miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression

https://doi.org/10.1016/j.bbrc.2019.11.040Get rights and content

Highlights

  • miR-145-5p was increased in hypertrophic scar and fibroblasts treated with TGF-β.

  • Smad2/3 were direct targets of miR-145-5p.

  • miR-145-5p overexpression inhibited fibroblasts proliferation, migration and induced apoptosis.

  • miR-145-5p decreased hypertrophic scar formation by reducing the expression of Smad2/3.

Abstract

The study was designed to explore the underlying mechanism of micro ribonucleic acids (miR)-145-5p in the process of hypertrophic scar (HS). The difference in the relative content of miR-145-5p between HS and adjacent normal skin collected from 5 patients was detected via RT-PCR. Expressions of Smad2 and Smad3 with or without TGF-β1 was detected by western blotting. Fibroblasts apoptosis rate was examined by Annexin V/Propidium Iodide double staining. HS fibroblasts (HSFs) were isolated from HS tissues, cultured and then divided into control group, miR-145-5p inhibitor group (transfected with miR-145-5p inhibitor) and miR-145-5p mimic group (transfected with miR-145-5p plasmid) based on different treatment methods. Next, CCK-8 was employed to examine the function of miR-145-5p in HSF proliferation. Luciferase assay was conducted to confirm whether Smad2/3 were direct targets of miR-145-5p, and RT-PCR was done to measure the expression of miR-145-5p, Smad2/Smad3 and fibrosis-related genes of fibroblasts in three groups. Wound injury mice model was established to determine the function of miR-145-5p in regulating scar formation. miR-145-5p was found lowly expressed in HS tissues. Compared with Control group, miR-145-5p mimic decreased the levels of Smad2/3, arrested the activation and proliferation of HSFs and induced HSFs apoptosis. Overexpressing miR-145-5p achieved the contrary results. Smad2/3 was confirmed as the target of miR-145-5p. Moreover, miR-145-5p mimic decreased the recruitment of fibroblasts in vivo and decreased the expression of fibrosis-related genes after wound injury. In conclusion, miR-145-5p arrests the development of fibrogenesis and decreases HS formation by reducing the expression of Smad2/3. miR-145-5p may be an optional novel molecular target for treating HS.

Introduction

Hypertrophic scar (HS), one of pathological scars, is the result of abnormal wound healing [1,2]. During the development of HS, abnormally enhanced activity and over-proliferation of many cells including fibroblasts are detected, leading to over-proliferation of dermal tissues and thus inducing HS [3,4]. Since the elasticity of scar tissues is reduced, the normal anatomical structure of skin tissues of the body surface is destroyed, which has bad effects on the good appearance and leads to different degrees of dysfunction of tissues and organs and even disability, thereby affecting the normal life and work of patients and damaging their physical and mental health status [5,6]. The etiology and molecular mechanisms leading to over-deposition of extracellular matrix (ECM) in HS remain unclear, which are great challenges for clinical treatment [[7], [8], [9]]. Currently, comprehensive treatments including surgery, silicone gel, drug therapy, laser therapy, radiotherapy, cryotherapy and compression therapy are adopted for the treatment of HS, but there is always no specific treatment [[10], [11], [12], [13], [14]]. Therefore, finding out specific treatments has been a major challenge in Department of Burns and Plastic Surgery. Recently, a lot of further studies on the genes and molecular mechanisms related to the pathogenesis of HS conducted by scholars have manifested that for the prevention and treatment of HS, the occurrence and development of abnormal scars should be avoided or reduced from the source, and finding out gene defects in HS and correcting from the genetic level are the premises of the breakthrough in the treatment of HS [[15], [16], [17], [18]].

Micro ribonucleic acids, a class of non-coding small-molecule RNAs with approximately 22 nucleotides in length, can identify and bind to related target genes in the form of complete or incomplete complementation [19], and adjust the expression quantities of genes and/or proteins at the post-transcriptional level via the cleavage or inhibition of the mRNA translation of target genes [[20], [21]]. Existing studies have demonstrated that miRNAs can modulate and control the occurrence of skin scars through various mechanisms [[22], [23], [24]]. MiR-145-5p functions an essential role in regulating the occurrence and development of various diseases [25,26], but its role in HS has not been studied.

The study aims to explore the function of miR-145-5p in HS. The results suggested that miR-145-5p directly targeted Smad2 and Smad3 and inhibited fibroblast activation and migration both in vivo and in vitro.

Section snippets

Patients

In this study, 5 pairs of HS in the proliferative stage definitely diagnosed in clinic and normal skin tissue samples were collected from patients in the hospital from May 2018 to May 2019. Five subjects of either gender from the Department of Burns and Plastic Surgery signed the informed consent and aged 20–28 years old. Besides, the site of HS was limbs, chest and abdomen after burning and scalding. The course of disease was 3–8 months (Table 1). The scar tissues in the affected area met the

miR-145-5p arrested fibroblast proliferation and migration

To explore the role of miR-145-5p in fibrogenesis, RT-PCR was employed to measure the level of miR-145-5p in HS and normal skin of patients. HS showed less content of miR-145-5p than that of normal skin (Fig. 1A). Additionally, a significant lower expression of miR-145-5p of fibroblasts was found after cultured with transforming growth factor beta (TGF-β) for 1 day (Fig. 1B), which indicates that miR-145-5p participates in the development of scar formation. Fibroblasts were transfected with

Discussion

Skin scar refers to the phenomenon that the skin heals after burns but still has pathological features, of which the most common is HS based on reports [27,28]. In the past few decades, the advancement in the acute burn care has decreased the mortality rate of burns, but a wide range of scars is usually found on the skin surface of patients with large-area burns, causing pain and itching, and even affecting movement [29,30]. Scars lead to dramatically lower quality of life of many patients and

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

None.

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