微小RNA-124通过靶向SERP1并抑制Wnt/β-连环蛋白信号通路来抑制伤口愈合。

MicroRNA-124 represses wound healing by targeting SERP1 and inhibiting the Wnt/β-catenin pathway.

作者信息

Zhang Guohui, Song Kunxiu, Yan Hongshan

机构信息

Department of Burn and Plastic Surgery, Binzhou Medical University Hospital, China.

Department of Hand and Microsurgery, Binzhou Medical University Hospital, China.

出版信息

Adv Clin Exp Med. 2019 Jun;28(6):711-718. doi: 10.17219/acem/94163.

DOI:10.17219/acem/94163

PMID:30740945

Abstract

BACKGROUND

Wound healing is a complex process which restores cellular structures and tissue layers after their destruction. Accumulating evidence has proven that microRNAs (miRs) are involved in wound healing.

OBJECTIVES

The aim of the study was to research the role of miR-124 in wound healing.

MATERIAL AND METHODS

Keratinocytes were respectively transfected with miR-124 mimic, scrambled miRNA (a negative control of miR-124 mimic: mimic NC), antisense oligonucleotides against miR-124 (ASOmiR124), or a negative control of ASO-miR-124 (ASO-NC), and then cell viability, colony formation, cell cycle, expression of cell cycle-associated proteins, and collagen content were all evaluated. The target gene of miR-124 was predicted using TargetScan and verified with luciferase assay. Subsequently, the effects of target gene overexpression on cell viability, colony formation and collagen synthesis were all evaluated. Finally, the expression levels of key kinases in the Wnt/β-catenin pathway were detected using western blot analysis.

RESULTS

Cell viability, colony formation, expression levels of cell cycle-associated proteins, and collagen content were all significantly reduced by miR-124 overexpression. As predicted using bioinformatics and validated with luciferase assay, stress-associated endoplasmic reticulum protein 1 (SERP1) is a target gene of miR-124. Meanwhile, the miR-124 mimic-induced decrease in cell viability, colony formation and collagen synthesis was reversed by SERP1 overexpression. Furthermore, the miR-124 mimic obviously upregulated glycogen synthase kinase 3β (GSK-3β) while downregulating β-catenin, T cell transcription factor 4 (TCF-4) and leukemia enhancer factor 1 (LEF-1). Additionally, all the effects of ASO-miR-124 were the opposite of those of the miR-124 mimic.

CONCLUSIONS

We found that miR-124 inhibited keratinocyte proliferation, collagen biosynthesis and activation of Wnt/β-catenin by targeting SERP1.

摘要

背景

伤口愈合是一个复杂的过程，可在细胞结构和组织层遭到破坏后对其进行修复。越来越多的证据表明，微小RNA（miR）参与伤口愈合过程。

目的

本研究旨在探究miR-124在伤口愈合中的作用。

材料与方法

分别用miR-124模拟物、乱序微小RNA（miR-124模拟物的阴性对照：模拟物NC）、抗miR-124反义寡核苷酸（ASOmiR124）或ASO-miR-124的阴性对照（ASO-NC）转染角质形成细胞，然后评估细胞活力、集落形成、细胞周期、细胞周期相关蛋白的表达以及胶原蛋白含量。使用TargetScan预测miR-124的靶基因，并通过荧光素酶测定进行验证。随后，评估靶基因过表达对细胞活力、集落形成和胶原蛋白合成的影响。最后，采用蛋白质印迹分析检测Wnt/β-连环蛋白信号通路中关键激酶的表达水平。

结果

miR-124过表达显著降低了细胞活力、集落形成、细胞周期相关蛋白的表达水平以及胶原蛋白含量。经生物信息学预测并通过荧光素酶测定验证，应激相关内质网蛋白1（SERP1）是miR-124的靶基因。同时，SERP1过表达逆转了miR-124模拟物诱导的细胞活力、集落形成和胶原蛋白合成的降低。此外，miR-124模拟物明显上调糖原合酶激酶3β（GSK-3β），同时下调β-连环蛋白、T细胞转录因子4（TCF-4）和白血病增强因子1（LEF-1）。此外，ASO-miR-124的所有作用与miR-124模拟物的作用相反。