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中华肺部疾病杂志(电子版) ›› 2025, Vol. 18 ›› Issue (02) : 220 -225. doi: 10.3877/cma.j.issn.1674-6902.2025.02.003

论著

miR-21 靶向环腺苷酸应答元件结合蛋白样蛋白2 加重放射性肺纤维化的作用机制
李杰1,2, 冉永红1, 郝玉徽1,()   
  1. 1. 400038 重庆,陆军军医大学军事预防医学系,重庆纳米医学工程研究中心,创伤与化学中毒国家重点实验室
    2. 125199 兴城,联勤保障部队兴城特勤疗养中心
  • 收稿日期:2025-02-08 出版日期:2025-04-25
  • 通信作者: 郝玉徽
  • 基金资助:
    重庆市自然科学基金(2022NSCQ-MSX467)重庆市教委科技研究计划项目(KJZD-K20231280)

miR-21 promotes radiation-induced pulmonary fibrosis by targeting CREBL2

Jie Li1,2, Yonghong Ran1, Yuhui Hao1,()   

  1. 1. State Key Laboratory of Trauma and Chemical Poisoning,Institute of Combined Injury,Chongqing Engineering Research Center for Nanomedicine,College of Preventive Medicine,Army Medical University,Chongqing 400038,China
    2. Joint Logistics Support Force Xingcheng Special Service Sanatorium Center,Xincheng 125199,China
  • Received:2025-02-08 Published:2025-04-25
  • Corresponding author: Yuhui Hao
引用本文:

李杰, 冉永红, 郝玉徽. miR-21 靶向环腺苷酸应答元件结合蛋白样蛋白2 加重放射性肺纤维化的作用机制[J/OL]. 中华肺部疾病杂志(电子版), 2025, 18(02): 220-225.

Jie Li, Yonghong Ran, Yuhui Hao. miR-21 promotes radiation-induced pulmonary fibrosis by targeting CREBL2[J/OL]. Chinese Journal of Lung Diseases(Electronic Edition), 2025, 18(02): 220-225.

目的

Ⅱ型肺泡上皮细胞(type Ⅱalveolar epithelial cells,AT2)功能障碍被认为是放射性肺纤维化(radiation-induced pulmonary fibrosis,RIPF)形成的关键病理过程之一,微小核糖核酸-21(microRNA,miR-21)介导上皮-间质转化(epithelial to mesenchymal transition,EMT)参与RIPF 形成,但中间分子尚待明确。 本文分析miR-21 与环腺苷酸应答元件结合蛋白样蛋白2(cAMP responsive element binding protein like 2,CREBL2)在RIPF 中的分子机制和关键作用。

方法

野生(wild type,WT)小鼠以及miR-21-/-小鼠的胸部被15Gy 钴源γ 射线辐照建立RIPF 模型。 通过活体肺功能检查,肺组织羟脯氨酸检测评估RIPF 小鼠的肺纤维化程度。 构建大鼠AT2 细胞系(rat type Ⅱalveolar epithelial cells line,RLE-6TN)辐射模型,采用RT-qPCR 和WB 对内质网应激(endoplasmic reticulum stress,ERS)以及EMT相关分子进行检测。 通过基因芯片阵列筛选差异表达基因并采用双荧光素酶实验进行验证。

结果

WT小鼠辐照后miR-21 表达增加14 倍,羟脯氨酸表达上升2 倍以上,CREBL2 降低50%;而miR-21-/-小鼠辐照后CREBL2 增加3 倍,相较WT 小鼠肺纤维化程度减轻,肺组织中ERS 和EMT 标志分子显著降低。双荧光素酶实验明确miR-21 对CREBL2 的特异性结合。

结论

肺组织AT2 细胞受到辐照导致miR-21增加和CREBL2 降低,进一步激活ERS 信号通路促进AT2 细胞发生EMT,最后导致RIPF 的形成。

Objective

Dysfunction of type Ⅱalveolar epithelial cells (AT2) is considered one of the key pathological processes underlying the development of radiation-induced pulmonary fibrosis (RIPF). miR-21 regulates epithelial to mesenchymal transition (EMT) during RIPF. However,the target gene through which miR-21 acts upon in the regulation of EMT remains to be confirmed. This study primarily investigates the molecular mechanisms and critical roles of miR-21 and CREBL2 in RIPF.

Methods

The chests of wild type(WT) mice and miR-21-/- mice were irradiated with 15Gy cobalt source γ-rays to establish a RIPF model.Through living lung function examination and lung hydroxyproline detection to evaluate the degree of pulmonary fibrosis in irradiated mice. Establishment of the rat type Ⅱalveolar epithelial cells line (RLE-6TN) irradiation model,the RT-qPCR and WB were used to detect the expression of endoplasmic reticulum stress (ERS) and EMT related markers in irradiated cells. Differentially expressed genes were identified through a microarray screen and validated using a dual-luciferase reporter assay.

Results

After irradiation,WT mice exhibited a 14-fold increase in miR-21 expression at 8 weeks post-irradiation. Additionally,hydroxyproline expression increased by more than 2-fold,while CREBL2 expression decreased by 50%. In contrast,miR-21-/- mice demonstrated an approximately 3-fold increase in CREBL2 expression following irradiation. The miR-21-/- mice exhibited a reduced degree of lung fibrosis compared to WT mice. The markers of ERS and EMT in the lung tissue of miR-21-/- mice were significantly reduced. Dual-luciferase reporter assays confirmed the specific binding of miR-21 to CREBL2.

Conclusion

Radiation-induced increase of miR-21 and decreased expression of its target gene CREBL2 in AT2 cells further activated the ERS signaling pathway,mediated EMT in AT2 cells,and finally led to the formation of RIPF.

图1 miR-21 基因敲除小鼠RIPF 压力-体积检查
图2 miR-21 靶基因CREBL2 调节ERS 和EMT。 图A 为CREBL2 3′-UTR WT/MUT 和miR-21 mimic/NC 混匀后分别转染HEK-293T 细胞,检测荧光值;图B 为辐照处理前对RLE-6TN 细胞进行CREBL2 过表达质粒(3 μg)转染且共孵育24 h,采用RT-qPCR 和WB 检测辐照48 h 后各分子的表达;图C 为辐照处理前对RLE-6TN 细加入CREB 抑制剂666-15(500 nM)处理1 h,辐照48 h RT-qPCR 和WB 检测各分子表达。 *P<0.05
图3 辐照引起ERS 促进EMT 导致RIPF。 图A 为RIPF 小鼠辐照0 周和16 周肺组织CHOP 免疫组化染色半定量统计。 图B、C 为RLE-6TN 细胞使用ERS 抑制剂4-PBA(1 mM)处理1 h 或CHOP 过表达质粒(3 μg)共培养24 h,8Gy 辐照后继续培养48 h,RT-qPCR 和WB 检测各分子表达。 使用t 检验进行两组间比较,*P<0.05
图4 4-PBA 注射WT 小鼠RIPF 模型压力-体积检查
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