Int J Clin Exp Pathol 2016;9(5):5505-5509www.ijcep.com /ISSN:1936-2625/IJCEP0022185

Original Article MMP9 and RECK expression in cutaneous lichen planus

Guoying Miao, Li Sun, Baoguo Liu, Zhixia Fan, Guijing Liu, Xiaohui Guo

Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan, Hebei, China

Received December 17, 2015; Accepted March 24, 2016; Epub May 1, 2016; Published May 15, 2016

Abstract: Basement membrane components damage is an important pathological feature of lichen planus. Matrix metalloproteinases (MMPs) can dissolve all types of extracellular matrix. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) can negatively regulate MMPs expression. This study intends to clarify the role of MMP-9 and RECK in lichen planus by detecting their expression. MMP-9 and RECK mRNA and protein expression in normal skin tissue, lichen planus lesional skin tissue, and lichen planus nonlesional skin tissue were detected by real time PCR and Western blot. MMP-9 mRNA and protein expression in lesional skin were obviously higher than that in non-lesional skin and normal tissue (P < 0.05), while they were slightly higher in nonlesional skin than normal skin (P < 0.05). RECK mRNA and protein expression in lesional skin were significantly lower than that in nonlesional skin and normal tissue (P < 0.05), while they were slightly lower in nonlesional skin than normal skin (P < 0.05). MMP-9 and RECK protein expression in lesional skin and nonlesional skin showed negative correlation (P < 0.05). MMP-9 and RECK expression was associated with lichen planus.

Keywords: Lichen planus, RECK, MMP-9

Introduction

Lichen planus (LP), also known as lichen ruber planus, is a kind of chronic inflammatory skin disease with unknown origin. It can occur in skin, hair follicles, nails, and mucous mem-brane, making the patients has occasional pru-ritus. The main histopathological features of LP include basal cell liquefaction degeneration, epidermal hyperkeratosis, and colloid body in dermal papilla layer. LP typical skin lesions appear polygonal fuchsia or atropurpureus flat papules mostly in flexural limb and present. LP presents repeated attack and protracted course that affects quality of life. Since its etiol-ogy and pathogenesis has not been fully eluci-dated, there is still lack of special treatment for LP. Investigating its etiology has important clini-cal significance to provide clues and basis for clinical treatment.

Matrix metalloproteinases (MMPs) is a highly conservative proteolytic enzyme family de- pends on the zinc ions. It can degrade most of basement membrane and extracellular matrix proteins. MMPs include gelatinases, collage-nase, and stromatolysis, which are secreted in

the form of inactive zymogen and need to be activated to obtain proteolytic enzyme activity. MMP-9, also known as gelatinase B, is an important member of MMPs family. Its main function is to degrade collagen type IV and layer adhesion proteins [1]. For the histopathologic features of LP is basement membrane element destruction, we have reason to believe that MMP-9 may play an important role in LP occur-rence. Current studies also showed MMPs had critical role in oral lichen planus (OLP) occur-rence and development. MMP-3 in serum and saliva was associated with OLP occurrence [2, 3]. MMP-7 mRNA and protein overexpressed in OLP lesional skin tissue compared with that in normal oral tissues [4]. In addition, MMP-9 level also elevated in OLP [5] and could be treated as biomarker for OLP diagnosis and prognosis [6]. However, most investigations about the rela-tionship between MMPs and LP were concen-trated in OLP. There was lack of report about the relationship between MMPs and cutaneous lichen planus (CLP). Therefore, this study aims to clarify the role of MMP-9 in CLP by detec- ting MMP-9 expression level in CLP patients. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) was first isolated in 1998.

MMP-9 and RECK in lichen planus

5506 Int J Clin Exp Pathol 2016;9(5):5505-5509

It has the function of inhibiting MMPs expres-sion and activity [7]. At present, RECK is believed to regulate at least three kinds of MMPs including MMP-9 at posttranscriptional level. Thus, this study also tested RECK expres-sion in CLP patients to elucidate its function in CLP occurrence. It may provide theoretical basis for CLP occurrence and clue for searching effective treatment.

Materials and methods

Main reagents and instruments

RNA extraction reagent Trizol (Invitrogen). Real time PCR kit (TaKaRa). Gel imaging system, Vii A7 real time PCR amplifier (ABI). Total protein extraction kit (BestBio). GIS-2020D gel image analysis system, vertical electrophoresis appa-ratus, SDS-PAGE, and PBST solution (Sigma). Coomassie brilliant blue protein determination kit (MajorBio). MMP-9, RECK, and GAPDH anti-bodies (Abcam).

