非小细胞肺癌组织外泌体PRPS2蛋白对肺癌细胞增殖和迁移的影响  

Effect of exosomal PRPS2 protein in non-small cell lung cancer tissues on proliferation and migration of lung cancer cells

作　　者：孙明飞[1] 李佩钰 潘丽红[1] 孟庆江[1] 郑先杰[1] 耿盛男[1] 张双林[1] SUN Mingfei;LI Peiyu;PAN Lihong;MENG Qingjiang;ZHENG Xianjie;GENG Shengnan;ZHANG Shuanglin(Department of Cardiovascular Surgery,the First Affiliated Hospital of Henan University,Kaifeng,Henan 475000,China)

机构地区：[1]河南大学第一附属医院心血管外科,河南开封475000

出　　处：《中华实用诊断与治疗杂志》2025年第1期9-20,共12页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省医学科技攻关计划省部共建项目(SBGJ202302087);开封市科技计划项目(2403007;2203022)。

摘　　要：目的筛选非小细胞肺癌(NSCLC)组织与癌旁组织外泌体差异表达蛋白,探讨外泌体PRPS2蛋白对NSCLC细胞增殖、迁移、侵袭的影响。方法取2022年2月—2023年2月河南大学第一附属医院20例NSCLC患者手术切除的癌组织和癌旁组织,采用超速离心法分离癌组织和癌旁组织外泌体,应用透射电镜观察外泌体形态特征,采用纳米颗粒追踪分析技术检测外泌体粒径,采用Western blot法检测外泌体特异性阳性蛋白标志物CD9、CD63、TSG101及阴性蛋白标志物钙连蛋白、细胞色素C表达情况;采用质谱法检测癌组织和癌旁组织外泌体蛋白,筛选出PRPS2、MCM6、PXDN、NT5C3A,对显著差异表达蛋白进行基因本体(GO)功能注释分析、京都基因和基因组百科全书(KEGG)通路富集分析。应用GEPIA和TCGA数据库分析218例NSCLC患者癌组织PRPS2、MCM6、PXDN、NT5C3A蛋白表达及5年生存情况,绘制Kaplan-Meier曲线分析PRPS2、MCM6、PXDN、NT5C3A与NSCLC患者5年总生存率的关系,选择与NSCLC患者中位总生存期相关的PRPS2蛋白进一步进行实验。取对数生长期NSCLC细胞A549、人肺正常上皮细胞BEAS-2B,采用Western blot法检测PRPS2蛋白相对表达量,采用实时荧光定量PCR法检测PRPS2 mRNA相对表达量。将对数生长期A549细胞分为si-NC组(转染si-NC)、si-PRPS2组(转染si-PRPS2),转染48 h采用Western blot法检测2组PRPS2蛋白相对表达量,转染后培养0、24、48、72、96 h时采用CCK-8法检测2组细胞增殖率,转染后培养48 h时采用细胞划痕实验检测2组细胞迁移率、采用Transwell小室实验检测2组侵袭细胞数。将转染成功的si-NC组、si-PRPS2组细胞分离外泌体,外泌体分离及鉴定方法同NSCLC癌组织、癌旁组织。取对数生长期A549细胞,分为NC-exo组和siPRPS2-exo组,分别加入分离的si-NC组、si-PRPS2组细胞外泌体,共培养48 h检测2组PRPS2蛋白相对表达量,共培养0、24、48、72、96 h时检测2组细胞增殖率,共培养48Objective To screen exosomal differentially expressed proteins between non-small cell lung cancer(NSCLC)tissues and paracancerous tissues,and to explore the effect of exosomal PRPS2 protein on the proliferation,migration and invasion of NSCLC cells.Methods Ultracentrifugation was used to isolate the exosomes of the cancerous tissues and its paracancerous tissues surgically removed from 20 NSCLC patients in the First Affiliated Hospital of Henan University from February 2022 to February 2023.The morphology of exosomes was observed with transmission electron microscopy,the particle size of exosomes was measured with nanoparticle tracking analysis technology,and the expressions of exosomal specific positive protein markers(CD9,CD63,TSG101)and negative protein markers(calnexin and cytochrome c)were detected with Western blot.The exosomal proteins of cancerous and paracancerous tissues were detected with mass spectrometry,and the differentially expressed proteins PRPS2,MCM6,PXDN and NT5C3A were identified.The Gene Ontology(GO)functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on differentially expressed proteins.The expressions of PRPS2,MCM6,PXDN and NT5C3A of cancerous tissues and 5-year survival in 218 NSCLC patients were analyzed with GEPIA and TCGA databases.Kaplan-Meier curves were drawn to analyze the relationships of PRPS2,MCM6,PXDN and NT5C3A with 5-year overall survival of NSCLC patients.The PRPS2 protein associated with the median overall survival of NSCLC patients was selected for further experiments.Human NSCLC cell line A549 and human normal lung epithelial cells BEAS-2B in logarithmic growth phase were selected,the relative expression of PRPS2 protein was detected with Western blot,and the relative expression of PRPS2 mRNA was detected with real-time fluorescence quantitative PCR.The A549 cells in logarithmic growth phase were divided into the si-NC group(transfected with si-NC)and the si-PRPS2 group(transfected with si-PRPS2).In two

关 键 词：非小细胞肺癌 A549细胞 外泌体 PRPS2 

分 类 号：R73[医药卫生—肿瘤]