Wu Jianguo, Huang Haozong, Yang Wenkai, Xue Tufeng, Wang Wenjuan, Zheng Guang-Di
Department of Cardiac and Macrovascular Surgery, Central People's Hospital of Zhanjiang, Guangdong province, China.
Heliyon. 2024 May 23;10(11):e31871. doi: 10.1016/j.heliyon.2024.e31871. eCollection 2024 Jun 15.
Transient receptor potential melastatin 4 (TRPM4) affects immune responses by regulating calcium homeostasis, but its role in calcific aortic valve inflammation remains unclear. This study aimed to assess the expression and function of TRPM4 in patients with or without calcific aortic valve disease (CAVD).
The mRNA and protein expression levels of TRPM4 and related factors in calcified and noncalcified tissues were measured using qRT-PCR and Western blot. The proteins interacting with TRPM4 were confirmed by RNA pull-down and RNA immunoprecipitation assays. Dual-Luciferase Reporter Assay was performed to confirm the m6A site of TRPM4.
The mRNA expression levels of TRPM4, TLR4, IL-6, MCP-1, TNF-α, and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues, and TRPM4 was significantly positively correlated with inflammation-related factors. The protein expression level of TRPM4, TLR4 and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues. N6-methyladenosine (m6A) modification of TRPM4 mRNA by METTL3-YTHDF1 up-regulated its expression in CAVD. And TRPM4 promoted the level of inflammation via activation of the JNK-MAPK signaling pathway, after knockdown TRPM4, the production of proinflammatory cytokines was significantly suppressed.
The results indicate the pivotal role of TRPM4 in CAVD and highlight METTL3-mediated m6A modification of TRPM4 in promoting inflammation through JNK-MAPK signaling pathway. This work provides potential therapeutic strategy to impede inflammation in CAVD.
瞬时受体电位香草酸亚家族成员4（TRPM4）通过调节钙稳态影响免疫反应，但其在钙化性主动脉瓣炎症中的作用尚不清楚。本研究旨在评估TRPM4在钙化性主动脉瓣疾病（CAVD）患者和非钙化性主动脉瓣疾病患者中的表达及功能。
采用qRT-PCR和蛋白质免疫印迹法检测钙化组织和非钙化组织中TRPM4及相关因子的mRNA和蛋白质表达水平。通过RNA下拉和RNA免疫沉淀试验确认与TRPM4相互作用的蛋白质。进行双荧光素酶报告基因检测以确认TRPM4的m6A位点。
钙化主动脉瓣组织中TRPM4、Toll样受体4（TLR4）、白细胞介素6（IL-6）、单核细胞趋化蛋白1（MCP-1）、肿瘤坏死因子-α（TNF-α）和核因子κB p65（NF-κB p65）的mRNA表达水平显著高于非钙化组织，且TRPM4与炎症相关因子显著正相关。钙化主动脉瓣组织中TRPM4、TLR4和NF-κB p65的蛋白质表达水平显著高于非钙化组织。METTL3-YTHDF1对TRPM4 mRNA的N6-甲基腺苷（m6A）修饰上调了其在CAVD中的表达。TRPM4通过激活JNK-丝裂原活化蛋白激酶（MAPK）信号通路促进炎症水平，敲低TRPM4后，促炎细胞因子的产生显著受到抑制。
结果表明TRPM4在CAVD中起关键作用，并突出了METTL3介导的TRPM4的m6A修饰通过JNK-MAPK信号通路促进炎症。这项工作为阻止CAVD中的炎症提供了潜在的治疗策略。
