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48 h时检测2组细胞迁移率、侵袭细胞数,检测方法同si-NC组和si-PRPS2组。结果(1)肺癌组织、癌旁组织分离的外泌体均呈圆形或椭圆形囊泡状结构,粒径大小和粒径峰值符合外泌体结构特征。癌组织和癌旁组织外泌体CD9、CD63和TSG101均呈阳性表达,钙连蛋白和细胞色素C均呈阴性表达。(2)癌组织与癌旁组织外泌体质谱分析共鉴定出3398种蛋白,其中差异表达蛋白549种,PRPS2、MCM6、PXDN、NT5C3A为显著差异表达蛋白。GO分析结果显示,4种显著差异表达蛋白生物过程主要集中在蛋白质羟化、核糖核酸加工、原肠胚形成等;细胞组分集中在层粘连蛋白复合体、黏附连接、线粒体膜等;分子功能集中在L-抗坏血酸结合、氧结合、有机酸结合等。KEGG分析结果显示,4种显著差异表达蛋白可能参与的代谢和信号通路主要包括氮代谢、ECM-受体相互作用、核蛋白合成等。(3)Kaplan-Meier生存分析结果显示,癌组织PRPS2蛋白表达与NSCLC患者5年总生存率相关(P=0.014),MCM6、PXDN、NT5C3A蛋白表达与NSCLC患者5年总生存率无相关性(P>0.05)。选择PRPS2蛋白进行后续实验。(4)A549细胞PRPS2 mRNA和蛋白相对表达量(12.725±2.132、0.934±0.021)均高于BEAS-2B细胞(1.025±0.023、0.051±0.004)(t=23.230,P<0.001;t=71.540,P<0.001)。转染48 h时,si-PRPS2组细胞PRPS2蛋白相对表达量(0.034±0.004)低于si-NC组(0.731±0.012)(t=95.440,P<0.001)。转染后培养72、96 h时,si-PRPS2组细胞增殖率[(1.127±0.101)%、(1.802±0.118)%]均低于si-NC组[(1.709±0.152)%、(2.495±0.156)%](t=7.146,P<0.001;t=8.508,P<0.001),转染后培养0、24、48 h时细胞增殖率与si-NC组比较差异均无统计学意义(P>0.05)。转染48 h时,si-PRPS2组细胞迁移率[(23.979±4.220)%]低于si-NC组[(38.851±4.981)%](t=4.946,P=0.009),侵袭细胞数[(26.000±6.557)个]少于si-NC组[(54.333±5.132)个](t=7.894,P<0.001)。(5)转染成功的si-NC组、si-PRPS组细胞分离的外泌体均呈圆形或椭圆形囊泡状结构,粒径大小和粒径峰值符合外泌体结构特征。共培养48 h时,siPRPS2-exo组PRPS2蛋白相对表达量(0.046±0.004)低于NC-exo组(0.334±0.012)(t=39.440,P<0.001)。共培养48、72、96 h时,siPRPS2-exo组细胞增殖率[(0.797±0.066)%、(1.249±0.092)%、(1.789±0.173)%]均低于NC-exo组[(1.089±0.090)%、(1.877±0.180)%、(2.859±0.221)%](t=4.536~8.610,P均<0.05),共培养0、24 h时细胞增殖率与NC-exo组比较差异均无统计学意义(P>0.05)。共培养48h时,siPRPS2-exo组细胞迁移率[(21.663±2.952)%]低于NC-exo组[(59.476±2.383)%](t=14.831,P<0.001),侵袭细胞数[(32.000±2.646)个]少于NC-exo组[(80.333±7.024)个](t=11.154,P<0.001)。结论NSCLC组织外泌体及A549细胞中PRPS2蛋白呈高表达,敲低其表达可抑制A549细胞的增殖、迁移与侵袭能力,PRPS2可通过外泌体途径促进A549细胞的恶性生物学行为。
Objective 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 groups,the relative expression of PRPS2 protein was detected with Western blot 48 h after transfection,the cell proliferation rate was detected with CCK-8 method after 0-,24-,48-,72-and 96-h culture;and the migration rate was detected with cell scratch assay and the number of invasive cells was detected by Transwell chamber assay after 48-h culture.Exosomes were isolated from successfully transfected cells in the si-NC and si-PRPS2 groups,and the methods of exosomal isolation and identification were the same as those of NSCLC cancerous and paracancerous tissues.The A549 cells in logarithmic growth phase were divided into the NC-exo group and the siPRPS2-exo group,which were added with isolated exosomes of si-NC group cells and si-PRPS2 group cells respectively.In two groups,the relative expression of PRPS2 protein was detected after 48-h coculture,the cell proliferation rate was detected after 0-,24-,48-,72-and 96-h coculture,and the migration rate and the number of invasive cells were detected after 48-h coculture.The detection methods were the same as those of the si-NC group and si-PRPS2 group.Results(1)The exosomes isolated from cancerous and paracancerous tissues presented spherical or oval vesicle-like structures.The particle size and particle peak value matched typical exosomal structural characteristics.The expressions of CD9,CD63 and TSG101 were positive and the expressions of calnexin and cytochrome c were negative in exosomes of cancerous and paracancerous tissues.