长链非编码RNA BC017398介导转录因子SP2调控MOP促进肝癌转移的机制研究

Although metastasis is the overwhelming causes of therapeutic failure and cancer-related mortality in patients with hepatocellular carcinoma (HCC), the molecular and cellular mechanisms remain largely unknown. Our studies demonstrated that mu-opioid receptor (MOP)-mediated epithelial-mesenchymal transition (EMT) promotes HCC metastasis, but the upstream of MOP needs to be elucidated. Our preliminary results showed that transcription factor SP2 may activate MOP transcription. RIP-seq and luciferase reporter assay further revealed long noncoding RNA (lncRNA) BC017398 may play a central role in the transcriptional activation of MOP through an interaction with SP2. The suppressed MOP expression caused by BC017398 knock-down was rescued by overexpressing SP2. Thus we speculate that lncRNA BC017398 cooperates with transcription factor SP2 to promote MOP-mediated hepatocellular carcinoma metastasis. In this study, the methods of ChIRP, RNA pull-down, ChIP, establishing stable expression cell lines, and subcutaneous and hepatic transplanted model with HCC cells are utilized to explore the mechanisms of BC017398/SP2/MOP axis in HCC. This project provides a new idea as well as theoretical basis on the better understanding and targeted therapy of HCC metastasis.

转移是导致肝癌治疗失败和死亡的主要原因，但其机制尚不明晰。申请人前期研究结果表明：mu-阿片受体（MOP）可通过EMT调控肝癌转移，然而其上游驱动因素有待进一步研究阐明。前期我们已经完成以下实验内容：1）转录因子SP2可能调控MOP的转录；2）通过RIP-seq和luciferase实验验证发现，长链非编码RNA（lncRNA）BC017398可能参与SP2对MOP的调控过程；3）敲低BC017398可以引起MOP的下调，但能够被过表达SP2所逆转。由此申请人提出科学假设：lncRNA BC017398介导SP2调控MOP促进肝癌转移。本项目拟采用ChIRP、RNA pull-down、ChIP、构建稳定细胞株、裸鼠皮下及肝脏原位肝癌移植瘤模型等实验方法，旨在解析BC017398/SP2/MOP轴在肝癌发生发展中的作用及具体机制，本研究的实施有望为肝癌的转移机制和靶向治疗提供新的理论依据。

转移是导致肝癌治疗失败和死亡的主要原因，但其机制尚不明晰。申请人研究结果表明：mu-阿片受体（MOP）可通过EMT调控肝癌转移，然而其上游驱动因素有待进一步研究阐明。我们结果发现长链非编码RNA BC017398在肝癌中表达上调并与不良预后有关。进一步通过我们通过构建其稳定过表达与稳定敲低的手段，发现LncRNA BC017398可调控肝癌细胞的增殖和转移；通过内源性RIP、外源性Pulldown及ChIRP实验对LncRNA BC017398、SP2与MOP的相互关系进行了考察，RIP和Pulldown实验表明LncRNA BC017398和SP2蛋白存在相互结合。LncRNA BC017398与SP2蛋白结合互作的核心区域位于100-149nt。进一步证实了LncRNA BC017398通过直接结合SP2蛋白，调控MOP的表达。本研究有望为肝癌的转移机制和靶向治疗提供新的理论依据。