Regulation function of MMP-1 downregulated by siRNA on migration of heat-denatured dermal fibroblasts.

Cutaneous wound healing is a complex physiological process that requires the efforts of various cell types and signaling pathways and often results in thickened collagen-enriched healed tissue called a scar. Therefore, the identification of the mechanism of cutaneous wound healing is necessary and has great value in providing better treatment. Here, we demonstrated that MMP-1 inhibition could promote cell proliferation in dermal fibroblasts via the MTT assay. Meanwhile, we investigated cell migration by flow cytometry and tested type I collagenase activity. We found that MMP-1 inhibition promoted cell proliferation and inhibited cell migration and type I collagenase activity. In conclusion, our study demonstrated that MMP-1 might be a potential therapeutic target in cutaneous wound healing.

Introduction

Skin fibroblasts are an important effector in wound repair and can affect wound tissue biological properties and ultimately wound healing. Cutaneous wound healing is a complex physiological process that requires the efforts of various cell types and signaling pathways. Current research have illustrated that the migration and proliferation of fibroblasts played key roles in the process of healing burned skin and that fibroblasts have been widely used for research on the underlying mechanisms of skin wound healing. In the research of Chen JC et al. dermal fibroblasts were used to investigate the role of nerve growth factor (NGF) in the healing of skin wounds in rats, and they demonstrated that fibroblast migration that was induced by NGF might contribute to the skin wound healing process. Even though the study of the healing mechanism and therapy of burned skin have made great progress, a deep understanding about the biological basis and underlying mechanism is still necessary.

Matrix metalloproteinases (MMPs) are a multi-gene endoproteinase family that comprises over 22 members which have ability to break down extracellular matrix. MMPs have been reported to play key roles in multiple biological processes, including tissue remodeling, cardiovascular diseases, obesity, and cancer. Previous studies found that MMP-1 was highly expressed in epidermal cells that had been subjected to burn or UV treatment. Piao MJ et al. reported that a high level of solar UVB irradiation induced the synthesis of MMPs in skin fibroblasts and caused photoaging and tumor progression. As a ubiquitous group of proteins, MMP upregulation affects the stability of the epidermis structure and may cause adverse effects during the wound healing process. In addition, in human dermal fibroblasts, tiron has been shown to inhibit the transcriptional activation of UVB-induced activator protein 1 (AP-1) binding sites on MMP-1 and MMP-3 promoters and was identified as a novel antioxidant for the prevention and treatment of skin photo aging that was induced by UV light exposure.

The current study was undertaken to explore the role of MMP-1 in the process of cutaneous burn healing for the first time. In this study, we used siRNAs to reduce the expression level of MMP-1 in human skin fibroblasts after heat stress treatment in vitro. Furthermore, we analyzed the effects of MMP-1 on cell proliferation, migration and related downstream protein expression. The results showed that the inhibition of MMP-1 promoted cell proliferation and decreased cell migration and type 1 collagenase expression. This study provided a new understanding of the regulatory mechanism of MMP-1 in burn wound healing and identified potential avenues for therapeutic intervention.

Results

siRNAs downregulated MMP-1 expression

We analyzed the MMP-1 mRNA expression level in dermal fibroblasts via real time PCR (RT-PCR). We monitored the relative levels of MMP-1 mRNA at 0 h, 24 h, and 72 h after heat treatment. As shown in Figure 1, the results suggested that heat treatment upregulated MMP-1 expression, while the transfection of siRNA effectively decreased MMP-1 expression.

Figure 1.

siRNA treatment successfully downregulated MMP-1 mRNA expression. An RT-PCR assay was performed to detect the mRNA expression of MMP-1. Untreated, untreated dermal fibroblasts without heat stress and transfection; NC, siRNA negative control.

The inhibition of MMP-1 promoted cell proliferation

We used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) to test the cell proliferation rate of fibroblasts after heat treatment. The results showed that, compared with the control group, heat treatment notably decreased cell proliferation, and cell proliferation was slightly increased after MMP-1-siRNA treatment, but the increase was not significant (Fig. 2).

