Lidocaine promotes fibroblast proliferation after thermal injury via up-regulating the expression of miR-663 and miR-486

Abstract

Thermal burn is a complex injury that induces pronounced inflammatory reactions and destruction of the microvasculature. Lidocaine, which is broadly used for pain relief, has been reported have the capacity to modulate wound healing processes. Seventeen burn injury patients with no treatment and nine controls were included in this study. The expression of 15 candidate miRNAs in the dermis samples were detected by qRT-PCR. The target genes were predicted using online bioinformatics tools and confirmed by dual luciferase assay and immunoblotting. The function of miR-486 and miR-663 on skin fibroblasts keratinocytes were determined by MTT assay and flow cytometry. The level of miR-486 and miR-663 was increased after burn injury, meanwhile, the highest level of miR-486 and miR-663 was found after lidocaine treatment. We identified that BCL2L14 was a direct target of both miR-486 and miR-663. Meanwhile, overexpression of miR-486 or miR-663 inhibit apoptosis and promoted proliferation of human skin fibroblasts keratinocytes. These results indicated that lidocaine treatment promoted the skin healing after thermal injury through up-regulating miR-486 and miR-663 expression, and partially explained how lidocaine modulates wound healing processes. This may provide an evidence for the therapeutic effect of lidocaine during skin healing process. However, the underlying mechanisms need to be further unveiled.

INTRODUCTION

Thermal burn is a significant cause of worldwide morbidity and mortality that can also lead to debilitating lifelong injuries. It is a complex injury that induces pronounced inflammatory reactions and destruction of the microvasculature. Proper and timely treatment are very helpful to obtain functional and emotional recovery. Lidocaine, a local anesthetic, is routinely used to prevent pain during minor surgery or for pain relief after injury. Meanwhile, lidocaine has been reported have the capacity to interfere with inflammation induced plasma extravasation and the release of cytokines which modulating wound healing processes. Recently, there are reports indicate that lidocaine treatment can change the miRNAs expression profile and plays a role during neurological impairment, ischemia-reperfusion injury, dorsal root ganglia apoptosis, and stem cell physiology, indicating the function of lidocaine in epigenetics level. However, the roles of lidocaine during skin thermal injury repair was not understood.

MicroRNAs (miRNAs) are a group of gene expression negative regulators which repress gene expression by binding to the 3′-untranslated region (UTR) of mRNA directly. miRNAs play important roles in maintaining normal human body physiologic conditions. Abnormal miRNA expressions have found to be related to human diseases spanning psychiatric disorders to malignant cancers. Emerging evidences shown that the skin miRNAs expression profile was changed during thermal burn injury and several miRNAs can inhibit or promote the growth of dermal fibroblasts during wound healing of the skin.

In this study, we detected 15 selected miRNAs in the denatured skin samples from 17 deep burn patients treated with lidocaine, 17 deep burn patients with no treatment, and 9 healthy skin samples. We found two miRNAs were significantly up-regulated which inhibit the apoptosis and promote the proliferation of fibroblasts and keratinocyte.

MATERIALS AND METHODS Clinical samples

Thirty four deep partial thickness burn patients were collected in Burn and Plastic Surgery Department, The Chinese PLA 98th Hospital from January 2016 to June 2017. Among them, 17 patients were treated by 1 mg/kg lidocaine through intravenous injection within 2 days after injury. The samples were collected during tangential excision of eschar and large sheets of split thickness auto graft with preservation of dermis within 2 days after burn. The samples from lidocaine group were obtained 24 hours after treatment. Nine normal skins were remnant donor skins taken from the trunk while the dermises were from extremities. Denatured dermis was distinguished as follows: the eschar and yellow necrotic tissue were debrided, then a layer of white dermis with a small amount of scattered tiny bleeding points was seen and confirmed as denatured dermis. The skin tissues were frozen in liquid nitrogen after surgical removal and stored at −80°C for further studies.

All participants provided written informed consent prior to participating in this study. This study was approved by Burn and Plastic Surgery Department, The Chinese PLA 98th Hospital ethics committee.

RNA extraction and quality determination

Total RNA was extracted from tissues and cells using Trizol reagent (Invitrogen, Carlsbad, California) according to the manufacturer's instruction. RNA concentration and purity were determined using a model ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware). Only samples with absorbance ratios 260/280 nm of ~2.0, and 260/230 nm of 1.9 to 2.2 were considered for inclusion in the study.

