miR-29a调控TGF-β1/Smad3通路在稀土氧化钕致肺纤维化过程中的作用
Research of miR-29a on TGF-β1/Smad3 pathway in pulmonary fibrosis induced by neodymium oxide
云玉丹 1王素华2
作者信息
- 1. 泰国格乐大学国际学院公共卫生系,泰国曼谷
- 2. 泰国格乐大学国际学院公共卫生系,泰国曼谷;包头医学院公共卫生学院,包头 014040
- 折叠
摘要
目的 探讨稀土氧化钕(Nd2O3)致小鼠肺纤维化过程中miR-29a对转化生长因子-β1(TGF-β1)/Smad同源物3(Smad3)通路的调控作用.方法 于2021年3月,选择SPF级C57/BL6J雄性小鼠72只,随机分为对照组、Nd2O3组、Nd2O3+miR-29a干预剂(agomir)组、Nd2O3+NC agomir组,每组18只.Nd2O3组、Nd2O3+miR-29a agomir组、Nd2O3+NC agomir组采用非暴露式气管滴注法染毒,染尘浓度为250 mg/ml,染尘体积为0.1 ml,对照组给予同体积生理盐水.染尘后,每3天Nd2O3+miR-29a agomir组小鼠尾静脉注射 0.1 ml(5 nmol)miR-29a agomir,Nd2O3+NC agomir 组小鼠尾静脉注射 0.1 ml NC agomir.染尘后第7、14、28天每组各处死6只小鼠,取小鼠肺组织,HE染色观察小鼠肺组织病理状况;ELISA法检测TGF-β1、结缔组织生长因子(CTGF)含量;qRT-PCR检测TGF-β1 mRNA的表达量;免疫荧光法检测小鼠肺组织中Smad3的表达量,利用TargetScan7、miRDB等网站工具预测miR-29a靶基因.计量资料满足正态分布时用(x)±s表示,组间比较用两独立样本t检验,两两比较方差齐时用LSD法检验.结果 HE染色显示,Nd2O3组小鼠肺组织初期出现明显的炎症细胞浸润、肺泡结构紊乱,28 d时,小鼠肺组织胶原纤维增多且肺组织呈纤维化蜂窝状变,Nd2O3+miR-29a agomir组小鼠肺组织纤维化程度明显减轻;与Nd2O3+NC agomir组比较,Nd2O3+miR-29a agomir组小鼠肺组织中TGF-β1、CTGF的含量较低,小鼠肺组织中TGF-β1 mRNA的相对表达量较低,细胞核中Smad3表达水平较低,差异均有统计学意义(P＜0.05).miR-29a 相关下游靶基因有152个,其中 FBN1、MAP2K6、KPNB1、COL1A2、SNIP1、LAMC1、SP1 可能与TGF-β1/Smad3通路的调控有关.结论 miR-29a可能通过调节TGF-β1/Smad3信号通路影响稀土 Nd2O3暴露所致的小鼠肺纤维化,过表达miR-29a可能抑制TGF-β1/Smad3信号通路而减轻小鼠肺纤维化程度.
Abstract
Objective To exploring the regulatory effect of miR-29a on the transforming growth factor-β1(TGF-β1)/Smad homolog 3(Smad3)pathway during the process of rare earth neodymium oxide(Nd2O3)induced pulmonary fibrosis in mice.Methods In March 2021,72 SPF grade C57/BL6J male mice were selected and randomly divided into a control group,Nd2O3 group,Nd2O3+miR-29a agomir group,and Nd2O3+NC agomir group,with 18 mice in each group.The Nd2O3 group,Nd2O3+miR-29a agomir group,and Nd2O3+NC agomir group were treated with non exposed tracheal instillation,with a dust concentration of 250 mg/ml and a dust volume of 0.1 ml.The control group was given the same volume of physiological saline.After exposure to Nd2O3,0.1 ml(5 nmol)of miR-29a agomir was injected into the tail vein of mice in the Nd2O3+miR-29a agomir group every 3 days,while 0.1 ml of NC agomir was injected into the tail vein of mice in the Nd2O3+NC agomir group.On the 7 th,14 th,and 28 th days after dust exposure,6 mice were killed in each group,and the lung tissue of the mice was taken out.HE staining was used to observe the pathological status of the mouse lung tissue;ELISA method was used to detect the levels of TGF-β1 and connective tissue growth factor(CTGF)in lung tissue;Use qRT-PCR detection method to detect the expression level of TGF-β1 mRNA;Using immunofluorescence assay to detect the expression level of Smad3 in mouse lung tissue;Use bioinformatics websites such as TargetScan7 and miRDB to predict the target gene of miR-29a.When the metrological date were satisfied with normal distribution,Mean±SD was used for comparison between groups,t test was used for two indepent samples,and LSD method was used when the variance was homogeneity in pairwise comparison.Results HE staining showed that the Nd2O3 group of mice showed obvious infiltration of inflammatory cells and structural disorder of alveoli in the early stage of lung tissue.At 28 days,the collagen fibers in the mouse lung tissue increased and the lung tissue showed fibrotic honeycomb like changes.The degree of pulmonary fibrosis in the Nd2O3+miR-29a agomir group of mice was significantly reduced;The content of TGF-β1 and CTGF in the lung tissue of mice in the Nd2O3+miR-29a agomir group was lower than that in the Nd2O3+NC agomir group(P＜0.05);The relative expression level of TGF-β1 in the lung tissue of mice in the Nd2O3+miR-29a agomir group was lower than that in the Nd2O3+NC agomir group(P＜0.05);The expression level of Smad3 in the nucleus of the Nd2O3+miR-29a agomir group was lower than that of the Nd2O3+NC agomir group(P＜0.05).The prediction results of bioinformatics websites have found 152 downstream target genes related to miR-29a,among which FBN1,MAP2K6,KPNB1,COL1A2,SNIP1,LAMC1,and SP1 genes may be related to the regulatory effect of miR-29a on TGF-β1/Smad3 signaling pathway.Conclusion miR-29a may affect lung fibrosis induced by rare earth Nd2O3 exposure in mice by regulating TGF-β1/Smad3 signaling pathway.Overexpression of miR-29a may inhibit TGF-β1/Smad3 signaling pathway and reduce the degree of pulmonary fibrosis in mice.
关键词
稀土元素/小鼠/稀土氧化钕/肺纤维化/转化生长因子-β1/Smad同源物3Key words
Lanthanoid series dements/Mice/Rare earth neodymium oxide/Pulmonary fibrosis/Transforming Growth Factor-β1/Smad homolog 3引用本文复制引用
基金项目
国家自然科学基金(81860575)
内蒙古自然科学基金(2022MS08045)
出版年
2024
NETL
NSTL
维普
万方数据