(2)Totally 3398 proteins were identified with mass spectrometry in exosomes of cancerous and paracancerous tissues,among which there were 549 differentially expressed proteins,and the significantly differentially expressed proteins were PRPS2,MCM6,PXDN and NT5C3A.GO analysis results showed that the biological processes were mainly focused on protein hydroxylation,RNA processing and gastrulation,the cellular components were focused on laminin complex,adherens junctions and mitochondrial membrane,and the molecular functions were focused on L-ascorbic acid binding,oxygen binding and organic acid binding in these four significantly differentially expressed proteins.KEGG analysis results showed that the metabolism and signaling pathways which possibly involved in these four significantly differentially expressed proteins mainly included nitrogen metabolism,ECM-receptor interactions and nuclear protein synthesis.(3)Kaplan-Meier survival analysis results showed that the expression of PRPS2 protein in cancerous tissues was correlated with the 5-year overall survival rate of NSCLC patients(P=0.014),and the expressions of MCM6,PXDN and NT5C3A proteins were not correlated with the 5-year overall survival rate of NSCLC patients(P>0.05).PRPS2 protein was selected for subsequent experiments.(4)The relative expressions of PRPS2 mRNA and protein were higher in A549 cells(12.725±2.132,0.934±0.021)than those in BEAS-2B cells(1.025±0.023,0.051±0.004)(t=23.230,P<0.001;t=71.540,P<0.001).The relative expression of PRPS2 protein was lower in the si-PRPS2 group(0.034±0.004)than that in the si-NC group(0.731±0.012)(t=95.440,P<0.001)after 48-h transfection.The cell proliferation rates were lower in the si-PRPS2 group[(1.127±0.101)%,(1.802±0.118)%]than those in the si-NC group[(1.709±0.152)%,(2.495±0.156)%](t=7.146,P<0.001;t=8.508,P<0.001)after 72-and 96-h culture,and showed no significant differences between two groups after 0-,24-and 48-h culture(P>0.05).The migration rate was lower in the si-PRPS2 group[(23.979±4.220)%]than that in the si-NC group[(38.851±4.981)%](t=4.946,P=0.009)and the number of invasive cells was less in the si-PRPS2 group(26.000±6.557)than that in the si-NC group(54.333±5.132)(t=7.894,P<0.001)after 48-h transfection.(5)The exosomes isolated from successfully transfected cells in the si-NC group and si-PRPS2 group presented spherical or oval vesicle-like structures.The particle size and particle peak value matched the typical exosomal structural characteristics.The relative expression of PRPS2 protein was lower in the siPRPS2-exo group(0.046±0.004)than that in the NC-exo group(0.334±0.012)(t=39.440,P<0.001)after 48-h coculture.The cell proliferation rates were lower in the siPRPS2-exo group[(0.797±0.066)%,(1.249±0.092)%,(1.789±0.173)%]than those in the NC-exo group[(1.089±0.090)%,(1.877±0.180)%,(2.859±0.221)%](t=4.536-8.610,all P values<0.05)after 48-,72-and 96-h coculture,and showed no significant differences between two groups after 0-and 24-h coculture(P>0.05).The migration rate was lower in the siPRPS2-exo group[(21.663±2.952)%]than that in the NC-exo group[(59.476±2.383)%](t=14.831,P<0.001),and the number of invasive cells was less in the siPRPS2-exo group(32.000±2.646)than that in the NC-exo group(80.333±7.024)(t=11.154,P<0.001)after 48-h coculture.Conclusions PRPS2 protein is highly expressed in exosomes of NSCLC tissues and A549 cells,and to knockdown its expression can inhibit the proliferation,migration and invasion ability of A549 cells.PRPS2 can accelerate the malignant biological behaviors of A549 cells via the exosomal pathway.
作者
孙明飞
李佩钰
潘丽红
孟庆江
郑先杰
耿盛男
张双林
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)
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)。