Quantification of selected miRNAs by quantitative RT-PCR

Quantitative RT-PCR analysis was used to determine the relative level of the 15 selected miRNAs. The levels were detected by TaqMan miRNA RT-Real Time PCR. Single-stranded cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, California) and then amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) together with miRNA-specific TaqMan MGB probes (Applied Biosystems). U6 small nuclear RNA was used for normalization. Each sample in each group was measured in triplicate and the experiment was repeated at least three times.

Western blotting

Protein extracts were boiled in sodium dodecyl sulfate/β-mercaptoethanol sample buffer, and 15 μg samples were loaded into each lane of 10% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer. The membrane was incubated with rabbit anti-BCL2L14 polyclonal antibody (Abcam, Cambridge, Massachusetts) and mouse anti-β-actin monoclonal antibody (Santa Cruz BiotechnologyInc., Santa Cruz, California) for 2 hours at 37°C.The specific protein-antibody complex was detected by using horseradish peroxidase conjugated rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using ECL kit (Pierce, Appleton, Wisconsin). The β-actin signal was used as a loading control.

Cell culture

Human embryonic kidney 293T cells, human skin fibroblasts HSF, and human keratinocyte cell line HaCaT were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS; Hyclone), 20 mmol/L HEPES, 100 IU/mL penicillin, and 100 μg/mL streptomycin.

Dual luciferase assays

To generate 3′-UTR luciferase reporter, a 532 bp segment of 3′UTR from BCL2L14 and 2545 bp segment of 3′UTR from ING5 were cloned downstream of the firefly luciferase gene in the pmirGLO plasmid (Promega, Madison, Wisconsin) separately. miRNA mimics and inhibitors were synthesized by GenePharma Co., Ltd (Shanghai, China). For the luciferase reporter assay, HEK293T human embryonic kidney cells were seeded in 48-well plates. Luciferase reporter vectors were co-transfected with miRNA mimic or inhibitor using lipofectamine 2000 (Invitrogen). Two days after transfection, cells were harvested and assayed with the Dual-Luciferase Assay kit (Promega). Each treatment was performed in triplicate in three independent experiments. The results are expressed as relative luciferase activity (Firefly Luciferase/Renilla Luciferase).

Statistical analysis

Data were analyzed using SPSS Statistical Package version 19 (SPSS Inc., Chicago, Illinois). The luciferase activities, cell apoptosis and proliferation assay were analyzed using paired t test. The expression of miRNAs were analyzed using un-paired t test. The findings were considered significant at a P-value <.05.

RESULTS

To explore the roles of disordered miRNAs during the process of skin wound healing after burn injury, we firstly detected the expression of 15 candidate miRNAs in a group of denatured dermis from 17 patients after lidocaine treatment, 17 patients with no treatment, and normal skin from 9 controls. The clinical characteristics were shown in Table . The miRNAs were selected according to the miRNA screening data in denatured skin after burn injury or skins during wound healing that reported by other researchers. We found the expression of miR-663 and miR-486 was significantly up-regulated after injury (Figure ). Meanwhile, the highest level of miR-663 and miR-486 was found in lidocaine treated group. To understand the function of the up-regulated miR-663 and miR-486, we predicted the direct targets of these two miRNAs using online bioinformatics tool: TargetScan (http://www.targetscan.org/vert_70/). A total of 193 potential targets of both miR-663 and miR-486 were subjected to gene ontology analysis using DAVID Bioinformatics Resources 6.8 (https://david-d.ncifcrf.gov/). We found eight genes were cell apoptosis activator and nine genes were cell proliferation inhibitor (Figure ).

