hnRNPK调控Wnt/β-catenin信号转导通路抑制乳腺癌细胞铁死亡

doi:10.19401/j.cnki.1007-3639.2024.10.003

摘要/Abstract

摘要：

背景与目的：核内不均一核糖核蛋白K（heterogeneous nuclear ribonucleoprotein K，hnRNPK）是一种调控基因表达和蛋白翻译的RNA结合蛋白，已被发现与多种肿瘤恶性进展密切相关，但其在乳腺癌中的作用尚未明确。本研究旨在探讨hnRNPK对乳腺癌细胞铁死亡的影响及其作用机制。方法：基于癌症基因组图谱（The Cancer Genome Atlas，TCGA）和基因表达综合（Gene Expression Omnibus，GEO）数据库，联合生信分析hnRNPK在乳腺癌组织及正常组织中的表达及与临床预后的关系；采用实时定量逆转录聚合酶链反应（real-time quantitative reverse transcription polymerase chain reaction，qRT-PCR）、蛋白质印迹法（Western blot）、免疫组织化学法检测hnRNPK在乳腺癌细胞及组织中的表达；将乳腺癌MDA-MB-231和MCF-7细胞进行siRNA转染，设置分组为对照组（control）、空载体组（NC）、干扰载体组（si-hnRNPK），采用细胞计数试剂盒（cell counting kit-8，CCK-8）和平板克隆实验检测细胞增殖能力；高通量RNA测序（RNA-sequencing，RNA-seq）分析hnRNPK富集的生物学功能及信号转导通路，Western blot及活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）、Fe^2＋试剂盒检测hnRNPK对铁死亡表型的影响，并利用铁死亡抑制剂（ferrostatin-1，Fer-1）检测敲低hnRNPK对铁死亡的回复作用；通过Western blot检测hnRNPK对Wnt/β-catenin通路蛋白表达的影响。结果：数据库结合生物信息学分析结果显示，hnRNPK在乳腺癌组织中表达上调（P<0.01），hnRNPK高表达组患者总生存期OS低于hnRNPK低表达组（P<0.05）；hnRNPK在乳腺癌组织及细胞中呈高表达，敲低hnRNPK后乳腺癌细胞增殖能力减弱（P<0.05）；RNA-seq分析发现hnRNPK显著富集铁死亡、凋亡、Wnt/β-catenin信号转导通路；敲低hnRNPK通过促进脂质ROS和MDA产生、Fe^2＋富集促进乳腺癌细胞铁死亡（P<0.05），且联合Fer-1有效抑制敲低hnRNPK对铁死亡的促进作用（P<0.05）；hnRNPK下调导致Wnt/β-catenin信号转导通路中β-catenin、c-Myc表达降低，CK1α、APC、GSK-3β复合物表达升高（P<0.05）。结论：hnRNPK在乳腺癌中呈高表达，敲低hnRNPK通过抑制Wnt/β-catenin信号转导通路促进乳腺癌细胞铁死亡，抑制乳腺癌恶性进展。

关键词: hnRNPK, 乳腺癌, 铁死亡, Wnt/β-catenin信号转导通路

Abstract:

Background and purpose: Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA special binding protein that participates in regulating the expression of related genes and protein translation. It has been linked to the malignant occurrence and development of various tumors, but its role in breast cancer remains unclear. The aim of this study was to investigate the effects of hnRNPK on ferroptosis in breast cancer cells and the underlying mechanisms. Methods: Based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, hnRNPK expression in breast cancer tissues and normal tissues and its relationship with clinical prognosis were analyzed by bioinformatics. We detected hnRNPK expression in breast cancer cells and tissues using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry staining diagnosis methods. MCF-7 and MDA-MB-231 breast cancer cells were transfected with siRNA, and divided into control group (control), empty body group (NC), and interference vector group (si-hnRNPK). Cell proliferation was detected by cell counting kit-8 (CCK-8) and plate clone formation assays. RNA-seq analysis was applied to explore potential targeted biological functions and signaling pathways affected by hnRNPK. Additionally, we investigated the impact of hnRNPK on ferroptosis phenotype using Western blot and commercial kits for reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and Fe²⁺, ferroptosis inhibitor (ferrostatin-1, Fer-1) was used to detect the rescue effect of hnRNPK knockdown on ferroptosis. The impact of hnRNPK on the expressions of Wnt/β-catenin pathway-related proteins were determined by Western blot. Results: The bioinformatics analyses indicated hnRNPK was upregulated in breast cancer tissues (P<0.01), and the overall survival of patients in the high expression group was poorer compared with those in the low expression group (P<0.05). hnRNPK was highly expressed in breast cancer tissues and cells, and knocking down hnRNPK weakened the proliferation ability of breast cancer cells (P<0.05). The RNA-seq analysis showed that hnRNPK was significantly enriched in ferroptosis, apoptosis, and the Wnt/β-catenin signaling pathway. Knocking down hnRNPK promoted ferroptosis in breast cancer cells by inducing lipid ROS and MDA, as well as Fe²⁺ accumulation (P<0.05). Interestingly, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the promotive effect of hnRNPK knockdown on ferroptosis (P<0.05). Downregulation of hnRNPK led to a decrease in the expressions of β-catenin and c-Myc in the Wnt/β-catenin signaling pathway, while expressions of CK1α, APC and the GSK-3β complex were elevated (P<0.05). Conclusion: hnRNPK is highly expressed in breast cancer, and knocking down hnRNPK promotes ferroptosis in breast cancer cells by inhibiting the Wnt/β-catenin signaling pathway, thereby suppressing the malignant progression of breast cancer.

Key words: hnRNPK, Breast cancer, Ferroptosis, Wnt/β-catenin pathway

中图分类号:

R735.3

马小兰, 王娟, 石斌, 王南, 田治翠, 曹佳. hnRNPK调控Wnt/β-catenin信号转导通路抑制乳腺癌细胞铁死亡[J]. 中国癌症杂志, 2024, 34(10): 931-943.

MA Xiaolan, WANG Juan, SHI Bin, WANG Nan, TIAN Zhicui, CAO Jia. hnRNPK regulates Wnt/β-catenin signaling pathway to inhibit ferroptosis in breast cancer[J]. China Oncology, 2024, 34(10): 931-943.

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