Object of study

A total of 32 cases of CLP patients diagnosed by clinic and pathology in Affiliated Hospital of Hebei University of Engineering between Jan 2014 and Jan 2015 were enrolled. There were 17 males and 15 females with the age at 20~57 years old. Another 32 patients in gen-eral surgery and plastic surgery with no system-ic disease or dermatosis were selected as nor-

mal control, including 17 males and 15 females aged 18~58 years old. No statistical differenc-es on gender or age were observed between two groups. Skin tissue was obtained and stored at -80°C. Inclusion criteria: 1, No gluco-corticoid, immune suppressants, or immune modulators usage with half a year; 2, no sys-temic disease; 3, complete clinical data.

The study protocol was approved by the Research Ethics Committee of our hospital, and all patients gave their informed consent before study commencement.

Real-time PCR

Total RNA was extracted by Trizol and identified by protein nucleic acid detector and 1% aga-rose gel electrophoresis. 1 μg RNA was reverse transcripted to cDNA according to the manu- al. Real-time PCR reaction system contains 2×SYBR Green Mixture 5 μl, cDNA 0.5 μl, prim-er (10 μM) 0.5 μl, and ddH2O 4 μl. The reaction consists one cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s on Vii A7 PCR amplifier. Each experiment had three replicates. β-actin was used as reference.

Western blot

Skin tissue was grinded in liquid nitrogen and total protein was extracted using the kit accord-ing to the manual. After centrifuged to obtain

Figure 1. Pathological examination of normal skin tissues and LP tissues. A. Normal skin tissues; B. LP skin tissues. Colloid bodies, read arrows.

MMP-9 and RECK in lichen planus

5507 Int J Clin Exp Pathol 2016;9(5):5505-5509

supernatant, protein was quantified by coo-massie brilliant blue protein determination kit and boiled for 5 min. 8% and 16% SDS-PAGE separation gels were prepared, and 4% SDS-PAGE spacer gel was added after solidification. The protein was concentrated at 12 mA and separated at 9 mA for 8 h. Then it was trans-ferred to NC membrane at 300 mA for 1.5 h. After blocked by 5% skim milk for 2 h, the mem-brane was incubated in primary antibody at 4°C over night. Then the membrane was incubated with secondary antibody at room temperature for 60 min and washed by PBST. At last, the membrane was treated with chemiluminescent agent and imaged on X-ray. GIS-2020D gel

image analysis system was applied to analyze MMP-9, RECK, and GAPDH protein band densi-ty to calculate the relative expression level.

Statistical analysis

SPSS 13.0 software was applied for data analy-sis. Data obeyed normal distribution were com-pared by t test or ANOVA. Bivariate normal dis-tribution correlation analysis was performed by Pearson correlation analysis, inspection level α = 0.05.

Results

Pathology of skin tissues

Skin is composed of epidermis, dermis and subcutaneous tissues. The epidermis consists of stratum corneum, stratum lucidum, granular layer, stratum spinosm and stratum basale (Figure 1A). When skin tissues had infection, injury or tumors, their histologic structure is often altered. LP is one common chronic inflam-matory skin disease with unknown reason. The pathological features of the skin tissues include liquidation/degradation of basal layer cells, over-keratinization of epidermis and the occur-rence of colloid bodies (Figure 1B).

MMP-9 and RECK mRNA expression level in skin tissue

Real time PCR was applied to test MMP-9 and RECK mRNA expression level in lesional skin tissue and nonlesional skin tissue from CLP patients and normal skin tissue from control. Relative expression level was calculated af ter 2-ΔΔCT transition (Figure 2). MMP-9 mRNA relative expression level was 2.13±0.13 and 1.19±0.12 in lesional skin tissue and nonle-sional skin tissue, respectively. MMP-9 mRNA level in lesional skin tissue was obviously high-er than that in nonlesional skin tissue and nor-mal control (P < 0.05), and it was slightly higher in nonlesional skin tissue compared with nor-mal control (P < 0.05). RECK mRNA relative expression level was 0.64±0.09 and 0.89±0.11 in lesional skin tissue and nonlesional skin tis-sue, respectively. RECK mRNA level in lesional skin tissue was significantly lower than that in nonlesional skin tissue and normal control (P < 0.05), and it was slightly lower in nonlesional skin tissue compared with normal control (P < 0.05).

Figure 2. MMP-9 and RECK mRNA expression level in skin tissue. *P < 0.05, compared with normal skin; #P < 0.05, compared with nonlesional skin.

Table 1. MMP-9 and RECK protein expression intensity in skin tissueGroup Cases MMP-9 RECKNormal skin 32 0.56±0.13 1.56±0.21Lesional skin 32 1.12±0.18 0.91±0.19Nonlesional skin 32 0.68±0.16 1.43±0.20F value 111.42 94.48P value < 0.001 < 0.001

Figure 3. MMP-9 and RECK protein expression in skin tissue.

