外泌体中ISG15在卵巢癌铂类药物耐药中的作用及初步机制探讨

doi:10.19401/j.cnki.1007-3639.2025.04.002

摘要/Abstract

摘要：

背景与目的：上皮性卵巢癌（epithelial ovarian cancer，EOC）患者预后较差，易对铂类药物产生耐受；目前认为外泌体可能参与卵巢癌铂类药物耐药，本研究主要探讨外泌体对卵巢癌铂类药物耐药的作用及机制。方法：通过超速离心提取外泌体，采用电镜、粒径分析和蛋白质印迹法（Western blot）对外泌体表征、外泌体蛋白及相关信号转导通路的表达水平进行检测。通过蛋白质组学对外泌体蛋白表达谱进行分析；通过与已有耐药数据集筛选关键差异蛋白。收集2023年8月—2024年8月在复旦大学附属肿瘤医院经病理学检查诊断为EOC并接受手术治疗符合入组标准的患者，以分析患者血清外泌体中干扰素刺激基因15（interferon-stimulated gene 15，ISG15）表达水平与铂类药物耐药的关系。通过酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）检测血清外泌体中相关蛋白的表达水平，采用受试者工作特征曲线（receiver operating characteristic，ROC）探究血清外泌体蛋白对卵巢癌耐药的预测价值。结果：本研究成功提取源自卵巢癌铂类药物敏感及耐药细胞的外泌体并进行蛋白组学分析，鉴定出9个与铂类药物耐药相关的差异蛋白，筛选出ISG15。与敏感细胞相比，卵巢癌耐药细胞外泌体中ISG15表达水平显著上调；外泌体示踪实验表明，外泌体能够被卵巢癌受体细胞摄取。在与耐药细胞外泌体共培养后，卵巢癌受体细胞ISG15表达水平升高。敲低细胞ISG15，外泌体中ISG15的表达下降；加入下调ISG15的外泌体后，卵巢癌细胞对铂类药物的耐受降低。且ISG15可通过磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）/核因子激活的B细胞的κ-轻链增强复合物（nuclear factor-kappa-light-chain-enhancer of activated B cells，NF-κB）信号转导通路调控多耐药蛋白1（multidrug resistance protein 1，MDR1）的表达促进卵巢癌细胞对铂类药物耐药。对87例患者血清标本检测结果发现，与卵巢癌敏感患者相比，耐药卵巢癌患者血清外泌体中ISG15表达升高；将血清外泌体ISG15作为区别敏感和耐药的指标绘制ROC的曲线下面积为0.779（P<0.05），Cutoff值为27.35 ng/mL，灵敏度为70.2%，特异度为76.4%。结论：外泌体中的ISG15可以通过PI3K/AKT/NF-κB信号转导通路调节卵巢癌细胞中MDR1的表达，促进卵巢癌细胞对铂类药物耐药。

关键词: 卵巢癌, 外泌体, ISG15, 铂类药物耐药

Abstract:

Background and purpose: Epithelial ovarian cancer (EOC) has a poor prognosis and is prone to developing resistance to platinum-based chemotherapy. Exosomes are currently believed to be involved in platinum resistance in ovarian cancer. This study explored the role and mechanism of exosomes on platinum resistance of ovarian cancer. Methods: Through ultracentrifugation, exosomes were isolated and analyzed using electron microscopy, particle size studies, and Western blot for detailed exosome analysis. We detected the expression levels of exosomal proteins and related signaling pathways. Exosomal protein expression profile was analyzed by proteomics. Key differential proteins were screened by intersecting with existing drug resistance datasets. Furthermore, patients diagnosed with EOC via pathological analysis at the Fudan University Shanghai Cancer Center from August 2023 to August 2024, who underwent surgery and fulfilled the eligibility requirements, were gathered to examine the link between the expression of interferon-stimulated gene 15 (ISG15) in serum exosomes and resistance to platinum medication. The expression levels of related proteins in serum exosomes were quantitatively detected by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curve was plotted to explore the predictive value of serum exosomal protein in ovarian cancer resistance. Results: Exosomes derived from platinum-sensitive and drug-resistant ovarian cancer cells were extracted and proteomic analysis was further performed. We identified 9 differential proteins associated with platinum-resistance and found the key molecule interferon-stimulated gene 15 (ISG15). Compared with sensitive cells, the expression of ISG15 in exosomes of drug-resistant ovarian cancer cells was significantly upregulated. Exosome tracer tests showed that exosomes were successfully taken up by ovarian cancer receptor cells. After coculture with drug-resistant exosomes, the expression level of ISG15 in ovarian cancer receptor cells was increased. Knockdown ISG15 in EOC cells decreased the expression of ISG15 in exosomes, and incubation of ISG15-knockdown exosomes decreased the cell viability on the condition of platinum drugs, indicating that ISG15 in exosomes regulated platinum resistance of ovarian cancer cells. Moreover, ISG15 could regulate the expression of multidrug resistance protein 1 (MDR1) through phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor-kappa-light-chain-enchancer of activated B cells (NF-κB) signaling pathway. This study included a total of 87 patients. Clinical serum samples also showed that the expression of ISG15 in exosomes was higher in platinum-resistant ovarian cancer patients than in sensitive ovarian cancer patients. Using serum exosomal ISG15 as an indicator to distinguish between sensitivity and resistance, the area under ROC curve was 0.779 (P<0.05), cutoff value was 27.35ng/mL, sensitivity was 70.2%, and specificity was 76.4%. Conclusion: ISG15 in exosomes can regulate the expression of MDR1 in ovarian cancer cells through PI3K/AKT/NF-κB signaling pathway, and promote platinum resistance in ovarian cancer cells.

