长链非编码RNA LINC00261对FOXA2基因的调控及其在Barrett食管发生中的作用和机制研究

The molecular mechanism of Barrett's esophagus (BE) remains unclear. Recently, LINC00261 was discovered to be highly expressed in BE tissues. Our previous experiment revealed that acid and bile exposure could lead to the upregulation of LINC00261 expression, and bioinformatics analysis indicates that LINC00261 maybe involved in the regulation of pioneer transcription factor FOXA2. CDX2 expression has been considered as playing a critical role in the development of BE. It has been determined that FOXA2 could recruit transcriptional repressors such as Rfx1 to inhibit the transcription of CDX2. Therefore, it is postulated that acid and bile exposure induced upregulation of LINC00261 results in the inhibition of FOXA2 transcription, which reduces the recruiting of Rfx1, and consequently in the interaction of inflammatory factors, the CDX2 expression in esophagus stem cells is induced. If this mechanism exists, it will enable the differentiation of esophagus stem cells into columnar epithelium and subsequently causes BE development. In this study, by in vitro and in vivo experiments, we will investigate the signal transduction pathway of LINC00261-FOXA2/Rfx1-CDX2 and its role in BE development which the columnar epithelium differentiates from esophagus stem cells. The results of this study will discover the potential molecular mechanism of BE development from lncRNA level, which may be very helpful for the prevention and therapy of BE.

Barrett食管（BE）的发生机制尚不清楚。LINC00261是新近发现在BE组织上调表达的lncRNA。我们前期研究发现酸暴露及胆酸可诱导LINC00261表达增高，生物信息学分析提示LINC00261可能参与先锋转录因子FOXA2表达的调控。CDX2表达是BE发生过程中的早期和必备事件。研究表明FOXA2可通过对负性转录调控因子Rfx1等的招募抑制CDX2转录。基于此，本课题提出：炎性因素诱导的LINC00261异常表达致FOXA2表达下调，消弱了其对Rfx1等的招募，由此在炎性因子等的共同作用下，食管干细胞CDX2表达开启，转分化为柱状上皮细胞，BE发生。本课题拟针对上述假设，进行体内外实验研究，探讨LINC00261-FOXA2/Rfx1-CDX2信号通路并参与食管干细胞转分化为柱状上皮细胞致BE发生的机制，其研究结果将从全新角度揭示BE发生的分子机制，为BE防治提供新的途径。

我们首先利用qRT-PCR技术检测LINC00261在中国人群正常食管鳞状上皮、Barrett食管及食管腺癌组织标本中的表达水平变化，结果显示在Barrett食管及食管腺癌组织标本中，LINC00261表达较正常食管鳞状上皮组织均显著上调；酸暴露及胆酸可诱导人胚胎干细胞LINC00261表达显著上调并抑制FOXA2蛋白表达水平，而LINC00261特异性反义寡核苷酸转染可削弱对FOXA2蛋白表达水平的这一抑制作用；上调FOXA2表达可阻断人胚胎干细胞在酸暴露及胆酸诱导下CDX2表达及细胞分化表型（柱状上皮标志物MUC2表达）的变化。进一步，通过RIP及CHIP实验发现LINC00261可与组蛋白甲基转移酶EZH2相结合，进而抑制FOXA2的转录；而在正常人胚胎干细胞中，通过CHIP、Co-IP、双荧光素酶报告基因实验等证实FOXA2可通过招募负性调控子Rfx1结合于CDX2启动子+7 kb区域，由此抑制CDX2的转录；慢病毒转染上调LINC00261表达或胆酸诱导的LINC00261表达上调均可导致FOXA2/Rfx1复合物与CDX2启动子+7 kb区域的结合降低。上述结果表明，FOXA2通过招募负性调控子Rfx1结合于CDX2启动子+7 kb区域，是干细胞对CDX2表达的一种负性调控机制，而LINC00261可抑制FOXA2的表达，由此参与了CDX2表达的调控。