Figure 2.

Effects of MMP-1 on cell proliferation. MTT analysis of cell proliferation in the dermal fibroblast cells that were transfected with MMP-1-siRNA or siRNA negative control for 0 h, 24 h, and 72 h after heat treatment. Untreated, untreated dermal fibroblasts without heat stress and transfection; NC, siRNA negative control.

The inhibition of MMP-1 downregulated cell migration

We evaluated the cell migration rate of fibroblasts after heat treatment and the inhibition of MMP-1. With the transwell assay, we found that the transfection of MMP-1-siRNA in fibroblasts could dramatically reduce the migration ratio (Fig. 3) (P < 0.05).

Figure 3.

Effects of MMP-1 on cell migration. Flow cytometry assay was used to test the cell migration rate in dermal fibroblast cells that had been transfected with MMP-1-siRNA or siRNA negative control after heat treatment. Untreated, untreated dermal fibroblasts without heat stress and transfection; NC, siRNA negative control; *, P < 0.05.

The inhibition of MMP-1 decreased type I collagenase activity

We evaluated the activity of type I collagenase, an important downstream molecule of MMP-1. The results (Fig. 4) demonstrated that the heat treatment increased the activity of type I collagenase, while the inhibition of MMP-1 via the transfection of siRNA significantly decreased the activity of type I collagenase (P < 0.05).

Figure 4.

Effects of MMP-1 on collagenase type 1 activity. Collagenase type 1 activity was assessed with a Type I Collagenase Activity Assay Kit after heat treatment and MMP-1-siRNA transfection. Untreated, untreated dermal fibroblasts without heat stress and transfection; NC, siRNA negative control; *, P < 0.05.

MMP-1 was downregulated and TIMP-1 was upregulated after heat treatment

MMP-1 and Tissue inhibitor of metalloproteinase 1 (TIMP-1) are a pair of mutual antagonism proteases, and we detected the expression levels of these two proteins via western blotting assay. As shown in Figure 5, the expression of TIMP-1 appeared to be improved, and the expression of MMP-1 was inhibited.

Figure 5.

The correlation of MMP-1 and TIMP-1 expression. The protein expression levels of MMP-1 and TIMP-1 were detected. The expression levels of MMP-1 and TIMP-1 were negatively related. Untreated, untreated dermal fibroblasts without heat stress and transfection; NC, siRNA negative control; *, P < 0.05.

Discussion

Technological advancement has led to various available burn wound products that help not only in healing and preventing infection but also in improving patients' comfort levels and reducing pain. However, patients who have suffered from burns are not only facing a physical recovery but also some complex problems that were caused by the injury, including pruritus, sleep disorders, pain, and psychological disorders. Among these, dermal repair and scar formation are still worldwide problems in the process of burn treatment. Skin scar formation is characterized by excessive collagen synthesis and aberrant collagen deposition during wound healing. Many biological factors have been indicated to act as potent post-transcriptional repressors of collagen synthesis in skin fibroblasts and have been shown to be potential therapeutic targets for scar reduction.

MMPs are a group of ubiquitous proteins with extensive roles that include extracellular matrix remodeling, blood-brain barrier disruption, cell migration, ischemic stroke pathophysiology and clinical outcome, etc. As a type of extracellular matrix protein factor, MMP and the tissue inhibitor TIMP families play important roles in cancer invasion and the regulation of homeostasis and have received widespread attention. In the study by Li J et al. the researchers provided novel evidence that p-cymene was an effective candidate for the prevention of tumor invasion and metastasis through the inhibition of MMP-9 expression and the augmentation of TIMP-1 production in tumor cells. Moreover, MMP and TIMP had an important role in the hair growth cycle; TIMP-1 and TIMP-2 expression were negatively correlated with MMP-9 and MMP-2 expression, respectively.

