TFE3基因易位相关性肾癌中TFE3融合蛋白亚细胞定位失调的机制研究.pdf

现代泌尿外科杂志基础研究TFE3基因易位相关性肾癌中 TFE3融合蛋白亚细胞定位失调的机制研究潘骏，陈怡2.3.4，杨磊2.3.4，刘宁5，逐艳文，马文亮，甘卫东，李冬梅2.3.4（1.南京大学医学院附属鼓楼医院泌尿外科，江苏南京2 10 0 0 8；2.南京大学医学院免疫与生殖生物学实验室，江苏南京210093；3.南京市江苏省医学分子技术重点实验室，江苏南京2 10 0 9 3；4.南京大学生命分析化学国家重点实验室，江苏南京210093；5.南京医科大学附属南京医院泌尿外科，江苏南京2 10 0 0 6）Mechanism of disordered subcellular localization of TFE3 fusion protein in TFE3translocation renal cell carcinomaPAN Jun,CHEN Yi?,YANG Lei?,LIU Ning,LU Yanwen,MA Wenliang,GAN Weidong,LI Dongmei?(1.Department of Urology,Affiliated Drum Tower Hospital of Medical School of Nanjing University,Nanjing 210008;2.Laboratory of Immunology and Reproduction Biology,Nanjing University School ofMedicine,Nanjing 210093;3.Jiangsu Key Laboratory of Molecular Medicine,Nanjing University,Nanjing210093;4.State Key Laboratory of Analytical Chemistry for Life Science,Nanjing University,Nanjing210093;5.Department of Urology,Nanjing First Hospital,Nanjing Medical University,Nanjing 210006,China)ABSTRACT:Objective To investigate the effects of the loss of exon 1 of TFE3 on nuclear localization of chimeric TFE3protein in TFE3 translocation renal cell carcinoma(TFE3 tRCC).Methods The localization of TFE3 protein in TFE3 tRCCand clear cell renal cell carcinoma(ccRCC)were detected with immunochemistry.The exon retention of TFE3 gene in TFE3tRCC was analyzed in databases and literatures.The plasmids containing TFE3 full-length and different-length of TFE3 exonswhich were constructed to pCDH-MCS-EGFP-Puro were transfected into HEK293T using Lipo FiterTM.The localization ofEGFP protein in HEK293T cells were detected with confocal microscopy.The localization of TFE3 protein and truncated TFE3protein were detected with Western blotting.The mRNA expression of the downstream genes of TFE3 protein were detectedwith q-PCR.Results Strong nuclear signal of TFE3 protein was observed in TFE3 tRCC,whereas TFE3 protein in ccRCCwas mainly localized in cytoplasm.The results of fluorescence imaging and Western blotting showed that TFE3 full-lengthprotein was expressed both in nucleus and cytoplasm,and the expression of truncated TFE3 protein was mainly localized innucleus.The q-PCR analysis demonstrated that the deletion of exon 1 in TFE3 gene led to a higher transcriptional level oftargeted genes of TFE3 protein.Conclusion The loss of exon 1 in TFE3 played a critical role in preventing TFE3 proteinfrom entering the nucleus.In TFE3 tRCC,the loss of exon 1 in TFE3 gene leads to the nuclear localization of TFE3 fusionprotein and activation of its downstream target genes.This mechanism promises to uncover the occurrence and development ofTFE3 tRCC.KEY WORDS:TFE3 fusion protein;exon;nuclear localization;TFE3 translocation renal cell carcinoma摘要：目的探究TFE3基因易位相关性肾癌（TFE3tRCC)中TFE3融合基因的TFE3片段1号外显子丢失对TFE3融合蛋白亚细胞定位的影响。