Clinical characteristics

	Patients without lidocaine treatment	Patients with lidocaine treatment	P value
Age (year)	48.6 ± 13.7	50.2 ± 11.4	.52
Gender (male/female)	10 / 7	11 / 6	.83
Burn surface area %	13.4 ± 8.4	11.2 ± 9.6	.81
Full thickness burn area %	6.4 ± 5.9	6.8 ± 6.2	.89
Staphylococcus aureus	4	5	.83
Acinetobacter baumannii	4	3	.81
Pseudomonas aeruginosa	3	4	.81
Candida albicans	1	3	.52
Escherichia coli	2	1	.67
Klebsiella pneumoniae	1	1	1.00

miR-663 and miR-486 were up-regulated after thermal injury and lidocaine treatment. Total RNA was extracted from clinical samples from 17 patients with burn injury and lidocaine treatment for 24 hours, 17 burn injury patients with no treatment, and 9 controls. The expression of 15 candidate miRNAs were detected by qRT-PCR. Results were analyzed by un-paired t test and P [less than] .05 was considered significant. *P [less than] .05, **P [less than] .01

Gene ontology analysis of predicted targets of both miR-633 and miR-486. The direct targets of miR-663 and miR-486 were predicted using online bioinformatics tool: TargetScan. A total of 193 potential targets of both miR-663 and miR-486 were subjected to gene ontology analysis using DAVID Bioinformatics Resources 6.8

Among these targets, eight genes (BCL2L14, ING5, GRIN2A, MAP3K9, PNMA3, PNMA5, SIAH1, and TRIM35) are positive regulators of apoptotic pathway. Since necrosis and apoptosis are two main mechanisms contribute to the cell death of fibroblasts in a burn wound. Necrosis is an irreversible process, whereas apoptosis is a programmed process and can be suppressed by inhibiting the apoptotic pathway. It is reported that apoptosis during burn injury is caused by mild hyperthermia in various types of cells including fibroblasts. Inhibiting the apoptosis pathway is a good choice to suppress burn wound progression. So we focusing on these apoptosis related genes. Among these genes, BCL2L14 and ING5 are two specific apoptosis inducer. In colorectal cancer cells, ING5 was identified to interact with p53 and enhance p53 activity. BCL2L14 also known as BCLG, is a BCL2 family member, and overexpression of BCL2L14 induces apoptosis. We did dual luciferase assay to examine the interaction between miR-486 or miR-663 and BCL2L14 or ING5, and found miR-486 and miR-663 can inhibit luciferase expression by targeting BCL2L14 but not ING5 (Figure S1).

Subsequently, we cloned the 3′UTR of BCL2L14 into pmirGLO vector following the firefly luciferase coding region and generated the wild type reporter vector (Figure A). Vectors with five replaced nucleotides were generated as mutant vectors (Figure A). HEK293T cells were transfected with BCL2L14 3′UTR reporter vector and the mimic of miR-663 or miR-486. Luciferase activities were detected 48 hours after transfection. As shown in Figure B, the luciferase activities were significantly repressed by miR-663 and miR-486 and increased by miR-663 and miR-486 inhibitor. When the predicted target site for miR-663 or miR-486 was mutated, the relative luciferase activity was not inhibited (Figure C), indicating that miR-663 and miR-486 repress luciferase expression via targeting the 3′UTR of BCL2L14. To further identify whether endogenous BCL2L14 was repressed by miR-663 and miR-486, human skin fibroblasts HSF, and keratinocyte cell line HaCaT were transient transfected with miR-663 or miR-486 mimic for 48 hours and the cell lysates were subjected to immunoblotting. As shown in Figure D, the protein level of BCL2L14 was reduced in the cells transfected with miR-663 or miR-486 mimic and increased in the cells treated by miR-663 or miR-486 inhibitor. To further confirm our findings in vivo, the mRNA level of BCL2L14 in patients and control skin samples was quantified by qRT-PCR. As shown in Figure A, we found BCL2L14 mRNA level was significantly reduced after lidocaine treatment. Although the BCL2L14 mRNA level slightly reduced in the skin after thermal injury when compared with control, the difference was not significant, suggesting multiple pathways may regulate the BCL2L14 expression. Meanwhile, immunoblotting was used to examine the BCL2L14 protein level in the skin samples from patients with thermal injury. As shown in Figure B, the BCL2L14 protein level was reduced in the lidocaine treated skins.