In this study, we established a skin fibroblast model in vitro and used siRNA to silence MMP-1 expression for the first time. The results showed that the inhibition of MMP-1 promoted cell proliferation and inhibited cell migration. We revealed that MMP-1 positively correlated with epidermal cell migration under a heat stress state.

Additionally, in the research of hypertrophic scar (HS) pathogenesis, the production of type I collagen was demonstrated to be inhibited by miR-181c inhibition or miR-10a overexpression in HSs, which resulted in an increased level of MMP-1. Therefore, in our following research, we investigated the correlation of MMP-1 and the activity of type 1 collagenase and found that the inhibition of MMP-1 significantly decreased the activity of type I collagenase.

Above all, our study found that the suppression of MMP-1 could promote cell proliferation, decrease cell migration, and decrease the activity of type I collagenase in heat-denatured dermal fibroblast cells. All of these findings suggested that MMP-1 might be involved in the progression of burn wound healing and could be a new therapeutic target for this disease. Meanwhile, animal models for further research might provide a new approach for burn treatment.

Materials and methods

Cell culture

Normal human skin was collected from patients without any other skin lesions who had undergone plastic surgery procedures in the hospital. Patients did not use any skin drug before the specimens were taken and did not have cancer or other serious illnesses. All of the patients provided informed consent, and the operations were approved by the Ethics Committee of the University. Primary human dermal fibroblasts were obtained as follows: first, skin tissues were dissected into pieces and then treated with trypsin-EDTA (Sigma, USA) for enzymatic digestion. Second, the isolated cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies, USA) containing 10% fetal bovine serum (FBS, Sigma), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, USA) at 37°C, 5% CO2. The medium was changed every 2 days, and the skin fibroblast cells were cultured and passaged after they were isolated from the tissue. Primary dermal fibroblasts from passages 5–8 were used throughout the experiments. Heat-denatured human dermal fibroblasts were obtained by placing the cell culture plates in a water bath at 52°C for 30 s; normal fibroblast cells were placed at 37°C for 30 s and acted as the control group (untreated).

Cell transfection

Briefly, three target sites that were located on the MMP-1 mRNA were chosen according to the siRNA design strategy that was developed by Elbashir et al. and the Silencer™ siRNA Construction Kit (Ambion, Austin, TX). The siRNA template sequences (T-1, T-2 and T-3) that were used for RNAi in this study are shown in Table 1. Three siRNA sequences were designed and tested. T-1 was the best one that was used in this study. The reverse sequence for the MMP-1 siRNA template T-1 was used to synthesize a corresponding siRNA, which served as a negative control (R). GAPDH DNA sense and antisense templates (Ambion), were used to synthesize in vitro GAPDH siRNA to assess specificity. The quality of the siRNA that was synthesized was evaluated with 4–15% PAGE electrophoresis.

Table 1.

siRNA templates designed to target MMP-1 mRNA.

Name	siRNA sequence	siRNA size
T-1	5′-A CAC AAG AGC AAG ATG TGG-3′	19 nt (MMP-1 mRNA nt 147–165)
T-2	5′-TA AGT ACT GGG CTG TTC AG-3′	19 nt (MMP-1 mRNA nt 1109–1127)
T-3	5′-AG TTG ATG CAG TTT TCA TG-3′	19 nt (MMP-1 mRNA nt 1345–1363)
R	5′-GGT GTA GAA CGA GAA CAC A-3′	19 nt, the reverse of T-1

T-1, T-2 and T-3, the siRNA template sequences for MMP-1; R, the reverse sequence for the MMP-1 siRNA template T-1.

The cells were harvested at 14 h after transfection with trypsin/EDTA, washed four times with 1× PBS, and fixed for 2 h in 2 ml of 2.5% paraformaldehyde/PBS (pH 7.4). The samples were analyzed at the Mayo Flow Cytometry/Optical Morphology Resource Core Facility using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with 488 nm excitation laser, a 530/30 bandpass filter for emission data collection, and Cell Quest Pro 4.0.2 analysis software.