方法免疫组织化学方法对TFE3tRCC和肾透明细胞癌（ccRCC)临床样本中TFE3蛋白进行定位观察；数据库和文献调研统计TFE3tRCC中TFE3基因外显子保留情况；pCDH-MCS-EGFP-Puro慢病毒过表达载体构建分别含TFE3片段全长及1号外显子缺失的增强绿色荧光蛋白（EGFP)融合重组质粒;LipoFiterTM试剂转染含不同TFE3片段重组质粒进入人胚肾细胞系HEK293T，共聚焦显微镜观察EGFP的亚细胞定位；蛋白免疫印迹法（Westernblotting）检测不同TFE3蛋白片段在胞浆胞核中的表达；实时荧光定量检测技术（q-PCR)检测不同TFE3蛋白片段对其下游靶基因转录的影响。结果病理切片显示TFE3融合蛋白在TFE3tRCC细胞核中定位；共聚焦显微镜观察及Wserternblotting结果显示全长TFE3蛋白平均分布在细胞质和细胞核，而缺失1号外显子的TFE3蛋白片段则主要定位于细胞核；q-PCR分析结果显示，TFE3蛋白的下游靶基因转录水平在缺失1号外显子的TFE3蛋白片段组显著上调。结论TFE3基因1号外显子在阻止TFE3蛋白进入细胞核的过收稿日期：2 0 2 2-0 9-0 2基金项目：江苏省自然科学基金（No.BK20221444）；江苏省卫生健康委医学科研项目（No.ZD2022013）通信作者：李冬梅，副教授。E-甘卫东，主任医师。E-mail：g w d n ju.e d u.c n，系共同通信作者作者简介：潘骏，硕士研究生在读。研究方向：肾肿瘤的致病机制。E-mail:陈怡，博士研究生在读。研究方向：肾肿瘤的致病机制。E-mail:DG，系共同第一作者2023年8 月第2 8 卷第8 期修回日期：2 0 2 2-11-2 5http:/;zgmnwk.cug.top713714程中发挥着重要作用。TFE3tRCC中，1号外显子丢失的TFE3片段导致融合型TFE3蛋白在细胞核定位，并激活其下游靶基因的表达，该机制有望解释TFE3tRCC肿瘤的发生和进展。关键词：融合型TFE3蛋白；外显子；核定位；TFE3基因易位相关性肾癌中图分类号：R737.11TFE3基因易位相关性肾癌（TFE3transloca-tion renal cell carcinoma,TFE3 tRCC)是一类发病率相对较低，但恶性程度极高的肾癌1-2。不同于常见肾癌，TFE3tRCC好发于45岁以下人群，且呈现出女性高发的特点3-4，提示该类肾癌具有不同于常见肾癌的致病机制。X染色体一区一带二亚带上的TFE3基因断裂，与其他染色体上的基因发生平衡易位并融合是导致TFE3tRCC发病的直接驱动因素。TFE3片段与含有强启动子的伙伴基因融合形成高表达的TFE3 融合蛋白成为该类肾癌的特有表征5。前期研究发现TFE3融合蛋白在细胞核内聚集并可持续激活下游靶基因的表达，进一步研究揭示TFE3融合蛋白脱离了野生型TFE3蛋白依赖哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）的胞质-胞核转运机制C6-8，并且也与线粒体自噬引起的TFE3人核机制无关9-10 。因此，有必要针对TFE3融合蛋白的人核机制进行更加深人的探究。已知，融合型TFE3基因中保留最长的TFE3片段为2-10 号外显子，保留最短的为7-10 号外显子1-12 ，据此推测1号外显子在TFE3融合蛋白的入核机制中扮演着重要角色。1资料与方法1.1材料人胚肾细胞系 HEK293T(Cat#CRL-3216）购自ATCC细胞库；DMEM培养基（Cat#31900608)购自维森特生物科技有限公司；胎牛血清(fetal bovine serum，FBS）（C a t#2 2 32 510）购自表1TFE3基因各外显子序列引物基因TFE3全长-FTFE3全长-RTFE32-10号外显子-FTFE32-10号外显子-RTFE33-10号外显子-FTFE33-10号外显子-RTFE34-10号外显子-FTFE34-10号外显子-RTFE35-10号外显子-FTFE35-10号外显子-RTFE36-10号外显子-FTFE36-10号外显子-RF：正向引物（forwardprimer);R：反向引物（reverseprimer)。J Mod Urol,Vol.28 No.8 Aug.2023文献标志码：AD0I:10.3969/j.issn.1009-8291.2023.08.015Gibco公司；pCDH-MCS-EGFP-Puro病毒过表达载体（VT8070)购自优宝生物；快速DNA胶回收试剂盒（Cat#D205-04)购自北京康润生物科技有限公司；无内毒素质粒抽提试剂盒（Cat#DP117)购自天根生物；片段重组试剂盒（Cat#C112）、逆转录试剂盒（C a t#R111-0 1/0 2）及实时荧光定量检测试剂盒(Cat#Q711-02)购自南京诺维赞生物科技有限公司;转染试剂LipoFiterTM（C a t#H B-L F-10 0)购自汉恒生物；抗荧光猝灭封片液（含DAPI)（P0 131）、细胞核蛋白和细胞浆蛋白抽提试剂盒（P0027）购自碧云天生物科技有限公司；TFE3抗体（354R-1)购自Sig-ma-Aldrich公司;GAPDH抗体（ACo02)购自爱博泰克公司；L