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

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 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.

关键词

miR-429 / 侵袭 / 迁移 / 铁死亡 / 肺腺癌 / HMOX1基因

Key words

microRNA-429 / Invasion / Migration / Ferroptosis / Lung adenocarcinoma / HMOX1 gene

参考文献

[1] Denisenko T V, Budkevich I N, Zhivotovsky B. Cell death-based treatment of lung adenocarcinoma[J/OL]. Cell Death Dis, 2018, 9(2): 117. https://www.nature.com/articles/s41419-017-0063-y. DOI: 10.1038/s41419-017-0063-y.

[2] Chen M, Zhang S, Wang F, et al. DLGAP5 promotes lung adenocarcinoma growth via upregulating PLK1 and serves as a therapeutic target[J/OL]. J Transl Med, 2024, 22(1): 209. https://doi.org/10.1186/s12967-024-04982-8.

[3] Shan J, An Y, Bues M, et al. Robust optimization in IMPT using quadratic objective functions to account for the minimum MU constraint[J]. Med Phys, 2018, 45(1): 460. DOI: 10.1002/mp.12674.

[4] Volovat S R, Volovat C, Hordila I, et al. MiRNA and LncRNA as potential biomarkers in triple-negative breast cancer: a review[J/OL]. Front Oncol, 2020, 10: 526850. https://doi.org/10.3389/fonc.2020.526850.

[5] Rogucki M, Buczyńska A, Krętowski A J, et al. The importance of miRNA in the diagnosis and prognosis of papillary thyroid cancer[J/OL]. J Clin Med, 2021, 10(20): 4737. https://doi.org/10.3390/jcm10204737.

[6] Xu J, Wu K J, Jia Q J, et al. Roles of miRNA and lncRNA in triple-negative breast cancer[J]. J Zhejiang Univ Sci B, 2020, 21(9): 673. DOI: 10.1631/jzus.B2000181.

[7] Murrone A, Cantini L, Pecci F, et al. BRCA-associated protein 1 (BAP1) and miR-31 combination predicts outcomes in epithelioid malignant pleural mesothelioma[J]. J Thorac Dis, 2021, 13(10): 5741. DOI: 10.21037/jtd-21-899.

[8] Zhang L, Liu Q, Mu Q, et al. MiR-429 suppresses proliferation and invasion of breast cancer via inhibiting the Wnt/β-catenin signaling pathway[J]. Thorac Cancer, 2020, 11(11): 3126. DOI: 10.1111/1759-7714.13628.

[9] Wei X, Li X, Hu S, et al. Regulation of ferroptosis in lung adenocarcinoma[J/OL]. Int J Mol Sci, 2023, 24(19): 14614. https://doi.org/10.3390/ijms241914614.

[10] Jiang M, Jike Y, Liu K, et al. Exosome-mediated miR-144-3p promotes ferroptosis to inhibit osteosarcoma proliferation, migration, and invasion through regulating ZEB1[J/OL]. Mol Cancer, 2023, 22(1): 113. https://doi.org/10.1186/s12943-023-01823-2.

[11] Bai T, Liang R, Zhu R, et al. MicroRNA-214-3p enhances erastin-induced ferroptosis by targeting ATF4 in hepatoma cells[J]. J Cell Physiol, 2020, 235(7/8): 5637. DOI: 10.1002/jcp.29502.

[12] Schabath M B, Cote M L. Cancer progress and priorities: lung cancer[J]. Cancer Epidemiol Biomarkers Prev, 2019, 28(10): 1563. DOI: 10.1158/1055-9965.EPI-19-0221.

[13] Wang J, Gong M, Fan X, et al. Autophagy-related signaling pathways in non-small cell lung cancer[J]. Mol Cell Biochem, 2022, 477(2): 385. DOI: 10.1007/s11010-021-04287-0.

[14] Jaszczuk I, Koczkodaj D, Kondracka A, et al. The role of miRNA-210 in pre-eclampsia development[J]. Ann Med, 2022, 54(1): 1350. DOI: 10.1080/07853890.2022.2070273.

[15] Lu Y, Liu Z, Zhang Y, et al. METTL3-mediated m6A RNA methylation induces the differentiation of lung resident mesenchymal stem cells into myofibroblasts via the miR-21/PTEN pathway[J/OL]. Respir Res, 2023, 24(1): 300. https://doi.org/10.1186/s12931-023-02604-7.

[16] Liao Y W, Tsai L L, Lee Y H, et al. miR-21 promotes the fibrotic properties in oral mucosa through targeting PDCD4[J]. J Dent Sci, 2022, 17(2): 677. DOI: 10.1016/j.jds.2021.11.009.

[17] Zhang W, Ji W, Zhao X. MiR-155 promotes anaplastic thyroid cancer progression by directly targeting SOCS1[J/OL]. BMC Cancer, 2019, 19(1): 1093. https://doi.org/10.1186/s12885-019-6319-5.

[18] Liu F, Kong X, Lv L, et al. MiR-155 targets TP53INP1 to regulate liver cancer stem cell acquisition and self-renewal[J]. FEBS Lett, 2015, 589(4): 500. DOI: 10.1016/j.febslet.2015.01.005.

[19] Jiang X, Stockwell B R, Conrad M. Ferroptosis: mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22(4): 266. DOI: 10.1038/s41580-020-00324-8.

[20] Doll S, Freitas F P, Shah R, et al. FSP1 is a glutathione-independent ferroptosis suppressor[J]. Nature, 2019, 575(7784): 693. DOI: 10.1038/s41586-019-1707-0.

[21] Wu L, Li N, Zhu L, et al. CircPDSS1 (hsa_circ_0017998) silencing induces ferroptosis in non-small-cell lung cancer cells by modulating the miR-137/SLC7A11/GPX4/GCLC axis[J/OL]. Toxicol In Vitro, 2024, 99: 105887. https://www.sciencedirect.com/science/article/abs/pii/S0887233324001176?via%3Dihub#section-snippets. DOI: 10.1016/j.tiv.2024.105887.

[22] Luo J, Song G, Chen N, et al. Ferroptosis contributes to ethanol-induced hepatic cell death via labile iron accumulation and GPx4 inactivation[J/OL]. Cell Death Discov, 2023, 9(1): 311. https://pmc.ncbi.nlm.nih.gov/articles/PMC10457354/. DOI: 10.1038/s41420-023-01613-x.

[23] Zhou Y, Zeng L, Cai L, et al. Cellular senescence-associated gene IFI16 promotes HMOX1-dependent evasion of ferroptosis and radioresistance in glioblastoma[J/OL]. Nat Commun, 2025, 16(1): 1212. https://pmc.ncbi.nlm.nih.gov/articles/PMC11785807/. DOI: 10.1038/s41467-025-56522-7.

[24] Loboda A, Damulewicz M, Pyza E, et al. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism[J]. Cell Mol Life Sci, 2016, 73(17): 3221. DOI: 10.1007/s00018-016-2223-0.

[25] Chau L Y. Heme oxygenase-1: emerging target of cancer therapy[J/OL]. J Biomed Sci, 2015, 22(1): 22. https://doi.org/10.1186/s12929-015-0130-z.

基金资助

*山东省自然科学基金面上项目(ZR2022MH311)

2023年国家级大学生创新创业训练计划项目(202310438044)

2024年潍坊市科技发展计划(软科学)项目(2024RKX007)

山东第二医科大学2024年校级教育教学改革与研究课题(2024YB039)