GSE202851
The m6A Methylated mRNA Pattern and the Activation of the Wnt Signaling Pathway under Hyper-m6A modifying condition in the keloid [RNA-seq]
Purpose: The present study was carried out to investigate the global m6A modified RNA pattern and possible mechanisms underlying the pathogenesis of keloid.     Method: 14 normal skin and 14 keloid tissue were firstly collected on clinic. Then 3 samples from each group were randomly selected to be verified with the Western Blot 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. With the 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 with the Western Blot and immunofluorescence staining.     Results: There was a higher protein level of WTAP and Mettl3 in the keloid than in the normal tissue. 21,020 unique m6A peaks with 6573 unique m6A-associated genetic transcripts appeared in the keloid samples. In the normal tissue, 4028 unique m6A peaks with 779-associated modified genes appeared in the keloid. 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 / ß-catenin pathway in keloid was verified with western blotting. From immunofluorescence staining results, we found that the accumulated fibroblasts were under a hyper-m6A condition in the keloid and the Wnt/ß-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 / ß-catenin pathway in the dermal fibroblasts might promote the pathogenesis of keloid. Overall design: Patients And Samples  This study was approved by the Ethics Committee of the First Affiliated Hospital of Jinan University, and informed consent was obtained from all patients and unaffected individuals.  14 keloid samples were collected from 14 patients at the Department of plastic surgery at the First Affiliated Hospital of Jinan University (Table 1). In addition, 14 individuals without a personal or family history of keloid were recruited from the Department of thoracic surgery and general surgery at the First Affiliated Hospital of Jinan University. Normal skin samples were obtained from these volunteers (Table 1). After being isolated, the samples were divided into two parts: One was immediately frozen and preserved in liquid nitrogen for the molecular analysis of MeRIP-Seq, RNA-Seq, and western blotting test. The other part was stored in the 4% formalin for the pathological change analysis of immunofluorescence staining.