RNA m^(6)A修饰在衰老相关特发性肺纤维化的作用研究  

Research on the mechanism of RNA m^(6)A modification in age-related idiopathic pulmonary fibrosis

作　　者：钱力[1] 孙梓越 韩永康 杜毓锋[1] 王小慧[1] 刘学军[1] 李丹[1,2] Qian Li;Sun Ziyue;Han Yongkang;Du Yufeng;Wang Xiaohui;Liu Xuejun;Li Dan(Department of Geriatrics,First Hospital of Shanxi Medical University,Taiyuan 030001,China;First Clinical Medical College,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院老年病科,太原030001 [2]山西医科大学第一临床医学院,太原030001

出　　处：《中华老年医学杂志》2025年第1期51-59,共9页Chinese Journal of Geriatrics

基　　金：山西省基础研究计划面上项目(20210302123248);山西省基础研究计划青年项目(202203021222373);山西省基础研究计划面上项目(20210302123249);山西省卫生健康委科研课题(2021051)。

摘　　要：目的探讨RNA m^(6)A修饰及其关键调节因子在衰老相关肺纤维化中的表达变化。方法通过收集特发性肺间质纤维化(idiopathic pulmonaryfibrosis,IPF)患者外周血标本,探讨老年IPF患者总RNA的m^(6)A修饰水平变化以及关键的调控因子,构建了青年与老年小鼠肺纤维化模型来进行验证。分别收集10例IPF患者及正常对照组(年龄均大于60岁)的外周血标本,进行RNA m^(6)A甲基化定量检测及实时定量PCR检测关键调节因子METTL3、METTL14及FTO mRNA的含量。16只10~12周龄C57BL/6雄性小鼠随机分为:A青年对照组、B青年肺纤维化组,16只6~7月龄小鼠随机分为:C老年对照组、D老年肺纤维化组,共4组,每组8只。肺纤维化组采用气管内注入博来霉素(5 mg/kg)制造肺纤维化模型,对照组则注入生理盐水,造模28 d后取肺组织,通过HE、Masson染色及羟脯氨酸含量测定检测小鼠肺组织病理变化,免疫组化检测肺组织(TGF-β1)、(α-SMA)的表达,通过RNA m^(6)A甲基化定量试剂盒(比色法)检测肺组织总RNA m^(6)A修饰水平,实时定量PCR和Western blot实验检测小鼠肺组织METTL3 mRNA和蛋白水平表达。结果老年IPF患者组(0.36±0.03)与健康对照组(0.25±0.02)相比m^(6)A修饰水平升高t=4.882(P<0.05);老年IPF患者组METTL3的表达较对照组显著升高t=6.082(P<0.05),METTL14的表达则显著降低t=17.58(P<0.05),FTO的表达水平无显著差异。老年小鼠肺纤维化程度较青年小鼠更加严重;免疫组化结果显示TGF-β1在肺纤维化组表达增高,D组TGF-β1表达高于B组(t=5.891,P<0.05),C组高于A组t=4.135(P<0.05)。α-SMA在肺纤维化小鼠模型中阳性面积占比明显高于对照组t=20.08(P<0.05)。两肺纤维化组与正常对照组相比m^(6)A修饰水平升高(P<0.05),而D组与B组比较无显著差异。肺纤维化组METTL3 mRNA和蛋白表达上调,而D组表达低于B组(P<0.05)。结论RNA m^(6)A修饰水平在肺纤维化中升高,METTL3在肺纤维化中表达上调,同�Objective This study aims to investigate the alterations in m^(6)A methylation associated with age-related idiopathic pulmonary fibrosis(IPF).MethodsBy collecting peripheral blood samples from IPF patients,we investigated the changes in m6A modification levels of total RNA and key regulatory factors in elderly IPF patients.Then,the pulmonary fibrosis models of young and old mice were constructed for verification.A total of 10 IPF patients and 10 healthy controls were selected for this study.The m^(6)A methylation quantitative kit was employed to assess the m^(6)A modification levels of total RNA.The expression levels of key m^(6)A methylation regulators,METTL3,METTL14,and FTO,were quantified using qRT-PCR.Additionally,thirty-two healthy male C57BL/6 mice,comprising 16 mice aged 10-12 weeks and 16 mice aged 6-7 months,were divided into four groups:young control(A),young pulmonary fibrosis(B),aged control(C),and aged pulmonary fibrosis(D),with 8 mice in each group.Mice in groups B and D were intratracheally administered bleomycin to establish a pulmonary fibrosis model,while those in groups A and C received normal saline.Twenty-eight days post-model establishment,the mice were euthanized,and lung tissues were collected for analysis.Histological evaluations were performed using hematoxylin and eosin(HE)staining,Masson staining,hydroxyproline content determination,and immunohistochemistry to assess the extent of pulmonary fibrosis.The m^(6)A methylation quantification kit was also utilized to measure the m^(6)A modification levels of total RNA in lung tissue.Furthermore,the mRNA and protein expression levels of the methyltransferase METTL3 were assessed by qRT-PCR and Western blot experiments.ResultsThe level of m^(6)A modification was significantly elevated in the aged IPF patient group(0.36±0.03)compared to the control group t=4.882(P<0.05).Furthermore,the expression of METTL3 was markedly higher in the aged IPF patients(t=6.082),while the expression of METTL14 was significantly lower t=17.58(P<0.05).In contrast,t

关 键 词：特发性肺纤维化 衰老 m^(6)A修饰 甲基转移酶样蛋白3 

分 类 号：R563[医药卫生—呼吸系统]