ALKBH5通过激活IRE1α-XBP1介导的内质网应激抑制YTHDF2-m6A介导的RCN1 mRNA降解，从而促进瘢痕疙瘩形成。

ALKBH5 Inhibits YTHDF2-m6A-Mediated Degradation of RCN1 mRNA to Promote Keloid Formation by Activating IRE1α-XBP1-Mediated ER Stress.

作者信息

Shi Min, Zhang Lu, Bi Fangfang, Zhou Zhuo

机构信息

School of Medicine, Xi'an Peihua University, Xi'an, Shaanxi, China.

Department of Obstetrics and Gynecology, Northwest University First Hospital, Xi'an, Shaanxi, China.

出版信息

J Cosmet Dermatol. 2025 Apr;24(4):e70177. doi: 10.1111/jocd.70177.

DOI:10.1111/jocd.70177

PMID:40214031

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11987481/

Abstract

BACKGROUND

Reticulocalbin 1 (RCN1) was reported to be upregulated in keloid, but its molecular mechanism remains unclear. The aim of this study is to investigate the role of RCN1 in keloid.

METHODS

The expression of RCN1 was detected in keloid tissues. Keloid fibroblasts were transfected with RCN1 overexpression vector. Cell viability, collagen production, apoptosis, and cell invasion were measured. Then, the m6A modification level of RCN1 mRNA was detected by methylated RNA immunoprecipitation (MeRIP), and the effect of overexpression of ALKB homolog5 (ALKBH5) on the m6A modification level of RCN1 mRNA was evaluated. Subsequently, the relationship between RCN1 and XBP1 was verified by co-immunoprecipitation (Co-IP) assay. pcDNA-RCN1 and XBP1 shRNA were transfected into keloid fibroblasts to for reversal experiments, and changes in the endoplasmic reticulum (ER) structure of keloid fibroblasts were observed by transmission electron microscopy (TEM). Finally, we established a mouse keloid model and injected mice with the RCN1 shRNA lentiviral vectors to monitor the keloid formation in mice.

RESULTS

RCN1 was highly expressed in keloid tissues and keloid fibroblasts. Overexpression of RCN1 significantly increased keloid fibroblast viability, collagen production, and invasion, but inhibited cell apoptosis. ALKBH5 upregulated RCN1 expression by reducing m6A-YTHDF2-mediated degradation of RCN1 mRNA, and RCN1 knockdown reversed the promoting effect of ALKBH5 overexpression on cell viability collagen production and invasion, and the inhibitory effect of ALKBH5 overexpression on apoptosis in keloid fibroblasts. Moreover, overexpression of RCN1 significantly upregulated the protein levels of XBP1, GRP78, and IRE1α, and promoted ER stress in keloid fibroblasts, but this change was eliminated by sh-XBP1 intervention. In vivo experiments showed that knockdown of RCN1 significantly inhibited keloid formation by alleviating cell apoptosis and ER stress in mice.

CONCLUSION

Our data revealed that RCN1 was upregulated by ALKBH5 to promote keloid formation by activating IRE1α-XBP1-mediated ER stress, RCN1 may be a potential biomarker for treatment of keloid.

摘要

背景

据报道，网钙蛋白1（RCN1）在瘢痕疙瘩中上调，但其分子机制仍不清楚。本研究旨在探讨RCN1在瘢痕疙瘩中的作用。

方法

检测瘢痕疙瘩组织中RCN1的表达。用RCN1过表达载体转染瘢痕疙瘩成纤维细胞。检测细胞活力、胶原蛋白产生、细胞凋亡和细胞侵袭。然后，通过甲基化RNA免疫沉淀（MeRIP）检测RCN1 mRNA的m6A修饰水平，并评估ALKB同源物5（ALKBH5）过表达对RCN1 mRNA m6A修饰水平的影响。随后，通过免疫共沉淀（Co-IP）试验验证RCN1与XBP1之间的关系。将pcDNA-RCN1和XBP1 shRNA转染到瘢痕疙瘩成纤维细胞中进行逆转实验，通过透射电子显微镜（TEM）观察瘢痕疙瘩成纤维细胞内质网（ER）结构的变化。最后，我们建立了小鼠瘢痕疙瘩模型，并给小鼠注射RCN1 shRNA慢病毒载体，以监测小鼠瘢痕疙瘩的形成。

结果

RCN1在瘢痕疙瘩组织和瘢痕疙瘩成纤维细胞中高表达。RCN1的过表达显著提高了瘢痕疙瘩成纤维细胞的活力、胶原蛋白产生和侵袭能力，但抑制了细胞凋亡。ALKBH5通过减少m6A-YTHDF2介导的RCN1 mRNA降解而上调RCN1表达，RCN1基因敲低逆转了ALKBH5过表达对瘢痕疙瘩成纤维细胞活力、胶原蛋白产生和侵袭的促进作用，以及ALKBH5过表达对细胞凋亡的抑制作用。此外，RCN1的过表达显著上调了XBP1、GRP78和IRE1α的蛋白水平，并促进了瘢痕疙瘩成纤维细胞的内质网应激，但这种变化被sh-XBP1干预消除。体内实验表明，RCN1基因敲低通过减轻小鼠细胞凋亡和内质网应激显著抑制瘢痕疙瘩形成。