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miR-429靶向HMOX1调控肺腺癌细胞侵袭、迁移和铁死亡的分子机制研究

Molecular Mechanism of microRNA-429 Targeting HMOX1 to Regulate Invasion, Migration, and Ferroptosis in Lung Adenocarcinoma Cells

    摘要
  • 摘要:
    目的 探讨microRNA-429（miR-429）对肺腺癌细胞侵袭、迁移及铁死亡的影响，为临床抑制肺腺癌侵袭迁移、促进铁死亡提供新思路。
    方法 利用starBase数据库分析miR-429在肺腺癌组织中的表达谱。采用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测miR-429在正常肺支气管上皮细胞BEAS-2B及肺腺癌细胞系A549、H1975中的表达水平，并评估质粒转染效率。通过5-乙炔基-2'-脱氧尿苷（EdU）增殖实验、划痕愈合实验和Transwell侵袭实验评估miR-429对A549细胞增殖、迁移和侵袭能力的影响。基于生物信息学数据库预测miR-429潜在靶向的铁死亡相关蛋白，利用双荧光素酶报告基因实验验证miR-429与血红素氧合酶1（heme oxygenase 1, HMOX1）的靶向结合关系。进一步通过上述功能实验验证miR-429靶向HMOX1对A549细胞恶性表型的影响。检测活性氧（reactive oxygen species, ROS）及脂质活性氧（lipid reactive oxygen species, L-ROS）水平，分析miR-429及HMOX1对肺腺癌细胞氧化应激状态的调节作用。测定丙二醛（malondialdehyde, MDA）、谷胱甘肽（glutathione, GSH）及Fe2+含量，评估miR-429和HMOX1对肺腺癌细胞铁死亡关键指标的影响。
    结果 miR-429在肺腺癌细胞系A549、H1975中的表达显著高于正常BEAS-2B细胞，其中A549细胞表达水平最高。功能实验表明，miR-429可促进A549细胞增殖、迁移和侵袭。双荧光素酶实验证实miR-429可直接靶向结合HMOX1，二者在肺腺癌细胞中表达呈负相关。HMOX1过表达可抑制A549细胞的增殖、侵袭和迁移，而miR-429过表达可逆转这一抑制效应。机制上，miR-429通过靶向抑制HMOX1，降低A549细胞内ROS、L-ROS、MDA及Fe2+水平，同时升高GSH含量，从而降低细胞对铁死亡的敏感性。
    结论 miR-429通过靶向抑制HMOX1，促进肺腺癌细胞侵袭和迁移，并抑制铁死亡的发生。

     

    Abstract:
    Objective To investigate the effects of microRNA-429 (miR-429) on the invasion, migration, and ferroptosis of lung adenocarcinoma (LUAD) cells, and to provide new insights into the clinical inhibition of LUAD invasion and migration and the promotion of ferroptosis.
    Methods The expression profile of miR-429 in LUAD tissues was analyzed using the starBase database. Quantitative real-time PCR (qRT-PCR) was performed to assess miR-429 expression levels in normal human bronchial epithelial cells (the BEAS-2B cell line) and LUAD cells (the A549 and H1975 cell lines), and to evaluate the plasmid transfection efficiency. The effects of miR-429 on A549 cell proliferation, migration, and invasion were assessed using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch wound healing assay, and Transwell invasion assay, respectively. Potential ferroptosis-related proteins targeted by miR-429 were predicted using bioinformatics databases, and the targeting relationship between miR-429 and heme oxygenase 1 (HMOX1) was validated by a dual-luciferase reporter assay. Further functional experiments were performed to examine the effect of miR-429 targeting HMOX1 on the malignant phenotypes of A549 cells. The levels of reactive oxygen species (ROS) and lipid ROS (L-ROS) were measured to analyze the regulatory effects of miR-429 and HMOX1 on oxidative stress in LUAD cells. The contents of malondialdehyde (MDA), glutathione (GSH), and ferrous ion (Fe2+) were measured to evaluate evaluate the effects of miR-429 and HMOX1 on key ferroptosis-associated indicators in LUAD cells.
    Results miR-429 expression was significantly higher in LUAD cell lines (A549 and H1975) compared with that in normal cells (BEAS-2B), with the highest expression level observed in A549 cells. Functional experiments demonstrated that miR-429 promoted A549 cell proliferation, migration, and invasion. The dual-luciferase assay confirmed that miR-429 directly targeted HMOX1, and their expression levels were negatively correlated in LUAD cells. HMOX1 overexpression inhibited the proliferation, invasion, and migration of A549 cells, while miR-429 overexpression reversed these inhibitory effects. Mechanistically, miR-429 targeted and inhibited HMOX1, which led to reduced intracellular levels of ROS, L-ROS, MDA, and Fe2+ and increased GSH levels in A549 cells, thereby decreasing cellular sensitivity to ferroptosis.
    Conclusion miR-429 promotes the invasion and migration of LUAD cells and inhibits ferroptosis by targeting and inhibiting HMOX1.

     

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