BCL2L14 is a direct target of miR-663 and miR-486. A, Schematic diagram of reporter vector construction for wild type or mutant BCL2L14 3′UTR. B, Dual luciferase assay using wild type BCL2L14 3′UTR reporter vector. C, Dual luciferase assay using mutant BCL2L14 3′UTR reporter vector. D, Immunoblotting

Lidocaine inhibit BCL2L14 after burn injury. A, BCL2L14 mRNA level in the skin from patients with thermal injury or healthy controls were determined by qRT-PCR. Results were analyzed by un-paired t test and P [less than] .05 was considered significant. *P [less than] .05, **P [less than] .01. B, BCL2L14 protein level in the skin from patients with thermal injury was examined by immunoblotting

miR-663 and miR-486 inhibit cell apoptosis and promote cell proliferation. A, miR-663 or miR-486 transfected into HSF and HaCaT cells for 48 hours and the level of miR-663 and miR-486 were detected by RT-qPCR. B, HSF and HaCaT cells were transfected with miR-663 and miR-486 mimics for 48 hours and the cell apoptosis was detected by flowcytometry after Annexin V and PI staining. C, HSF and HaCaT cells were transfected with miR-663 and miR-486 mimics for 48 hours and the cell viability was detected by MTT assay. Results were analyzed by student's t-test and P [less than] .05 was considered significant. *P [less than] .05, **P [less than] .01

BCL2L14 is predominantly localized to cytosolic organelles and considered to be an apoptosis activator. To further understand the function of overexpressed miR-663 and miR-486 during skin burn injury, we transfected miR-663 or miR-486 mimics into HSF and HaCaT cells. The overexpression of miR-663 and miR-486 was confirmed by qRT-PCR (Figure A). We found the number of apoptotic cells was significantly reduced in cells treated by the mimic of miR-663 or miR-486 (Figure B). Meanwhile, the cell viabilities were increased by miR-663 and miR-486 mimics (Figure C).

DISCUSSION

Lidocaine, which is broadly used for pain relief, has been reported have the capacity to modulate wound healing processes. To investigate the function of lidocaine in epigenetics level, In the present study, we examined the expression of 15 candidate miRNAs in the dermis samples from 17 patients with burn injury and lidocaine treatment for 24 hours, 17 burn injury patients with no treatment, and 9 controls. We found the level of miR-486 and miR-663 was increased after burn injury, meanwhile, the highest level of miR-486 and miR-663 was found in the lidocaine group. After target prediction and GO analysis, we found eight targets are positive regulators of cell apoptosis process and nine targets are negative regulators of cell proliferation (Figure A-C). These results suggest that lidocaine promote miR-486 and miR-663 expression which may contribute the dermal healing process after thermal injury.

The previous fibroblast denaturation model by thermal damage has shown that fibroblasts that are exposed to thermal injury can complete the recovery process over a period of time and researchers found that the dermal fibroblasts differentially express more than 100 miRNAs when exposed to hyperthermia. In the present study, we found miR-486 and miR-663 were overexpressed in the thermal injured dermis. Meanwhile, we identified BCL2L14, an apoptosis activator, is a direct target of miR-486 and miR-663, so we propose that miR-486 and miR-663 are important regulator of the recovery process after skin thermal injury. There are reports indicated that miR-663 may be a multifunction molecule whose function is tissue specific. miR-486 has been identified as an oncogene promotes tumor cell proliferation, migration, invasion, and relates to poor prognosis via targeting PTEN, PIK3R1, and TGF-β2. In this study, we confirmed miR-486 and miR-663 directly targeted BCL2L14 in human dermal fibroblasts and keratinocytes for the first time which unveiled the function of these two miRNAs during skin thermal injury healing.

Local anaesthetics are known to affect a variety of cell functions including cell proliferation. Martinsson T and colleagues examined the effect of lidocaine on proliferation of human fibroblasts, endothelial cells, and keratinocytes. They found lidocaine inhibits these cells proliferation when these cells were cultured with calf serum or 40% human serum. However, no significant inhibition was obtained with lidocaine when these cells were cultured in 5% human serum suggesting the function of lidocaine on cell proliferation may be different in altered conditions. In this study, we found lidocaine treatment could induce the expression of miR-486 and miR-663, which further inhibit apoptosis and promoted cell proliferation. This may provide an evidence for the therapeutic effect of lidocaine during skin healing process. However, these results were from in vitro cultured cells. Which need to be further confirmed by more in vivo evidences in the further.

Lidocaine, a local anesthetic, which has been found can change the miRNAs expression profile. Through comparing with normal controls and skin samples after thermal injury, we found lidocaine can significantly up-regulate the expression of miR-486 and miR-663.This may provide an evidence for the therapeutic effect of lidocaine during skin healing process. However, the underlying mechanisms need to be further unveiled.

CONFLICT OF INTEREST

All authors declare no conflict of interest.