| 方少芬,冯阳,张琦,等.RNA m6A甲基转移酶促进中波紫外线辐射诱导皮肤损伤的作用研究[J].中华放射医学与防护杂志,2024,44(7):555-561.Fang Shaofen,Feng Yang,Zhang Qi,et al.Study on the role of RNA m6A methyltransferase in promoting ultraviolet B radiation-induced skin injury[J].Chin J Radiol Med Prot,2024,44(7):555-561 |
| RNA m6A甲基转移酶促进中波紫外线辐射诱导皮肤损伤的作用研究 |
| Study on the role of RNA m6A methyltransferase in promoting ultraviolet B radiation-induced skin injury |
| 投稿时间：2024-01-08 |
| DOI：10.3760/cma.j.cn112271-20240108-00006 |
| 中文关键词: 中波紫外线 辐射皮肤损伤 METTL14 RNA m6A甲基化修饰 |
| 英文关键词:Ultraviolet B Radiation-induced skin injury METTL14 RNA m6A methylation |
| 基金项目:国家自然科学基金(U1967220,81872552,12075165);国家重点研发计划项目(2022YFC2503700,2022YFC2503703) |
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| 中文摘要: |
| 目的 研究RNA m6A甲基转移酶(METTL14)在中波紫外线(UVB)辐射诱导皮肤损伤中的调控作用,初步探讨靶向抑制METTL14干预UVB诱导皮肤损伤的可能性。方法 C57BL/6J小鼠经150 mJ/cm2 UVB暴露构建辐射皮肤损伤模型,通过HE染色和Masson染色观察皮肤损伤情况,并进行病理评分。采用人永生化角质形成细胞HaCaT和人皮肤成纤维细胞WS1分别暴露于10和30 mJ/cm2 UVB照射,构建UVB辐射损伤细胞模型。采用RNA m6A甲基化比色法定量检测上述小鼠模型皮肤组织及细胞模型受照后的m6A水平,并通过Western blot法检测照射后细胞中m6A相关蛋白的表达情况。采用重组腺病毒载体构建过表达METTL14的HaCaT和WS1细胞系,并通过Western blot法检测过表达效果。检测上述细胞系的m6A水平;通过克隆形成实验检测上述细胞UVB暴露后的增殖能力,流式细胞术检测细胞凋亡的变化。采用UVB辐射皮肤损伤小鼠模型,给药组于照前和照后连续两次皮下注射METTL14抑制剂S-腺苷半胱氨酸(SAH)溶液(1、5 mg/kg),通过HE染色和Masson染色观察照射后皮肤损伤情况,进行损伤评分比较。结果 150 mJ/cm2 UVB照射后第4天小鼠皮肤组织发生显著损伤,主要病理改变表现为皮肤炎症浸润,组织结构紊乱,胶原纤维降解,损伤评分达到极点。在10、30 mJ/cm2照射后24 h, HaCaT、WS1细胞存活率均显著降低(t = 7.64、7.15, P ＜ 0. 05)。UVB照射后第4天,小鼠皮肤组织中m6A水平下调(t = 3.07, P ＜ 0.05)。受照后24 h,在HaCaT和WS1细胞中检测到m6A水平下调(t = 4.78、4.36, P ＜ 0.05),METTL14蛋白表达呈时间依赖性表达下调(t = 6.39、4.76, P ＜ 0.05)。成功构建过表达METTL14的HaCaT和WS1细胞系,过表达METTL14上调m6A水平(t = 7.66、3.67, P ＜ 0.05),抑制细胞UVB照射后的克隆形成能力(t = 6.29、3.84、5.37、5.44, P ＜ 0.05),显著增加细胞凋亡发生率(t = 3.48、9.54, P ＜ 0.05)。在UVB辐射皮肤损伤小鼠模型中,与生理盐水组相比,给药组(5 mg/kg SAH)的小鼠皮肤损伤病理评分明显减轻(t = 3.21、4.27、5.81, P ＜ 0.05),皮肤炎症浸润减少,组织结构有序,胶原纤维降解减少。结论METTL14促进皮肤细胞对UVB辐射敏感性,靶向抑制METTL14可有效缓解UVB辐射引起的皮肤损伤,有可能成为UVB辐射皮肤损伤的潜在治疗新靶点。 |
| 英文摘要: |
| Objective To investigate the regulatory role of RNA m6A methyltransferase (METTL14) in ultraviolet B (UVB) radiation-induced skin injury, and to preliminarily explore the potential of targeted inhibition of METTL14 for treating UVB-induced skin injury. Methods A UVB radiation-induced skin injury model was established by exposing C57BL/6J mice to 150 mJ/cm2 UVB, and was assessed and scored with HE staining and Masson staining. UVB radiation-induced cell injury models were established by exposing human immortalized keratinocytes (HaCaT) and human skin fibroblasts (WS1) to 10 and 30 mJ/cm2 UVB, respectively. The m6A levels in the mouse skin and cell models after UVB exposure were quantified by colorimetric assay, and m6A-related enzymes in cells were measured by Western blot. HaCaT and WS1 cell lines overexpressing METTL14 were constructed using recombinant adenoviral vectors, and the overexpression effects were tested by Western blot. The METTL14 overexpression cells were examined for their m6A levels, proliferative abilities after UVB exposure (by clone formation assay), and changes in apoptosis (by flow cytometry). The model mice with UVB-induced skin injury in the treatment groups received subcutaneous injection of the METTL14 inhibitor S-adenosylhomocysteine (SAH) solution (1 mg/kg, 5 mg/kg) twice consecutively before and after irradiation; and the mice were assessed and scored for skin injury with HE staining and Masson staining. Results On the 4th day after 150 mJ/cm2 UVB irradiation, the mice showed remarkable skin injury, pathologically featuring inflammatory infiltration, tissue structure disorganization, and collagen fiber degradation, reaching the maximum score; and the m6A level in the skin was significantly downregulated (t = 3.07, P ＜ 0.05). At 24 h after 10 and 30 mJ/cm2 irradiation, HaCaT and WS1 cells showed significantly reduced survival rates (t = 7.64, 7.15, P ＜ 0.05), significantly downregulated m6A levels (t = 4.78, 4.36, P <0.05), and significantly time-dependent downregulation of METTL14 protein expression (t = 6.39, 4.76, P ＜ 0.05). In HaCaT and WS1 cells, METTL14 overexpression significantly up-regulated m6A levels (t = 7.66, 3.67, P ＜ 0.05), significantly inhibited the clone-forming ability of cells after UVB irradiation (t = 6.29, 3.84, P ＜ 0.05), and significantly increased the rate of cell apoptosis (t = 3.48, 9.54, P ＜ 0.05). Compared with those in the normal saline group, the model mice with UVB-induced skin injury in the SAH treatment group (5 mg/kg) showed significantly decreased pathological scores of skin injury (t = 3.21, 4.27, 5.81, P ＜ 0.05), with milder inflammatory infiltration, more orderly tissue structure, and less collagen fiber degradation. Conclusions METTL14 can increase the sensitivity of skin cells to UVB radiation, and targeted inhibition of METTL14 can effectively alleviate UVB radiation-induced skin injury, which may be a potential new target for the treatment of UVB radiation-induced skin injury. |
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