pancRNA调控CTCF活性对胃癌E26转化特异性因子1表达的影响及机制研究

Recent evidence shows that promoter-associated non-coding RNAs (pancRNAs) are able to impact the activity of transcription regulatory proteins through RNA-protein interaction, and subsequently regulate gene expression. Our previous studies have indicated that E26 transformation-specific-1 (Ets-1) facilitates the invasion, metastasis, and angiogenesis of gastric cancer. The Ets-1 promoter-associated non-coding RNA (pancEts-1) might bind transcription factor CCCTC-binding factor (CTCF) and inhibit the CTCF-mediated repression on Ets-1 expression in gastric cancer. However, the underlying mechanisms still remain to be determined. This project is proposed to further observe the regulatory effects of CTCF-mediated recruitment of histone deacetylases and chromosome remodeling on Ets-1 expression, and elucidate the interaction domains between pancEts-1 and CTCF in gastric cancer. The impacts of pancEts-1 on the binding of CTCF on Ets-1 promoter and Ets-1 gene transcription will also be explored in gastric cancer. In addition, the in vitro and in vivo invasion, metastasis, and angiogenesis of gastric cancer cells will be studied through stable over-expression or knockdown of pancEts-1 and Ets-1 rescue experiments. This study will elucidate the crucial roles of endogenous pancRNA in regulating the Ets-1 expression, and will provide a novel approach and theoretical basis for the therapeutics of gastric cancer.

新近研究显示：启动子相关非编码RNA(pancRNA)能以RNA-蛋白质相互作用方式，影响转录调节蛋白的活性，调控基因表达。我们前期发现：E26转化特异性因子1(Ets-1)能促进胃癌侵袭转移及血管生成；Ets-1启动子区域存在的非编码RNA(pancEts-1)能结合转录因子CCCTC结合因子(CTCF)，阻遏CTCF对胃癌中Ets-1表达的抑制作用，但其内在机制不明。本项目拟进一步观测CTCF招募组蛋白去乙酰化酶导致的染色质重塑对胃癌Ets-1表达的调控作用，解析pancEts-1与CTCF相互作用的结构域，明确pancEts-1对CTCF结合至Ets-1启动子、Ets-1基因转录的影响，观测pancEts-1稳定过表达/抑制、Ets-1表达回补体系对胃癌细胞体内外侵袭转移及血管生成的影响，旨在从新型内源性pancRNA角度阐明Ets-1表达调控机理，为胃癌的治疗提供新途径和理论基础。

我国是胃癌发病率和死亡率最高的国家之一，70%以上胃癌早期无症状，确诊时已发生浸润、转移，预后较差，探究其内在调控机制将是胃癌诊治的一个突破点。E26转化特异性因子1(Ets-1)是促进胃癌侵袭、转移及血管生成的关键因子，但其表达调控机制不明。本研究发现：转录因子CCCTC结合因子(CTCF)、EST相关基因(ERG)在胃癌组织和细胞中分别下调、上调，与Ets-1表达水平负相关、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：CTCF、ERG能靶向结合至Ets-1启动子区域，分别抑制、促进Ets-1基因的表达；启动子相关非编码RNA(pancEts-1)在胃癌组织和细胞中高表达，与Ets-1表达水平正相关；RNA免疫沉淀、RNA pull-down、RNA EMSA、免疫共沉淀、染色质免疫沉淀检测发现pancEts-1能结合CTCF蛋白，阻遏CTCF招募组蛋白去乙酰化酶，促进癌细胞中Ets-1表达；pancEts-1亦能促进含非POU域八聚体结合蛋白(NONO)与ERG的相互作用，激活转录因子ERG作用于Ets-1启动子的调控位点，上调胃癌细胞中Ets-1基因转录和表达水平。将pancEts-1过表达及敲低载体稳定转染入胃癌细胞株，发现过表达或敲低pancEts-1能分别促进、抑制胃癌细胞的体内外增殖、侵袭及转移活性，在胃癌恶性进展中发挥重要作用。因此，本项目率先在国内外揭示了pancEts-1对胃癌Ets-1表达的调控作用及机制，丰富了Ets-1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。