RT-PCR

cDNA synthesis was completed by using 1 μg of total RNA and priming with oligo dT. Two microliters of the cDNA synthesis reaction were amplified in a total volume of 25 μl that contained 1× PCR buffer, 0.2 μM dNTPs, 1.5 mM MgCl2, 0.2 pM of each primer, and 1.25 U of thermostable Taq DNA polymerase (Roche). After an initial denaturation at 94°C for 5 min, cycling conditions were 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final extension and incubation at 72°C for 7 min. The PCR products were resolved on 1.2% agarose gel, and the corresponding individual gene band densities were analyzed using NIH Image software (Scion) and were normalized relative to GAPDH.

MTT assay

Cell proliferation was determined via the MTT assay. Briefly, dermal fibroblasts that had undergone heat treatment were cultured in 96-well plates and transfected with MMP-1 siRNAs. Then, the transfected cells (Heat + MMP-1-siRNAs), normal cultured cells (Control) and heat treated cells that had not been transfected as a negative control (Heat + NC) were subjected to heat treatment at 52°C for 30 s, and 20 μl (0.5 mg/ml) of MTT was added to each well, followed by incubation for another 4 h at 37°C. Subsequently, 100 μl of dimethyl sulfoxide (DMSO, Sigma) was added to dissolve the formazan crystals, and the absorbance at a wavelength of 490 nm was determined using an ELISA reader (Bio-Rad, USA).

Cell migration assay

Transwells were used to evaluate the migration of Control, Heat + NC and Heat + MMP-1-siRNAs groups. First, the cells in the logarithmic growth phase were suspended with culture medium containing 0.5% FBS after conventional digestion. A total of 100 μl of cell suspension (cell density 1.0 × 105/mL) were added into the upper chamber, and 500 μl of purpose medium containing 10% FBS were added into the lower chamber. Then, the upper chamber was removed and washed with PBS 3 times after 16 h of culture. The cells in the upper membrane were wiped gently using cotton, fixed with 4% paraformaldehyde for 30 min, then stained with crystalline violet for 15 min. Finally, the number of cells were moved to the lower membrane under an inverted microscope for the calculation of the number of vertically migrating cells.

Type I collagenase activity assay

A collagenase activity assay was performed as per the manufacturer's instructions (Type I Collagenase Activity Assay Kit, Chemicon International, Inc.). Briefly, serum-free conditioned media concentrates were activated at 37°C for 5 h. Then, the samples were incubated with a biotinylated collagenase substrate at 37°C for 2 h, followed by the addition of an enhancer and a 30 min incubation. This mixture was transferred to a biotin-binding plate, incubated for 30 min, and washed with assay buffer. Streptavidin-enzyme conjugate was added then washed, followed by the addition of a substrate solution. The activity of the sample was calculated by comparing the degradation of biotinylated collagen substrate to the serially diluted MMP-1 standard.

Western blotting

Briefly, 5 × 104 dermal fibroblasts were lysed using Laemmli sample buffer (S3401, Sigma) and subsequently 15 µl of total cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane. To detect protein expression, antibodies against MMP-1 (ab38929, Abcam), TIMP-1 (ab1827, Abcam), GAPDH (ab9484, Abcam) and secondary HRP- conjugated (Dako) antibodies were used. TIMP-1 and MMP-1 (both R&D Systems) protein concentration in culture supernatants were measured by ELISA according to the manufacturer's protocol. Signal development was performed using HRP/Streptavidin and the OPD substrate (Sigma-Aldrich) at room temperature. Fluorescence was measured using a plate reader (Tecan, Sunrise). Samples were run in duplicate, and serial dilution was performed in order to obtain concentrations that fell within the detection limits of the assay (0–80 ng/ml).

Statistics analysis

All of the collected data were tested for normal distribution using the one-sample K-S test. Enumeration data were analyzed by chi-square test or rank-sum test. Measurement data were tested by Student's t-test (for two groups) or the analysis of variance (ANOVA, for more than three groups). Further between-group-comparisons were then performed by post hoc Tukey test. Statistical significance was defined when P was < 0.05.