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| TOP2A激活PI3K/AKT轴促进上皮间充质转化并影响卵巢癌顺铂耐药性的机制研究 |
| Research on the mechanism by which TOP2A activates the PI3K/AKT axis, promotes epithelial - mesenchymal transition and affects cisplatin resistance in ovarian cancer |
| 投稿时间：2025-03-07 修订日期：2026-01-20 |
| DOI： |
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| 中文关键词: 上皮性卵巢癌 TOP2A PI3K/AKT通路 EMT 顺铂耐药 |
| 英文关键词:Epithelial Ovarian Cancer TOP2A PI3K/AKT Pathway EMT Platinum Resistance |
| 基金项目:2μm连续波和飞秒脉冲激光辅助下对卵巢癌荷瘤大鼠光动力治疗的研究 |
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| 中文摘要: |
| [摘? 要] 目的：研究拓扑异构酶Ⅱα（Topoisomerase Ⅱ alpha，TOP2A）在上皮性卵巢癌组织中的表达及其对耐药上皮性卵巢癌细胞的生物学行为和顺铂敏感性的影响，并探究其作用机制。方法：采用免疫组化检测TOP2A在上皮性卵巢癌（epithelial ovarian cancer,EOC）组织中的表达，蛋白质印迹分析（Western Blot）筛选TOP2A高表达的卵巢癌细胞株（OVCAR3、SKOV3）及其相应铂耐药细胞株（SKOV3/DDP、OVCAR3/DDP）作为研究对象，随后分别慢病毒构建TOP2A低表达的耐药细胞稳转株用于后续研究。实验分为空转对照组（sh-NC组简称NC）、TOP2A低表达组1（sh-TOP2A#1简称sh-1）及TOP2A低表达组2（sh-TOP2A#2组简称sh-2）三组，上述两种细胞系均按此分组通过细胞计数试剂盒-8实验（cell counting kit-8,CCK8）、平板克隆实验、细胞划痕实验、细胞侵袭实验（Transwell实验）、反转录实时定量PCR（quantitative real-time polymerase chain reaction,qRT-PCR）以及Western Blot实验检测细胞活力、增殖能力、迁移及侵袭能力、TOP2A在mRNA及蛋白水平表达情况和磷脂酰肌醇3-激酶-蛋白激酶B（Phosphatidylinositol3 - Kinase-Protein Kinase B，PI3K/AKT）轴、上皮-间充质转化（epithelial-mesenchymal transition，EMT）的相关蛋白表达情况。随后使用PI3K激动剂（740Y-P）处理低表达TOP2A的耐药细胞稳转株（SKOV3/DDP、OVCAR3/DDP），分组均为空转对照组（NC）、TOP2A低表达组1（sh-1）及TOP2A低表达组1+740Y-P组（sh-1+740Y-P）三组，Western Blot检测PI3K、p-PI3K及EMT相关标志蛋白的表达，进一步验证TOP2A通过PI3K/AKT影响耐药上皮性卵巢癌细胞恶性生物行为及耐药性。结果：上皮性卵巢癌组织和卵巢癌耐药细胞中TOP2A表达水平升高，,其表达程度与分期、分级及淋巴结转移呈正相关（p<0.05），与患者的年龄未见明显关系（p=0.36）；在两种卵巢癌耐药细胞系中，与NC组相比，sh-1组、sh-2组细胞活力、增殖能力、侵袭和迁移能力、TOP2A mRNA表达水平、TOP2A蛋白表达水平均下降（p<0.01），铂敏感性均上升（p<0.01），p-PI3K、p-AKT蛋白表达均下降（p<0.01）、E-cadherin蛋白表达均上升、N-cadherin、Snail、Vimentin蛋白表达均下降（p<0.01）；加入PI3K激动剂740Y-P后，NC组、sh-1组、sh-1+740Y-P组互为对照，sh-1+740Y-P组E-cadherin蛋白表达低于sh-1组（p<0.01），相近但高于NC组（p<0.01），sh-1+740Y-P组p-PI3K、p-AKT、N-cadherin、Snail、Vimentin蛋白表达高于sh-1组（p<0.01），相近但低于NC组（p<0.01）。结论：TOP2A在原发上皮性卵巢癌组织中异常高表达并通过调控PI3K/AKT轴调节EMT过程从而影响肿瘤细胞恶性生物学行为及EMT过程，进而影响卵巢癌顺铂敏感性。 |
| 英文摘要: |
| [Abstract]Objective: To investigate the expression of topoisomerase II alpha (TOP2A) in epithelial ovarian cancer (EOC) tissues and its impact on the biological behavior and cisplatin sensitivity of drug-resistant EOC cells, as well as to explore the underlying mechanism. Methods:Immunohistochemistry was used to detect TOP2A expression in EOC tissues. Western Blot was employed to screen EOC cell lines with high TOP2A expression (OVCAR3, SKOV3) and their corresponding cisplatin-resistant counterparts (SKOV3/DDP, OVCAR3/DDP) as research objects. Lentiviral vectors were then used to construct stable cisplatin-resistant cell lines with low TOP2A expression for subsequent experiments. The cells were divided into three groups: empty vector control group (sh-NC, referred to as NC), TOP2A low-expression group 1 (sh-TOP2A#1, referred to as sh-1), and TOP2A low-expression group 2 (sh-TOP2A#2, referred to as sh-2). For both cell lines, cell viability, proliferative capacity, migration and invasion abilities were evaluated using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, and Transwell invasion assay, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot were performed to detect the expression levels of TOP2A at mRNA and protein levels, as well as the expression of proteins related to the Phosphatidylinositol 3-Kinase-Protein Kinase B (PI3K/AKT) axis and epithelial-mesenchymal transition (EMT). Subsequently, the stable TOP2A low-expression cisplatin-resistant cells (SKOV3/DDP, OVCAR3/DDP) were treated with the PI3K agonist 740Y-P, and divided into three groups: NC group, sh-1 group, and sh-1 + 740Y-P group. Western Blot was used to detect the expression of PI3K, phosphorylated PI3K (p-PI3K), and EMT-related marker proteins, so as to further verify that TOP2A affects the malignant biological behaviors and drug resistance of cisplatin-resistant EOC cells through the PI3K/AKT axis.Results:TOP2A expression was significantly upregulated in EOC tissues and cisplatin-resistant EOC cells. Its expression level was positively correlated with tumor stage, grade, and lymph node metastasis (p<0.05), but showed no significant correlation with patient age (p=0.36). In both cisplatin-resistant cell lines, compared with the NC group, the sh-1 and sh-2 groups exhibited decreased cell viability, proliferative capacity, migration and invasion abilities, as well as reduced TOP2A mRNA and protein expression levels (all p<0.01), while cisplatin sensitivity was significantly increased (p<0.01). Additionally, the protein expression levels of p-PI3K and p-AKT were downregulated (p.01), E-cadherin was upregulated, and N-cadherin, Snail, and Vimentin were downregulated in the sh-1 and sh-2 groups (all p<0.01). After treatment with 740Y-P, compared with the sh-1 group, the sh-1 + 740Y-P group showed lower E-cadherin expression (p<0.01) and higher expression levels of p-PI3K, p-AKT, N-cadherin, Snail, and Vimentin (all p<0.01); these expression levels in the sh-1 + 740Y-P group were close to but still significantly different from those in the NC group (all p<0.01). Conclusion: TOP2A is abnormally highly expressed in primary EOC tissues. It regulates the EMT process by modulating the PI3K/AKT axis, thereby affecting the malignant biological behaviors of tumor cells and ultimately influencing the cisplatin sensitivity of ovarian cancer. |
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