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

Mechanism of disordered subcellular localization of TFE3 fusion protein in TFE3 translocation renal cell carcinoma

目的探究TFE3基因易位相关性肾癌(TFE3 tRCC)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TFE3 tRCC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TFE3 tRCC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;LipoFiter TM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(Western blotting)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TFE3 tRCC细胞核中定位;共聚焦显微镜观察及Wsertern blotting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程中发挥着重要作用。TFE3 tRCC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TFE3 tRCC肿瘤的发生和进展。

Objective To investigate the effects of the loss of exon 1 of TFE3 on nuclear localization of chimeric TFE3 protein in TFE3 translocation renal cell carcinoma(TFE3 tRCC).Methods The localization of TFE3 protein in TFE3 tRCC and clear cell renal cell carcinoma(ccRCC)were detected with immunochemistry.The exon retention of TFE3 gene in TFE3 tRCC was analyzed in databases and literatures.The plasmids containing TFE3 full-length and different-length of TFE3 exons which were constructed to pCDH-MCS-EGFP-Puro were transfected into HEK293T using Lipo Fiter TM.The localization of EGFP protein in HEK293T cells were detected with confocal microscopy.The localization of TFE3 protein and truncated TFE3 protein were detected with Western blotting.The mRNA expression of the downstream genes of TFE3 protein were detected with q-PCR.Results Strong nuclear signal of TFE3 protein was observed in TFE3 tRCC,whereas TFE3 protein in ccRCC was mainly localized in cytoplasm.The results of fluorescence imaging and Western blotting showed that TFE3 full-length protein was expressed both in nucleus and cytoplasm,and the expression of truncated TFE3 protein was mainly localized in nucleus.The q-PCR analysis demonstrated that the deletion of exon 1 in TFE3 gene led to a higher transcriptional level of targeted genes of TFE3 protein.Conclusion The loss of exon 1 in TFE3 played a critical role in preventing TFE3 protein from entering the nucleus.In TFE3 tRCC,the loss of exon 1 in TFE3 gene leads to the nuclear localization of TFE3 fusion protein and activation of its downstream target genes.This mechanism promises to uncover the occurrence and development of TFE3 tRCC.