长链非编码RNA ITPR1-AS1调控小细胞肺癌侵袭转移的机制研究

By using microarray analysis, we identified ITPR1-AS1 as a novel long non-coding RNA (lncRNA), which is highly expressed in small cell lung cancer (SCLC) metastatic lymph nodes (LN). Our pre-experiments demonstrated that ITPR1-AS1 was significantly overexpressed in SCLC metastatic lymph nodes, positively correlated with clinical stages, and negatively correlated with prognosis. SiRNA-mediated silence of ITPR1-AS1 significantly inhibited migration and invasion of SCLC cells. Bioinformatic analysis, literature search and pre-experimentation revealed that ITPR1-AS1 could specifically bind with RNA binding proteins, DDX3X, which promoted Rac1 translation at the post-transcriptional level. Subsequently, when silencing ITPR1-AS1 by siRNA, we found that ITPR1-AS1 was closely related to the MAPK and Wnt signaling pathways. Based on the preliminary data, we therefore hypothesized that ITPR1-AS1 could promote SCLC invasion and metastasis via binding DDX3X, which encourages Rac1 translation at the post-transcriptional level, activation of MAPK and Wnt pathway. This project aims to validate that ITPR1-AS1 could promote the invasion and metastasis of SCLC in vitro and in vivo firstly. Secondly, the mechanism of ITPR1-AS1 promoting SCLC progression at post-transcriptional level will be deeply validated by the experiments of RIP, RNA pull down the assay. Finally, the correlation between ITPR1-AS1, DDX3X proteins, Rac1 and SCLC progression will be further validated in a large number of samples of clinical patients. There has been no report about ITPR1-AS1 function so far, thus，this study may provide a new target for the prevention and treatment of SCLC.

ITPR1-AS1（简称IT）是我们基于原发及转移灶配对的人小细胞肺癌（SCLC）组织、经高通量筛选获得、显著高表达于转移灶的长非编码RNA。前期发现：IT表达越高，SCLC患者分期越晚、预后越差；体外沉默IT显著抑制SCLC细胞侵袭转移。生物信息学分析、文献检索及预实验提示：IT可绑定“促癌”RNA结合蛋白DDX3X，在转录后水平促进Rac1翻译，并激活Rac1已知下游MAPK、Wnt信号通路。故提出“IT可绑定DDX3X转录后促进Rac1翻译、激活MAPK及Wnt通路、从而促进SCLC侵袭转移”的科学假说。本项目拟通过临床样本及体内外实验证实IT促进SCLC侵袭转移；结合过表达/沉默策略、设计拯救实验，阐明IT绑定DDX3X调控SCLC侵袭转移的分子机制；临床评价IT/DDX3X通路与SCLC侵袭转移的相关性。有关IT调控SCLC侵袭转移的功能机制未见报道，本研究将为其防治提供新思路。

ITPR1-AS1（简称IT）在人小细胞肺癌（SCLC）转移淋巴结组织中高表达，且IT表达越高，SCLC患者分期越晚、预后越差；体外过表达IT可显著促进SCLC细胞侵袭转移，沉默IT显著抑制SCLC细胞侵袭转移。生物信息学分析及预实验提示：IT可在转录后水平绑定“促癌”RNA结合蛋白DDX3X。在分别过表达及干扰IT后，敲除DDX3X表达，可以部分拯救IT对SCLC侵袭转移能力的促进作用。RNA-seq提示沉默IT后，差异表达基因主要与Wnt信号通路密切相关。故提出“IT可在转录后水平绑定DDX3X，激活下游Wnt信号通路，调控SCLC细胞侵袭转移过程”。在过表达IT后，Wnt信号通路基因TCF4、TCF7L1、FZD4表达水平明显上调，敲除DDX3X表达，TCF4、TCF7L1、FZD4表达上调作用被部分拯救，干扰IT后，敲除DDX3X表达，Wnt信号通路基因TCF4、TCF7L1、FZD4表达水平进一步下降。在体内实验中，过表达IT后，裸鼠肺部成瘤及转移瘤形成能力增强，肿瘤标本中DDX3X及TCF4、TCF7L1、FZD4表达水平出现上调，而抑制IT后，肿瘤标本中DDX3X及TCF4、TCF7L1、FZD4表达水平出现下调。