长链非编码RNA ANRIL下调PLZF表达介导胃癌细胞上皮-间充质转化的机制研究

PLZF是一种肿瘤抑制基因，其缺失或低表达导致肿瘤的发生和侵袭，但具体机制尚不明确。前期工作显示PLZF在胃癌中普遍低表达，而沉默ANRIL和EZH2可以恢复PLZF的表达；过表达PLZF抑制胃癌细胞增殖、增加E-cadherin的表达，表明PLZF对胃癌发病及侵袭具有重要作用。本研究拟采用IP检测PLZF与PRC2组分之间相互作用，RNA-RIP检测PLZF、PRC2组分与ANRIL的相互作用，同时检测EZH2诱导的H3K27me3与PLZF表达的关系，阐明ANRIL绑定PRC2引起组蛋白修饰沉默PLZF表达的机制；然后观察PLZF对胃癌细胞EMT的的影响，阐明PLZF低表达促进肿瘤发生的机制；最后在体内验证PLZF在胃癌的表达及调控机制。本课题拟从以上几方面探讨PLZF在胃癌低表达的表观调控机制及对EMT的影响，增加对胃癌发病机制的认识，为胃癌的诊断和治疗提供新的生物标志物和理论依据。

Promyelocytic leukemia zinc finger protein (PLZF), a tumor suppressor gene, its deletion or low expression often results in tumorigenesis and invasion, but the mechanism is still obscure. Our preliminary work has verified that PLZF is generally low expressed in gastric cancer cells and overexpression of PLZF inhibits gastric cancer cell proliferation, increases expression of the epithelial cell marker E-cadherin, suggesting that PLZF plays an important role in tumorigenesis and progression of gastric cancer. According to our preliminary work and reported literature, we screened a panel of long noncoding RNAs (lncRNAs) from those known differential expression in gastric cancer and found that INK4 locus antisense non-coding RNA (ANRIL) was colsely associated with low expression of PLZF, and silencing ANRIL and EZH2 could increase PLZF expression. Therefore, our study intends to explore regulatory mechanism and effect on epithelial-mesenchymal transformation (EMT) of the PLZF deletion in gastric cancer. Firstly, we will perform immunoprecipitation and RNA-RIP techniques to detect interaction of PLZF and PRC2 components SUZ12 and EZH2 and interaction of PLZF, PRC2 and ANRIL, Western blot to detect the EZH2-induced H3K27 trimethylation. In light of these, we will clarify the mechanism of ANRIL binding PRC2 to silence PLZF expression. Next, to clarify the mechanism of tumorigenesis by PLZF, we will observe the effect of overexpressing PLZF on EMT in gastric cancer cells. Finally, PLZF expression, H3K27-methylated status and its effect on tumor EMT will be detected by above same techniques in gastric cancer xenografts on nude mice to monitor regulatory mechanism of PLZF on gastric cancer in vivo. Therefore, this project intends to study why PLZF is low expressed in gastric cancer and its impact on tumor EMT from above several aspects. It will strongly increase the understanding of tumor pathogenesis and provide a new biomarker and rationale for the diagnosis and treatment of gastric cancer.