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

IF 2.3 4区医学 Q2 DERMATOLOGY

Journal of Cosmetic Dermatology Pub Date : 2025-04-11 DOI:10.1111/jocd.70177

Min Shi, Lu Zhang, Fangfang Bi, Zhuo Zhou

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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.

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ALKBH5 通过激活 IRE1α-XBP1 介导的ER应激，抑制 YTHDF2-m6A 介导的 RCN1 mRNA 降解，从而促进瘢痕疙瘩的形成

据报道，网状定位蛋白1 （RCN1）在瘢痕疙瘩中上调，但其分子机制尚不清楚。本研究的目的是探讨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。测定细胞活力、胶原蛋白生成、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，通过共免疫沉淀（Co-IP）实验验证RCN1与XBP1之间的关系。将pcDNA-RCN1和XBP1 shRNA转染瘢痕疙瘩成纤维细胞进行逆转实验，透射电镜（TEM）观察瘢痕疙瘩成纤维细胞内质网（ER）结构的变化。最后，我们建立小鼠瘢痕疙瘩模型，注射RCN1 shRNA慢病毒载体，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过表达显著增加瘢痕疙瘩成纤维细胞活力、胶原生成和侵袭，但抑制细胞凋亡。ALKBH5通过降低m6a - ythdf2介导的RCN1 mRNA降解而上调RCN1表达，RCN1敲低逆转了ALKBH5过表达对瘢痕疙瘩成纤维细胞活力、胶原生成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的过表达可显著上调XBP1、GRP78和IRE1α的蛋白水平，并促进瘢痕疙瘩成纤维细胞的内质网应激，但sh-XBP1干预可消除这种变化。体内实验表明，敲低RCN1可通过减轻小鼠细胞凋亡和内质网应激，显著抑制瘢痕疙瘩的形成。结论ALKBH5上调RCN1，通过激活ire1 α- xbp1介导的内质网应激促进瘢痕疙瘩形成，RCN1可能是治疗瘢痕疙瘩的潜在生物标志物。

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来源期刊

Journal of Cosmetic Dermatology DERMATOLOGY-

CiteScore

4.30

自引率

13.00%

发文量

818

审稿时长

>12 weeks

期刊介绍： The Journal of Cosmetic Dermatology publishes high quality, peer-reviewed articles on all aspects of cosmetic dermatology with the aim to foster the highest standards of patient care in cosmetic dermatology. Published quarterly, the Journal of Cosmetic Dermatology facilitates continuing professional development and provides a forum for the exchange of scientific research and innovative techniques. The scope of coverage includes, but will not be limited to: healthy skin; skin maintenance; ageing skin; photodamage and photoprotection; rejuvenation; biochemistry, endocrinology and neuroimmunology of healthy skin; imaging; skin measurement; quality of life; skin types; sensitive skin; rosacea and acne; sebum; sweat; fat; phlebology; hair conservation, restoration and removal; nails and nail surgery; pigment; psychological and medicolegal issues; retinoids; cosmetic chemistry; dermopharmacy; cosmeceuticals; toiletries; striae; cellulite; cosmetic dermatological surgery; blepharoplasty; liposuction; surgical complications; botulinum; fillers, peels and dermabrasion; local and tumescent anaesthesia; electrosurgery; lasers, including laser physics, laser research and safety, vascular lasers, pigment lasers, hair removal lasers, tattoo removal lasers, resurfacing lasers, dermal remodelling lasers and laser complications.