsa-miR-19a were designed based on miRBase (http://www.mirbase.org). DNA amplification was performed by 40∼50 cycles of denaturation for 5 sec at 95 ◦ C and annealing for 20 sec at 60 ◦ C. The levels of miR19a in hair roots were normalized by U6 levels in the same samples.
Statistical analysis Differences between 2 groups were compared using MannWhitney’s U test. Correlations were assessed by Pearson’s correlation coefficient. p
