AXL在鼻咽癌中的功能及表达调控的分子机制

Function of AXL and molecular mechanisms in regulation of nasopharyngeal carcinoma

目的:鼻咽癌(nasopharyngeal carcinoma,NPC)是一种高侵袭性的上皮源性恶性肿瘤,具有独特的地理和种族分布特征,多发于中国南方和东南亚地区,其治疗主要依靠放射治疗和化学治疗。但NPC通常发现于晚期,且常出现局部复发和远处转移,患者预后较差。受体酪氨酸激酶AXL在多种肿瘤中表达上调,参与肿瘤增殖、迁移、侵袭等过程,与肿瘤不良预后相关。本研究旨在检测AXL在NPC细胞系及组织中的表达,探讨AXL在NPC中的生物学功能及表达调控的分子机制。方法:利用基因表达综合(Gene Expression Omnibus,GEO)数据库中的GSE68799、GSE12452、GSE53819三个数据集分析AXL在正常鼻咽上皮组织及NPC组织中的表达水平,利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析AXL与头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSC)患者预后的关系,包括总生存时间(overall survival,OS)、无病生存期(disease-free interval,DFI)、疾病特异性生存期(disease-specific survival,DSS)及无进展生存期(progression-free interval,PFI)4个预后指标;采用蛋白质印迹法检测AXL在正常鼻咽上皮细胞系及NPC细胞系中的蛋白质表达水平,免疫组织化学实验检测AXL在正常鼻咽上皮组织及NPC组织中的表达;利用慢病毒干扰载体包装病毒感染5-8F和Fadu细胞建立稳定敲低AXL的细胞系,利用慢病毒表达载体包装病毒感染C666-1和HK-1细胞建立稳定过表达AXL的细胞系,并通过real-time PCR及蛋白质印迹法检测稳转细胞系的干扰和过表达效率;利用CCK-8、平板克隆形成实验及Transwell实验检测敲低或过表达AXL对NPC细胞增殖、迁移及侵袭能力的影响,利用裸鼠皮下成瘤实验检测敲低AXL对裸鼠体内肿瘤生长的影响;利用UCSC在线数据库查询并获取AXL上游2.0 kb启动子区域的碱基序列,将序列输入PROMO在线数据库中以容错率0%为条件预测AXL的转录因子,并利用JASPAR在线数据库预测ETS1与AXL的结合位点;通过real-time PCR及蛋白质印迹法检测ETS1表达改变对AXL蛋白及mRNA表达水平的影响;将AXL上游2.0 kb启动子区域划分为8个片段,每个片段长度为250 bp,分别针对8个片段设计引物,利用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)实验检测ETS1与AXL启动子区域的结合,明确ETS1对AXL的直接调控关系;利用功能回复实验检测ETS1是否通过AXL影响NPC细胞的增殖、迁移及侵袭能力。结果:生物信息学分析发现AXL在NPC组织中高表达(P<0.05),且AXL高表达与HNSC患者更短的OS、DFI、DSS及PFI呈正相关。蛋白质印迹及免疫组织化学实验结果显示:相对于正常鼻咽上皮细胞系及组织,AXL在NPC细胞系及组织中高表达。Real-time PCR及蛋白质印迹实验结果显示:稳转细胞系的干扰及过表达效率满足后续实验要求。CCK-8、平板克隆形成实验、Transwell实验及裸鼠皮下成瘤实验结果表明:AXL表达下调显著抑制NPC细胞的增殖、迁移、侵袭及肿瘤生长(均P<0.05),AXL表达上调显著促进NPC细胞的增殖、迁移及侵袭(均P<0.05)。PROMO及JASPAR在线数据库预测结果显示:ETS1是AXL的转录因子,且与AXL启动子区域存在多个结合位点。Real-time PCR及蛋白质印迹法结果显示:敲低或过表达ETS1能够下调或上调AXL蛋白及mRNA的表达水平。ChIP实验结果显示:ETS1能与AXL的启动子区域结合,直接调控AXL的表达。功能回复实验结果显示:AXL能够回复ETS1对NPC细胞增殖、迁移及侵袭能力的影响(P<0.05)。结论:AXL在NPC细胞系及组织中高表达,能促进NPC的恶性进展,其表达受转录因子ETS1的调控。

Objective:Nasopharyngeal carcinoma(NPC)is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics.NPC is mostly found in south China and Southeast Asia,and its treatment mainly depends on radiotherapy and chemotherapy.However,NPC is usually found in the late stage,and local recurrence and distant metastasis are common,leading to poor prognosis.The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation,migration,invasion,and other processes,which are associated with poor prognosis of tumors.This study aims to detect the expression of AXL in NPC cell lines and tissues,and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC.Methods:The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799,GSE12452,and GSE53819 data sets based on Gene Expression Omnibus(GEO)database.The Cancer Genome Atlas(TCGA)database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma(HNSC).The indicators of prognosis included overall survival(OS),disease-free interval(DFI),disease-specific survival(DSS),and progression-free interval(PFI).Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines.Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues.Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector,and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector.Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines.The effects of AXL knockdown or overexpression on proliferation,migration,and invasion of NPC cells were detected by CCK-8,plate colony formation,and Transwell assays,and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay.The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database.The PROMO online database was used to predict AXL transcription factors with 0%fault tolerance,and the JASPAR online database was used to predict the binding sites of ETS1 to AXL.Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and m RNA expression.The AXL upstream 2.0 kb promoter region was divided into8 fragments,each of which was 250 bp in length.Primers were designed for 8 fragments.The binding of ETS1 to AXL promoter region was detected by chromatin immunoprecipitation(Ch IP)assay to determine the direct regulatory relationship between ETS1and AXL.Rescue assay was used to determine whether ETS1 affected the proliferation,migration,and invasion of NPC cells through AXL.Results:Bioinformatics analysis showed that AXL was highly expressed in NPC tissues(P<0.05),and AXL expression was positively correlated with OS,DFI,DSS,and PFI in HNSC patients.Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues.Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments.The results of CCK-8,plate colony formation,Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation,migration,invasion of NPC cells and tumor growth(all P<0.05),and the up-regulation of AXL significantly promoted the proliferation,migration,and invasion of NPC cells(all P<0.05).As predicted by PROMO and JASPAR online databases,ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region.Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and m RNA expression levels.Ch IP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression.Rescue assay showed that AXL rescued the effects of ETS1 on proliferation,migration and invasion of NPC cells(P<0.05).Conclusion:AXL is highly expressed in NPC cell lines and tissues,which can promote the malignant progression of NPC,and its expression is regulated by transcription factor ETS1.