Comparative genome analysis of exosomal mRNA derived from Tspan8-overexpressing and Tspan8-negative rat pancreas tumors

We recently described that tumor-derived exosomes containing the tetraspanin Tspan8 induce angiogenesis. However, not much is known about exosome-endothelial cell interactions and there are no clues on the role of tetraspanins, which are a constitutive component of exosomes. We used the stably Tspan8 expressing rat adenocarcinoma cell line AS-Tspan8, to explore how exosomal Tspan8 supports angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes. A genome-wide microarray analysis revealed that both, AS- and AS-Tspan8-exosomes contain about 1500 transcripts, from which 285 are over 3 fold enriched in AS-Tspan8-exosomes. We confirmed high levels mRNA of transcription factors MycN and Spi-C, elongation factor B(SIII), TCEB-1, fibroblast growth factor binding protein 1 (FGFBP1) and growth and differentiation factor-3 (GDF3) in AS-Tspan8-exosomes compared to AS-exosomes and to the parental cells by RT-PCR. VEGF mRNA was not enriched in AS-Tspan8 cells or exosomes. Expression of CCL20, prothrombin, bone morphogenetic protein-2, several Ras and MAPK family members and a phosphatase2A subunit was below the detection level in exosomes.本课题组此前已报道，携带四跨膜蛋白（tetraspanin）Tspan8的肿瘤来源外泌体可诱导血管生成。然而，目前关于外泌体与内皮细胞的相互作用机制仍尚不明确，且作为外泌体组成型成分的四跨膜蛋白其具体功能亦缺乏相关研究线索。本研究采用稳定表达Tspan8的大鼠腺癌细胞系AS-Tspan8，探究外泌体携带的Tspan8如何促进血管生成。实验结果显示，Tspan8可介导蛋白质与信使核糖核酸（mRNA）选择性招募进入外泌体。全基因组微阵列分析表明，AS细胞及AS-Tspan8细胞来源的外泌体均携带约1500种转录本，其中285种转录本在AS-Tspan8外泌体中的富集倍数超过3倍。我们通过逆转录聚合酶链反应（RT-PCR）验证发现，相较于AS细胞来源外泌体及亲本细胞，AS-Tspan8外泌体中高表达转录因子MycN、Spi-C、延伸因子B(SIII)、TCEB-1、成纤维细胞生长因子结合蛋白1（FGFBP1）以及生长分化因子3（GDF3）的mRNA。血管内皮生长因子（VEGF）的mRNA在AS-Tspan8细胞及其外泌体中均未出现富集。外泌体中CCL20、凝血酶原、骨形态发生蛋白2、多种Ras家族与丝裂原活化蛋白激酶（MAPK）家族成员以及蛋白磷酸酶2A亚基的表达量均低于检测限。