SPRY4-IT1介导SMD途径靶向降解TCEB1调控乳腺癌干细胞特性的分子机制

Breast cancer stem cells (BCSCs) are responsible for the chemoresistance and recurrence of breast cancer. The regulation of long non-coding RNAs (LncRNAs) on the characteristics of BCSCs attracts researchers' more interest. We found that SPRY4 interionic transcript 1 (SPRY4-IT1) was specifically up-regulated in MCF-7 mammary sphere cells (MCF7 MS) with characteristics of BCSCs by LncRNA-Chip detection. Furthermore, in breast cancer tissues, we also found that the high expression of SPRY4-IT1 was positively correlated with the expression of p65, and negatively correlated with the expression of TCEB1, a key adaptor in E3 ubiquitin ligase complex, contributed to the HIF-1α/2α ubiquitination. Bioinformatic analysis showed that there were four binding sites between p65 and the promoter of SPRY4, and there was base-pairing between the Alu element with SPRY4-IT1 and the Alu element with the 3'UTR of TCEB1 mRNA. Can SPRY4-IT1 be activated by p65, and then regulate the characteristics of BCSCs through (STAU1)-mediated mRNA decay (SMD) degrading TCEB1 mRNA? This remains to be elucidated. Therefore, in this project, we plan to detect and analyze the changes of SPRY4-IT1 degrading TECB1 mRNA after interfering the STAU1 expression, and clarify the SMD mechanisms of SPRY4-IT1 degrading TCEB1. Furthermore, after confirming the direct key binding sites between p65 and SPRY4 promoter and translation activation of p65 on the SPRY4-IT1, our study will clarify the mechanism of SPRY4-IT1 activated by p65 and regulating the characteristics of BCSCs through degrading TCEB1 mRNA with SMD, and will propose a new idea and target for deep researching the mechanism of maintaining the BCSCs characteristics.

BCSCs是乳腺癌耐药复发的根源，LncRNA调控BCSCs特性备受关注。课题组通过LncRNA芯片检测到BCSCs中SPRY4-IT1特异高表达，并与P65表达正相关，与降解HIF-1α/2α的E3泛素连接酶关键因子TCEB1负相关；且预测到SPRY4-IT1存在与P65结合位点及与TCEB1-3'UTR区Alu元件互补。SPRY4-IT1是否被P65激活、进而通过STAU1介导mRNA降解（SMD）靶向抑制TCEB1调控BCSCs特性呢？有待阐明。本项目通过检测干扰STAU1表达后SPRY4-IT1降解TCEB1的变化，明确SPRY4-IT1降解TCEB1mRNA的SMD机制；通过确认p65与SPRY4-IT1启动子关键位点结合调控转录，阐明SPRY4-IT1被p65激活、进而介导SMD途径靶向降解TCEB1调控BCSCs特性的分子机制。为深入研究BCSCs特性机制提供新思路和新靶点。

侵袭转移是目前影响肿瘤患者预后和生存质量的最大危险因素之一，阐明肿瘤发生侵袭转移的机制并有效的预防肿瘤侵袭转移是延长患者生存期和提高患者生存质量的重要手段。研究表明SPRY4-IT1可促进多种肿瘤的侵袭转移，因此探究其促进肿瘤侵袭转移的机制对多种肿瘤均有重要价值。本项目拟探讨SPRY4-IT1对乳腺癌、大肠癌和卵巢癌细胞TCEB1表达的调控作用及其介导的侵袭转移。通过检测不同侵袭迁移能力乳腺癌、大肠癌和卵巢癌细胞中的SPRY4-IT1表达水平，发现高侵袭的细胞系SPRY4-IT1高表达，低侵袭的肿瘤细胞系SPRY4-IT1低表达；在高侵袭的细胞系中沉默SPRY4-IT1的表达可抑制肿瘤细胞侵袭迁移能力，在低侵袭的细胞系中过表达SPRY4-IT1可促进肿瘤细胞侵袭迁移；基因芯片结果显示，过表达SPRY4-IT1的表达后可下调TCEB1的表达水平；生物信息学预测、荧光素酶报告基因分析证实SPRY4-IT1可与TCEB1mRNA-3‘UTR结合，RIP实验证实该结合可招募STAU1蛋白。过表达SPRY4-IT1，可下调HIF-1α的表达水平，TCEB1的过表达会逆转SPRY4-IT1对HIF-1α的调控作用。采用结肠癌细胞构建裸鼠移植瘤模型，SPRY4-IT1过表达的HCT116细胞相对于亲本的HCT116细胞更容易发生肝脏和肺部转移，且小鼠生存期更短；同时检测了肿瘤患者组织中SPRY4-IT1、TCEB1和HIF-1α的表达水平，结果显示指标之间的表达关系与细胞水平一直；综上，SPRY4-IT1能过通过与TCEB1mRNA-3’UTR相互作用，下调TCEB1的表达，进而解除其对下游HIF-1α的降解，从而介导的肿瘤侵袭转移作用，其有望成为抑制肿瘤侵袭转移的新靶点。

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