Key words: Ovarian cancer, Exosome, ISG15, Platinum resistance

田亚楠, 郑慧, 严天晴, 张珩, 卢仁泉, 郭林. 外泌体中ISG15在卵巢癌铂类药物耐药中的作用及初步机制探讨[J]. 中国癌症杂志, 2025, 35(4): 346-354.

TIAN Yanan, ZHENG Hui, YAN Tianqing, ZHANG Heng, LU Renquan, GUO Lin. Preliminary study on the role and mechanism of ISG15 in exosomes in platinum resistance in ovarian cancer[J]. China Oncology, 2025, 35(4): 346-354.

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参考文献 19

[1]	SIEGEL R L, MILLER K D, WAGLE N S, et al. Cancer statistics, 2023[J]. CA A Cancer J Clinicians, 2023, 73(1): 17-48.
[2]	STEWART C, RALYEA C, LOCKWOOD S. Ovarian cancer: an integrated review[J]. Semin Oncol Nurs, 2019, 35(2): 151-156. doi: S0749-2081(19)30012-9 pmid: 30867104
[3]	LI B L, LU W, QU J J, et al. Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis[J]. J Cell Physiol, 2019, 234(3): 2943-2953.
[4]	DOYLE L M, WANG M Z. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis[J]. Cells, 2019, 8(7): 727.
[5]	ZHANG Y, BI J Y, HUANG J Y, et al. Exosome: a review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications[J]. Int J Nanomedicine, 2020, 15: 6917-6934.
[6]	LIU Y X, ZHU K Y, GUAN X L, et al. TTK is a potential therapeutic target for cisplatin-resistant ovarian cancer[J]. J Ovarian Res, 2021, 14(1): 128. doi: 10.1186/s13048-021-00884-z pmid: 34598710
[7]	ZHENG H, ZHANG M Q, MA S, et al. Identification of the key genes associated with chemotherapy sensitivity in ovarian cancer patients[J]. Cancer Med, 2020, 9(14): 5200-5209.
[8]	LIU J, ZHU M Y, FENG Y, et al. The multidrug resistance can be reversed for the decrease of P-gp and LRP by inhibiting PI3K/AKT/NF-κB signal pathway in nasopharynx carcinoma[J]. Biosci Rep, 2020, 40(5): BSR20190239.
[9]	SANDERCOCK J, PARMAR M B, TORRI V, et al. First-line treatment for advanced ovarian cancer: paclitaxel, platinum and the evidence[J]. Br J Cancer, 2002, 87(8): 815-824.
[10]	ROMA-RODRIGUES C, FERNANDES A R, BAPTISTA P V. Exosome in tumour microenvironment: overview of the crosstalk between normal and cancer cells[J]. Biomed Res Int, 2014, 2014: 179486.
[11]	MAIORINO L, DAßLER-PLENKER J, SUN L J, et al. Innate immunity and cancer pathophysiology[J]. Annu Rev Pathol, 2022, 17: 425-457.
[12]	PADARIYA M, SZNARKOWSKA A, KOTE S, et al. Functional interfaces, biological pathways, and regulations of interferon-related DNA damage resistance signature (IRDS) genes[J]. Biomolecules, 2021, 11(5): 622.
[13]	NGUYEN H M, GAIKWAD S, OLADEJO M, et al. Interferon stimulated gene 15 (ISG15) in cancer: an update[J]. Cancer Lett, 2023, 556: 216080.
[14]	PERNG Y C, LENSCHOW D J. ISG15 in antiviral immunity and beyond[J]. Nat Rev Microbiol, 2018, 16(7): 423-439.
[15]	DESAI S D, WOOD L M, TSAI Y C, et al. ISG15 as a novel tumor biomarker for drug sensitivity[J]. Mol Cancer Ther, 2008, 7(6): 1430-1439. doi: 10.1158/1535-7163.MCT-07-2345 pmid: 18566215
[16]	CHEN R H, DU Y, HAN P, et al. ISG15 predicts poor prognosis and promotes cancer stem cell phenotype in nasopharyngeal carcinoma[J]. Oncotarget, 2016, 7(13): 16910-16922.
[17]	DORAYAPPAN K D P, WAGNER V, PARK D, et al. ISG15 mediates the function of extracellular vesicles in promoting ovarian cancer progression and metastasis[J]. J Extracell Biol, 2024, 3(2): e92. doi: 10.1002/jex2.92 pmid: 38939897
[18]	OYAMA Y, SHIGETA S, TOKUNAGA H, et al. CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: a potential role of CHD4 inhibition as a combination therapy with platinum agents[J]. PLoS One, 2021, 16(6): e0251079.
[19]	QIU F H, HOU T Y, HUANG D H, et al. Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing[J]. Mol Med Rep, 2015, 12(5): 7749-7755. doi: 10.3892/mmr.2015.4417 pmid: 26460178