增强子RNA对胃癌肝素酶基因表达的调控作用及其机制研究

Recent evidence shows that enhancer RNA (eRNA), a novel kind of endogenous noncoding RNA, can enhance gene expression through facilitating the assembly of transcription initiation complex on promoter via RNA-protein interaction. Our previous studies show that heparanase (HPA) is correlated with the invasion, metastasis, and angiogenesis of gastric cancer. In addition, we initially discovered the enhancer element upstream the HPA promoter, and found the over-expression of its transcribed eRNA in gastric cancer tissues and cell lines, which may participate in the regulation of HPA expression. Based on these findings, the current study will explore the roles of enhancer region and its binding transcription factor heat shock factor 1 (HSF1) in regulating the expression of HPA and eRNA in gastric cancer tissues and cell lines. To elucidate the underlying mechanisms for eRNA-medicated HPA expression, the interaction of eRNA with transcription initiation complex component TFIIB, and its regulatory effects on the binding of TFIIB with HPA promoter and HPA transcription will be investigated. After stable over-expression or knockdown of HPA enhancer-derived eRNA, the invasion, metastasis, and angiogenesis of gastric cancer cells will be studied in vitro and in vivo. This study will elucidate the roles of endogenous noncoding eRNA in HPA gene expression, and will provide a novel strategy and theoretical basis for the therapeutics of gastric cancer.

新近研究显示，内源性非编码增强子RNA（eRNA）能以RNA-蛋白质相互作用方式，调控转录起始复合物在启动子区域的募集，促进基因表达。我们前期研究表明：肝素酶（HPA）与胃癌侵袭、转移及血管生成密切相关；HPA启动子上游存在增强子元件，其转录生成的eRNA在胃癌组织和细胞中显著上调，且参与HPA基因表达调控。本项目拟在此基础上，解析增强子区域及其结合的转录因子热休克因子1（HSF1）对胃癌组织和细胞中HPA、eRNA表达的调控作用；观测胃癌组织和细胞中eRNA与转录起始复合物组分TFIIB的相互作用及其对TFIIB锚定至HPA启动子、HPA基因转录的影响，探讨eRNA调控HPA基因表达的分子机制；观测稳定过表达或抑制HPA基因增强子生成的eRNA对胃癌细胞体内外侵袭、转移及血管生成活性的影响，旨在从新型内源性非编码RNA角度阐明HPA基因表达调控机理，为胃癌的治疗提供新策略和理论依据。

胃癌是我国发病率和死亡率最高的消化道恶性肿瘤之一，70%以上胃癌早期无症状，确诊时已发生浸润、转移，预后较差。解析胃癌侵袭转移的分子机制及防治策略一直是我们致力追求的目标。新近研究显示：内源性非编码增强子RNA（eRNA）能以RNA-蛋白质相互作用方式，调控转录起始复合物在启动子区域的募集，促进基因表达。本研究发现：在人类肝素酶（HPSE）启动子临近的基因组区域存在增强子，能募集热休克因子1（HSF1），转录因子HSF1在胃癌组织和细胞中高表达，与HPSE表达水平正相关；荧光素酶-报告基因、染色质免疫沉淀、核连缀转录实验证实：HSF1能通过靶向结合至HPSE增强子区域，促进eRNA生成；RNA免疫沉淀、染色质免疫沉淀检测发现：胃癌组织和细胞中高表达的eRNA能结合核不均一核糖核蛋白U（hnRNPU），促进hnRNPU与p300蛋白的相互作用，进而募集转录因子IIB（TFIIB）、早期生长反应基因1（EGR1）作用于HPSE启动子的调控位点，上调胃癌中HPSE基因转录和表达水平。通过建立稳定过表达或拮抗eRNA的胃癌亚克隆细胞株，进一步发现eRNA能通过靶向上调HPSE基因表达，促进胃癌细胞的体内外增殖、侵袭转移、血管生成活性，而抑制eRNA表达能阻遏胃癌的恶性进展。因此，本项目率先在国内外揭示了eRNA对胃癌HPSE表达的调控作用及机制，丰富了HPSE基因表达调控机理，为胃癌的治疗提供了新途径和研究基础，具有广阔的应用前景和潜在的社会、经济效益。