口腔肿瘤干细胞样细胞中锌指蛋白750的潜在靶蛋白筛选  

Screening of potential target proteins of zinc finger protein 750 in oral cancer stem cell-like cells

作　　者：姜博 于水 杨一昆 徐聪[1] 杨红莉[1] 陈海英[1] JIANG Bo;YU Shui;YANG Yikun;XU Cong;YANG Hongli;CHEN Haiying(Central Laboratory,Liaocheng People's Hospital,Liaocheng 252000,China;不详)

机构地区：[1]聊城市人民医院中心实验室,山东聊城252000 [2]聊城市人民医院样本库

出　　处：《山东医药》2023年第15期38-42,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81773759)。

摘　　要：目的筛选锌指蛋白750(ZNF750)在口腔肿瘤干细胞样细胞(CSC-like cell)中的潜在靶蛋白。方法(1)CSC-like cell的富集:采用肿瘤球形成实验富集口腔鳞癌(OSCC)细胞系TCA-83及CAL-27细胞中CSC-like cell细胞。(2)CSC-like cell细胞中ZNF750潜在靶蛋白筛选:将CSC-like cell分为oe-Con组(转导空载体慢病毒)及oeZNF750组(转导过表达ZNF750慢病毒),采用TMT标记定量蛋白质组学技术筛选两组差异表达蛋白,对差异表达蛋白进行生物信息学分析[GO、KEGG及IPR(结构域)分析],从差异表达蛋白中选择与ZNF750关系密切的候选蛋白[角鲨烯合酶(FDFT1)、与糖代谢相关的糖原合成酶(GYS1)、与核糖体相关的核糖体蛋白RPL15及40S核糖体蛋白RPS7以及具有结构分子活性的角蛋白6A、17(KRT6A、KRT17)]进行实时荧光定量PCR(qPCR)验证。从与ZNF750关系密切的候选蛋白中选择FDFT1(与类固醇生物合成有关兼具酶转运活性)作为兴趣蛋白,采用qPCR检测CSC-like cell、口腔鳞癌(OSCC)细胞、正常口腔上皮细胞中FDFT1 mRNA的相对表达量,蛋白质印迹法检测过表达或敲减ZNF750基因的CSC-like cell中FDFT1蛋白相对表达量。结果(1)转导过表达ZNF750基因的CSC-like cell细胞中差异表达蛋白的筛选结果:与oe-Con组相比,oe-ZNF750组中差异表达蛋白共26个,其中上调9个,下调17个。(2)差异表达蛋白生信分析结果及其中与ZNF750关系密切的差异表达蛋白验证结果:GO分析结果显示,差异表达蛋白主要位于细胞内和细胞内非膜结合细胞器,参与生物合成及生物大分子的合成过程;涉及的生物功能包含结构分子活性、糖原合成酶活性、糖脂转运及糖脂结合功能;KEGG信号通路分析结果显示,差异表达蛋白显著富集在类固醇生物合成及糖代谢过程中;IPR分析证明富集的结构域包含糖脂转移蛋白结构域。qPCR结果显示,ZNF750可降低FDFT1、GYS1、RPL15、RPS7、KRT6A、KRT17基因的表达(P均<0.05)。(3)CSC-like Objective To screen the potential target proteins of zinc finger protein 750(ZNF750)in oral cancer stem cell-like cells(CSC-like cells).Methods①Enrichment of CSC-like cells:CSC-like cells were enriched from oral squamous-cell carcinoma(OSCC)cell lines TCA-83 and CAL-27 by tumor sphere formation assay.②Screening of the po⁃tential target protein of ZNF750 in CSC-like cells:CSC-like cells were divided into the oe-Con group(transduced with empty lentivirus)and oe-ZNF750 group(transduced with overexpressed ZNF750 lentivirus).The differentially expressed proteins(DEPs)between the two groups were identified by TMT-based proteomics technology,and then were analyzed by GO,KEGG and IPR(structural domain)analysis.The candidate proteins which were closely related to ZNF750 were se⁃lected from DEPs and were verified by real time fluorescent quantitative PCR(qPCR),such as squalene synthase(FDFT1),glycogen synthase related to glucose metabolism(GYS1),ribosome-related ribosome protein RPL15 and 40S ri⁃bosome protein RPS7,keratins 6A and 17(KRT6A,KRT17)with structural molecule activity.FDFT1 which was related to steroid biosynthesis and enzyme transport activity was selected as the interested protein from the candidate proteins which were closely related to ZNF750.Using qPCR to detect the relative expression of FDFT1 mRNA in CSC-like cells,OSCC cells,and normal oral epithelial cells.Western blotting was used to detect the relative expression level of FDFT1 in CSC-like cells with the ZNF750 gene overexpression or knocking down.Results①The screening results of DEPs in CSC-like cells transfected with ZNF750 gene:Compared with the oe-Con groups,there were 26 DEPs in the oe-ZNF750 group,of which,9 were up-regulated and 17 were down-regulated.②Bioinformatics analysis results for DEPs and the ver⁃ification results of DEPs which were closely related to ZNF750:GO analysis results showed that DEPs were mainly located in intracellular part and intracellular non-membrane-bounded organelle,which were mainly involved in the bio

关 键 词：锌指蛋白750 角鲨烯合酶 胆固醇代谢 肿瘤干细胞 蛋白组学测序技术 同位素标记定量技术 口腔鳞癌 

分 类 号：R739.8[医药卫生—肿瘤]