光老化成纤维细胞中m6A甲基化修饰的差异表达  

Differential Expression of m6A-Methylated mRNA in Photoaged Human Fibroblasts

作　　者：钱雯 李敏 骆丹[2] QIAN Wen;LI Min;LUO Dan(Departments of Dermatology,Children′s Hospital of Nanjing Medical University,Nanjing 210008,China;Departments of Dermatology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学附属儿童医院皮肤科,江苏南京210008 [2]南京医科大学第一附属医院皮肤科,江苏南京210029

出　　处：《中国皮肤性病学杂志》2025年第3期265-272,共8页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金项目(81972961)。

摘　　要：目的研究中波紫外线(UVB)诱导的光老化成纤维细胞及正常细胞中mRNA的N6-甲基腺苷(m6A)甲基化修饰水平的差异表达。方法体外培养人成纤维细胞,用UVB照射建立光老化模型。将细胞分为对照组和照光组,对照组给予常规培养,照光组给予多次重复照射UVB(总剂量51 mJ/cm^(2))。采用CCK-8和流式法检测细胞活性及细胞周期,β-半乳糖苷酶检测细胞衰老,Western blot和qRT-PCR检测衰老标志物p16、p21和p53的蛋白及mRNA表达水平,m6A微阵列芯片检测m6A差异表达的mRNA谱,并通过基因本体论(GO)分析和京都基因与基因组百科全书(KEGG)通路分析进行结果分析。结果通过UVB照射成功建立光老化模型。与对照组相比,照光组中m6A甲基化修饰差异表达的mRNA共2749个,表达上调的有1163个,表达下调的有1586个(P<0.05且Fold change≥1.5)。GO富集分析发现差异表达的基因主要参与DNA重组修复、有丝分裂、应激、代谢等生物学过程;KEGG通路富集分析发现在p53、肿瘤坏死因子(TNF)、核转录因子kappa B(NF-κB)等通路明显富集。结论UVB诱导的光老化成纤维细胞与正常细胞之间存在m6A甲基化修饰差异表达的mRNA,这些差异表达的mRNA可以为后续临床研究提供思路。Objective To analyze the differential expression of N6-methyladenosine(m6A)methylated mRNA in ultraviolet B(UVB)induced photoaged and non-photoaged human skin fibroblasts(HSFs).Methods Human fibroblasts were cultured in vitro and the photoaging model was established by UVB irradiation.Fibroblasts were divided into control group and UVB-irradiated group.The control group was given normal culture.The UVB-irradiated group was given repeated UVB irradiation(total dose 51 mJ/cm^(2)).Cell count kit-8(CCK-8)and cell cycle assay were used to detect the viability and cell cycle.β-galactosidase staining was used to observe the senescence of HSFs.The expression of protein and mRNA of p16,p21 and p53 were detected by Western blot and qRT-PCR.The m6A microarray analysis was used to detect the differential expression of m6A-methylated mRNAs,and the results were performed by GO analysis and KEGG analysis.Results The photoaging model was successfully constructed by UVB irradiation.Compared with the control group,there were 2749 abnormally expressed mRNAs in the UVB group,in which 1163 mRNAs were up-regulated while 1586 mRNAs were down-regulated(P<0.05 and Fold change≥1.5).GO enrichment analysis showed that these differential mRNAs were mainly involved in biological processes such as DNA recombination repair,mitosis,stress and metabolism.KEGG analysis showed that signal pathways such as p53,TNF,and NF-κB were significantly enriched.Conclusion There are differentially expressed m6A-methlyted mRNAs between UVB-induced photoaged human fibroblasts and normal fibroblasts,which can provide ideas for subsequent clinical research.

关 键 词：光老化 成纤维细胞 N6-甲基腺苷甲基化 MRNA 

分 类 号：R751[医药卫生—皮肤病学与性病学]