MMP-9 and RECK in lichen planus

5508 Int J Clin Exp Pathol 2016;9(5):5505-5509

MMP-9 and RECK protein expression in skin tissue

We further used Western blot to test MMP-9 and RECK protein expression level in lesional skin tissue and nonlesional skin tissue from CLP patients and normal skin tissue from con-trol (Table 1; Figure 3). Similar with mRNA results, MMP-9 protein expression level in lesional skin tissue was markedly higher than that in nonlesional skin tissue and normal con-trol (P < 0.05), while it was slightly higher in nonlesional skin tissue compared with normal control (t = 3.29, P < 0.05). RECK protein expression level in lesional skin tissue was sig-nificantly lower than that in nonlesional skin tis-sue and normal control (P < 0.05), and it was slightly lower in nonlesional skin tissue com-pared with normal control (t = 2.54, P < 0.05).

MMP-9 and RECK protein expression correla-tion analysis

Pearson correlation analysis was applied to test MMP-9 and RECK protein expression le- vel in lesional skin and nonlesional skin. The results showed that both MMP-9 and RECK pro-tein level presented negative correlation in lesional skin and nonlesional skin (r = -0.561, P < 0.01; r = -0.603, P < 0.01).

Discussion

LP pathogenesis and etiology are still contro-versial. A variety of theories exist, including heredity, infection, autoimmune, drug, endo-crine and metabolic disorders, and mental neu-rology. Current study mostly focused on the immune theory, thus LP studies concentrated on immune related molecules, such as COX-2 [4], PGE2 [8], Wnt5a [9], IFN-γ [10], and IL-6 [11]. Meanwhile, LP is also considered as an autoimmune disease. MMPs expression is low in normal status, while significantly increases when stimulated by inflammatory cytokines, hormones, and growth factors. For example, MMP-2 and MMP-9 showed dose-response elevation in corneal epithelium cells stimulated by TNF-α [12, 13]. Furthermore, they also pre-sented similar phenomena stimulated by IL-1 [14]. For MMPs expression had closely associa-tion with autoimmune status, we have reason to believe that MMPs played an important role in LP. As a suppressor gene of MMPs, RECK may also participate in LP.

This study used real-time PCR and Western blot to detect MMP-9 and RECK mRNA and protein expression level in lesional skin and nonlesion-al skin from CLP patients and normal tissue from control. The results revealed that MMP-9 mRNA and protein expression in lesional skin were obviously higher than that in nonlesional skin and normal tissue, while they were slightly higher in nonlesional skin than normal skin. On the contrary, as the suppressor gene of MMP-9, RECK level showed negative tendency with MMP-9. RECK mRNA and protein expression in lesional skin were significantly lower than that in nonlesional skin and normal tissue, while they were slightly lower in nonlesional skin than normal skin. The results suggested that CLP onset may be related to MMP-9 and RECK. It was reported that Wnt5a silence markedly downregulated MMP-9 expression [15, 16], indicating that MMP-9 may be regulated by Wnt5a. Current study showed that Wnt5a level significantly enhanced in CLP [9], which verified our results from one side. Similarly, MMP-9 expression changes also participated in OLP onset [17].

MMP-9 is regulated by RECK [18], and this study also confirmed that MMP-9 presented negative correlation with RECK. Moreover, it was found that RECK level decreased in lesion-al skin in CLP patients, suggesting that MMP-9 overexpression may be caused by RECK decli-nation. Though there is still lack of reports about RECK on LP onset, it was discovered that tissue inhibitor of metalloproteinase (TIMP), the natural inhibitor of MMPs, expression changes was related to OLP onset [19, 20].

Our study found that MMP-9 and RECK expres-sion changes may be involved in CLP onset. MMP-9 expression elevation may be caused by RECK downregulation. In vitro experiments are needed to verify. Furthermore, larger scale trial is needed to clarify the role of MMP-9 and RECK in CLP.

Acknowledgements

Important medical funded projects of Hebei province health department (No. zd2013095); Hebei province department of funded projects (No. 10276105D-63).

Disclosure of conflict of interest

None.

MMP-9 and RECK in lichen planus

5509 Int J Clin Exp Pathol 2016;9(5):5505-5509

Address correspondence to: Dr. Li Sun, Department of Dermatology, Affiliated Hospital of Hebei Univer- sity of Engineering, 81 Cong Tai Road, Handan 056029, Hebei, China. Tel: +86-18031028096; E-mail: sunlidas@sina.com

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