To investigate the downstream target gene of YAP1 in enzalutamide-resistant (EnzaR) cells and the effects of extracellular vehicles (EVs) derived from EnzaR cells on enzalutamide-sensitive cells by next-generation sequencing analyses. To investigate the downstream target gene of YAP1 in enzalutamide-resistant (EnzaR) cells and the effects of extracellular vehicles (EVs) derived from EnzaR cells on enzalutamide-sensitive cells by next-generation sequencing analyses

Our study has identified that yes-associated protein 1 (YAP1) is overexpressed in enzalutamide-resistant (EnzaR) cells. Furthermore, enzalutamide-induced YAP1 expression is mediated through the function of chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII) at the transcriptional and the post-transcriptional levels. Furthermore, both YAP1 and COUP-TFII have proven to be cargoes of EVs by our study. Next, treatment with EnzaR-EVs induces the abilities of enzalutamide resistance in its parental cells while depletion of YAP1 or COUP-TFII in EnzaR-EVs attenuated it. In order to systemically investigate the downstream target gene of YAP1 and the expression profiles changed by EnzaR-EVs from control, YAP1 and COUP-TFII depletions in prostate cancer cells, we have performed RNA-seq analyses to pursuit the research directions of those questions. Overall design: RNA samples were isolated from EnzaR cells treated with siRNA against YAP1 and control siRNA (n=2), and from LNCaP cells treated without or with control, YAP1- and COUP-TFII-depleted EnzaR-EVs (n=1) for 48 hours.

本研究证实，Yes相关蛋白1（yes-associated protein 1, YAP1）在恩扎卢胺耐药（enzalutamide-resistant, EnzaR）细胞中呈过表达状态。进一步研究发现，恩扎卢胺诱导的YAP1表达，是通过鸡卵清蛋白上游启动子转录因子2（chicken ovalbumin upstream promoter transcription factor 2, COUP-TFII）在转录及转录后水平发挥调控作用实现的。本研究还证实，YAP1与COUP-TFII均可作为细胞外囊泡（Extracellular Vesicles, EVs）的cargo。后续实验显示，EnzaR-EVs可诱导其亲本细胞产生恩扎卢胺耐药性；而在EnzaR-EVs中敲降YAP1或COUP-TFII，则会削弱该效应。为系统探究YAP1的下游靶基因，以及前列腺癌细胞分别经对照组来源EnzaR-EVs、YAP1敲降EnzaR-EVs及COUP-TFII敲降EnzaR-EVs处理后发生的表达谱变化，我们开展了RNA测序（RNA-seq）分析以探寻相关研究方向。总体实验设计如下：我们提取了经靶向YAP1的小干扰RNA（siRNA）及对照siRNA处理的EnzaR细胞的RNA样本（n=2）；同时提取了经未处理、对照EnzaR-EVs、YAP1敲低EnzaR-EVs及COUP-TFII敲低EnzaR-EVs处理48小时的LNCaP细胞的RNA样本（n=1）。