Polo样激酶4调控口腔鳞状细胞癌侵袭、增殖和凋亡的分子机制研究

doi:10.19401/j.cnki.1007-3639.2025.04.004

摘要/Abstract

摘要：

背景与目的：Polo样激酶4（Polo-like kinase 4，PLK4）为细胞周期调控蛋白，与多种恶性肿瘤的发生、发展关系密切。本研究旨在探究PLK4对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）增殖、凋亡和侵袭的影响。方法：利用基于基因表达水平值的交互式分析平台2数据库（gene expression profiling interactive analysis 2，GEPIA2）和阿拉巴马大学伯明翰分校癌症数据分析门户网站数据库（the University of Alabama at Birmingham Cancer Data Analysis Portal，UALCAN）在线数据平台分析PLK4在头颈部鳞癌及周围正常组织中的表达，并分析OSCC细胞系中PLK4的mRNA表达和蛋白水平。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）检测进一步分析敲低和过表达PLK4后对磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）、磷酸化磷脂酰肌醇3-激酶（phosphorylated phosphoinositide 3-kinase，p-PI3K）、蛋白激酶B（protein kinase B，AKT）、磷酸化蛋白激酶B（phosphorylated protein kinase B，p-AKT）、生存素（survivin）、细胞周期蛋白依赖性激酶D1（cyclin D1）的mRNA表达和蛋白水平的影响。通过细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验、流式细胞术、transwell侵袭实验分析PLK4对细胞的增殖、凋亡和侵袭活动的影响。此外，通过将12只4周龄SPF级BALB/c nude雌性裸鼠通过随机数字表法分为sh-NC组（n=6）和sh-PLK4组（n=6）。分别在裸鼠右前腋下注射sh-NC/sh-PLK4细胞进行裸鼠皮下移植瘤模型，观察统计两组裸鼠体质量、瘤体积和质量，并对剥离的瘤体组织进行H-E染色病理学诊断。动物实验已获得山东第二医科大学动物实验中心的批准（编号：2024SDL840）。结果：PLK4在OSCC中相较于正常组织呈现出高表达。此外，PLK4在OSCC细胞系（HN6、Cal-27、SCC-4、SCC-9、SCC-25）中相较于口腔黏膜上皮细胞高表达（P<0.05）。Western blot结果显示，对OSCC细胞进行敲低和过表达PLK4后，PI3K/AKT信号转导通路蛋白p-PI3K、p-AKT、cyclin D1及survivin分别表达降低和增高（P<0.05）；细胞增殖活力及穿膜细胞数在敲低和过表达PLK4后分别出现降低和增高，而细胞的凋亡率则分别增高和降低（P<0.05）。此外，相较于sh-NC组，sh-PLK4组裸鼠肿瘤的体积和质量降低（P<0.05），而两组裸鼠的质量差异无统计学意义（P > 0.05）。sh-PLK4组裸鼠肿瘤组织相较于sh-NC组肿瘤组织核异形性减少。结论：Polo样激酶4可介导PI3K/AKT信号转导通路参与OSCC的侵袭、增殖和凋亡活动，有望成为治疗OSCC的新靶点。

关键词: Polo样激酶4, 口腔鳞状细胞癌, PI3K/AKT信号转导通路, 增殖, 凋亡, 侵袭

Abstract:

Background and purpose: Polo-like kinase 4 (PLK4) is a cell cycle regulatory protein, which is closely related to the occurrence and development of a variety of malignant tumors. The aim of this study was to investigate the role of PLK4 in the proliferation, invasion and apoptosis of oral squamous cell carcinoma (OSCC). Methods: Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) were used to analyze the expression of PLK4 in head and neck squamous cell carcinoma and surrounding normal tissues. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were employed to evaluate the mRNA and protein expression of PLK4 in OSCC cells. We further analyzed PLK4, phosphoinositide 3-kinase (PI3K), phosphorylated phosphoinositide 3-kinase (p-PI3K), protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), survivin, and cyclin D1 protein expression. The effects of PLK4 on cell proliferation, apoptosis and invasion were analyzed by cell counting kit-8 (CCK-8) assay, flow cytometry and transwell assay. In addition, 12 4-week-old SPF BALB/c female nude mice were divided into sh-NC group (n=6) and sh-PLK4 group (n=6) by random number table method. The sh-NC/sh-PLK4 cells were injected into the right anterior armpit of nude mice for subcutaneous tumor formation. The body weight, tumor volume and tumor weight of the two groups of nude mice were observed, and the stripped tumor tissues were analyzed by H-E staining. The animal experiments were approved by the Animal Experiment Center of Shandong Second Medical University (No: 2024SDL840). Results: The results of GEPIA2 and UALCAN online databases showed that PLK4 was highly expressed in OSCC compared with normal tissues. In addition, PLK4 was highly expressed in OSCC cell lines (HN6, Cal-27, SCC-4, SCC-9, SCC-25) compared with oral mucosal epithelial cells (P<0.05). Western blot results showed that the expression levels of PI3K/AKT signaling pathway proteins p-PI3K, p-AKT, cyclin D1 and survivin were decreased after PLK4 knockdown and increased after PLK4 overexpression in OSCC cells (P<0.05). The cell proliferation activity and the number of transmembrane cells were positively correlated with the decrease and increase of PLK4 expression (P<0.05), while the cell apoptotic rate was negatively correlated (P<0.05), indicating that cell proliferation and apoptosis were both affected. In addition, compared with the sh-NC group, the tumor volume and weight of the nude mice in the sh-PLK4 group were decreased (P<0.05), while there was no significant difference in the body weight of the nude mice between the two groups (P>0.05). Moreover, the nuclear atypia of tumor tissues in sh-PLK4 group was lower than that in sh-NC group (P<0.05). Conclusion: PLK4 can regulate the invasion, proliferation and apoptosis of OSCC cells, potentially through the activation of PI3K/AKT signaling pathway.

Key words: PLK4, Oral squamous cell carcinoma, PI3K/AKT signaling pathway, Proliferation, Apoptosis, Invasion

秦欢, 王洁, 杨杰, 华英杰, 曲会娟, 冀洪海. Polo样激酶4调控口腔鳞状细胞癌侵袭、增殖和凋亡的分子机制研究[J]. 中国癌症杂志, 2025, 35(4): 365-375.

QIN Huan, WANG Jie, YANG Jie, HUA Yingjie, QU Huijuan, JI Honghai. Mechanistic study on PLK4 regulation of invasion, proliferation and apoptosis in oral squamous cell carcinoma[J]. China Oncology, 2025, 35(4): 365-375.

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sh-PLK4	Target sequence (5’-3’)
sh-PLK4-1	GCTGTGTGGGTTCAGTTTAAT
sh-PLK4-2	GAAATCCAGCAGATCGTTTAA
sh-NC	CCTAAGGTTAAGTCGCCCTCG