[摘 要] 目的：探讨钙蛋白酶小亚基1（CAPN4）调控肺腺癌顺铂耐药性及肿瘤干细胞干性的作用机制，为通过靶向干性逆转 耐药提供实验依据。方法：收集2023年1月至2024年1月期间在福建省肿瘤医院手术切除的10例肺腺癌患者的组织标本，通过 免疫组织化学（IHC）法检测5例对顺铂耐药与5例敏感肺腺癌组织中CAPN4的表达差异，并进行组织化学评分（H评分）。利用 癌症基因组图谱（TCGA）数据库和基因表达谱交互分析（GEPIA）平台进行肺癌CAPN4基因表达相关的生存分析。另收集2例 对顺铂耐药、2例敏感的肺腺癌组织标本，建立肺腺癌类器官（PDO）模型，采用H-E、IHC染色评估PDO与原发肿瘤形态学的一致 性。采用慢病毒介导的shRNA技术敲减CAPN4基因表达，运用qPCR和WB法检测PDO中干细胞标志物ALDH1A1、CD133、 Nanog和SOX9的基因和蛋白表达水平。通过腺苷三磷酸（ATP）法检测CAPN4下调后肺腺癌PDO对顺铂的敏感性变化，使用半 胱氨酸蛋白酶-3（caspase-3）活性检测法评估CAPN4下调的肺腺癌PDO经顺铂处理后的凋亡情况。结果：IHC检测结果表明，耐 药肺腺癌患者组织中CAPN4蛋白的表达水平显著上调（P < 0.05）。在TCGA数据库肺腺癌患者队列中，CAPN4的高表达与肺腺 癌患者的不良预后（OS缩短）显著相关（HR = 1.4，P < 0.05）。耐药患者来源的PDO中CAPN4蛋白表达、干细胞标志物的基因 和蛋白表达水平均显著上调（均P < 0.05）。在顺铂敏感性测试中，耐药患者来源PDO的IC50 值显著高于敏感患者来源的 PDO（P < 0.05）。敲低CAPN4后，耐药患者来源的PDO的干细胞标志物表达显著降低，顺铂敏感性增强（P < 0.05）。结论：敲低 CAPN4可降低肺腺癌PDO的干细胞标志物表达，显著增强其对顺铂的敏感性，为逆转肺癌顺铂耐药提供了潜在治疗靶点。

[Abstract] Objective: To investigate the mechanisms by which calpain small subunit 1 (CAPN4) regulates cisplatin resistance and cancer stem cell (CSC) stemness in lung adenocarcinoma, and to provide experimental evidence for reversing drug resistance through targeting CSC stemness. Methods: Tissue samples were collected from 10 lung adenocarcinoma patients who underwent surgical resection at Fujian Cancer Hospital from January 2023 to January 2024. Immunohistochemistry (IHC) stining was used to detect the differential expression of CAPN4 in five cisplatin-resistant and five cisplatin-sensitive lung adenocarcinoma tissues, followed with a histological scoring (H-score). CAPN4 gene expression-related survival analysis in lung cancer patients was conducted using The Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA) platform. Additionally, tissue samples from two cisplatin-resistant and two cisplatin-sensitive lung adenocarcinoma cases were collected to establish lung adenocarcinoma organoid (PDO) models. H-E and IHC staining were used to assess the morphological consistency between PDOs and the primary tumors. CAPN4 gene expression was silenced using lentivirus-mediated shRNA transduction. The expression levels of stem cell markers ALDH1A1, CD133, Nanog, and SOX9 were detected at both the gene and protein levels using quantitative polymerase chain reaction (qPCR) and Western blotting (WB), respectively. The sensitivity of CAPN4-knockdown PDOs to cisplatin was evaluated using the adenosine triphosphate (ATP) assay, and the apoptosis was assessed using the caspase-3 assay. Results: IHC results showed that CAPN4 protein expression was significantly upregulated in cisplatin-resistant lung adenocarcinoma tissues (P < 0.05). TCGA cohort analysis revealed that high CAPN4 expression was significantly associated with poor prognosis (reduced OS) in lung adenocarcinoma patients (HR = 1.4, P < 0.05). PDOs derived from cisplatin-resistant patients exhibited significant upregulation in CAPN4 protein and stemness markers at both gene and protein levels (all P < 0.05). Cisplatin sensitivity assays demonstrated that PDOs derived from cisplatin-resistant patients had significantly higher IC50 values than those from cisplatin-sensitive patients (P < 0.05). After CAPN4 knockdown, the expression of stem cell makers in PDOs derived from cisplatin-resistant patients were significantly reduced, and their sensitivity to cisplatin was enhanced (P < 0.05). Conclusion: Knockdown of CAPN4 reduces stem cell marker expression and enhances cisplatin sensitivity in lung adenocarcinoma PDOs, providing a potential therapeutic target for reversing cisplatin resistance in lung cancer.

[基金项目] 福建省自然科学基金（No. 2022J05075）；福建省卫生健康中青年骨干人才培养项目（No. 2022GGA030）； 福建创新联合基金项目（No. 2021Y9198）；福建肿瘤医院高层次人才培养项目（No. 2022YNG03）