The m6A-methylated mRNA pattern and the activation of the Wnt signaling pathway under the hyper-m6A-modifying condition in the keloid

Can-Xiang Lin; Zhi-Jing Chen; Qi-Lin Peng; Ke-Rong Xiang; Du-Qing Xiao; Ruo-Xi Chen; Taixing Cui; Yue-Sheng Huang; Hong-Wei Liu

doi:10.3389/fcell.2022.947337

The m⁶A-methylated mRNA pattern and the activation of the Wnt signaling pathway under the hyper-m⁶A-modifying condition in the keloid

Front Cell Dev Biol. 2022 Oct 3:10:947337. doi: 10.3389/fcell.2022.947337. eCollection 2022.

Authors

Can-Xiang Lin¹, Zhi-Jing Chen¹, Qi-Lin Peng², Ke-Rong Xiang¹, Du-Qing Xiao³, Ruo-Xi Chen¹, Taixing Cui⁴, Yue-Sheng Huang⁵, Hong-Wei Liu¹

Affiliations

¹ Key Laboratory of Regenerative Medicine, Ministry of Education, Department of Plastic Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, China.
² The Research Center of Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
³ Department of Thoracic Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, China.
⁴ Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, United States.
⁵ Department of Wound Repair, Institute of Wound Repair and Regeneration Medicine, Southern University of Science and Technology Hospital, Southern University of Science and Technology School of Medicine, Shenzhen, China.

Abstract

Purpose: The present study was carried out to investigate the global m⁶A-modified RNA pattern and possible mechanisms underlying the pathogenesis of keloid. Method: In total, 14 normal skin and 14 keloid tissue samples were first collected on clinics. Then, three samples from each group were randomly selected to be verified with the Western blotting to determine the level of methyltransferase and demethylase. The total RNA of all samples in each group was isolated and subjected to the analysis of MeRIP sequencing and RNA sequencing. Using software of MeTDiff and htseq-count, the m⁶A peaks and differentially expressed genes (DEGs) were determined within the fold change >2 and p-value < 0.05. The top 10 pathways of m⁶A-modified genes in each group and the differentially expressed genes were enriched by the Kyoto Encyclopedia of Genes and Genomes signaling pathways. Finally, the closely associated pathway was determined using the Western blotting and immunofluorescence staining. Results: There was a higher protein level of WTAP and Mettl3 in the keloid than in the normal tissue. In the keloid samples, 21,020 unique m⁶A peaks with 6,573 unique m⁶A-associated genetic transcripts appeared. In the normal tissue, 4,028 unique m⁶A peaks with 779 m⁶A-associated modified genes appeared. In the RNA sequencing, there were 847 genes significantly changed between these groups, transcriptionally. The genes with m⁶A-methylated modification and the upregulated differentially expressed genes between two tissues were both mainly related to the Wnt signaling pathway. Moreover, the hyper-m⁶A-modified Wnt/β-catenin pathway in keloid was verified with Western blotting. From the immunofluorescence staining results, we found that the accumulated fibroblasts were under a hyper-m⁶A condition in the keloid, and the Wnt/β-Catenin signaling pathway was mainly activated in the fibroblasts. Conclusion: The fibroblasts in the keloid were under a cellular hyper-m⁶A-methylated condition, and the hyper-m⁶A-modified highly expressed Wnt/β-catenin pathway in the dermal fibroblasts might promote the pathogenesis of keloid.

Keywords: RNA sequencing; fibroblasts; keloid; m6A modification; the Wnt signaling pathway.

Grants and funding

Funding for this study was provided by the National Nature and Science Foundation, China (81871563 and 81804153), the Medical Research Foundation of Guangdong Province (A2021165), the Fundamental Research Funds for the Central Universities (21619350), and the Guangdong Foundation for Basic and Applied Basic Research (grant no. 2019A1515110161).