Epithelial adherens junctions regulate colon cell behavior through RNAi and lncRNAs

项目总结 上皮完整性受损是胃肠道异常的标志，例如炎性肠炎。 疾病和结肠癌，这是第三种最流行和第二致命的癌症形式。高潮 这些疾病的发病率表明，我们仍然没有完全了解其潜在的机制。 最近，我们发现了一种将上皮组织完整性与RNA干扰(RNAi)联系起来的机制 机械和miRNA调节。我们已经证明了粘着连接，这是一个基本的 细胞的结构组件，招募微处理器和RNAi诱导的沉默复合体(RISC)， RNAi机制的核心组件，以及结肠上皮细胞中的一组特定的miRNAs。这 相互作用通过PLEKHA7发生，PLEKHA7是E-钙粘蛋白细胞-细胞黏附复合体的新伙伴。PLEKHA7 丢失会导致上皮完整性受损，一组miRNAs水平和功能下降，以及 非锚定生长增加，这是上皮转化的一个指标。我们的初步数据显示 PLEKHA7在结肠癌细胞系和肿瘤患者样本中的广泛错误定位或丢失。有趣的是， RNA片段实验和RNA测序揭示了PLEKHA7与长非编码的关联 RNA(LncRNAs)。LncRNAs可以通过多种方式与miRNAs和RNAi机制相互作用 其中一些人与肠道疾病有牵连。然而，我们对lncRNA调控和功能的了解 仍然是有限的。我们的初步数据显示，PLEKHA7缺失会导致其中一些基因表达的改变 LncRNAs，包括MIR17HG的上调，MIR17HG是一种已知的细胞转化促进剂。我们假设 黏附连接招募和调节RNAi机制和lncRNAs以维持结肠上皮 动态平衡。我们将在两个目标上检验我们的假设，这将决定：1)PLEKHA7是否抑制 MIR17HG通过RISC和粘连连接处的miRNAs表达；2)PLEKHA7维持正常结肠 通过miRNAs和MIR17HG检测上皮表型。这项研究意义重大，因为它提供了一个缺失的 上皮结构和细胞行为之间的机制联系和一种新的意外的局部调控 RNAi和IncRNAs。这项拟议的工作是创新的，因为它将两个以前不相关的领域--细胞-细胞--联系在一起 黏附和非编码RNA生物学。这项研究的影响是，它将促进我们对 肠道疾病的潜在机制原因，将为系统讯问奠定基础 新发现的粘着连接、RNAi机制和非编码RNA之间的联系。

PROJECT SUMMARY Compromised epithelial integrity is a hallmark of gastrointestinal abnormalities, such as inflammatory bowel disease and colon cancer, which is the third most prevalent and second most lethal form of cancer. The high incidence rates of these diseases suggest that we still don’t fully understand the underlying mechanisms. Recently, we discovered a mechanism that links epithelial tissue integrity with the RNA interference (RNAi) machinery and with miRNA regulation. We have shown that the adherens junctions, which is an essential architectural component of the cell, recruit the microprocessor and the RNAi-induced silencing complex (RISC), the core components of the RNAi machinery, as well as a specific set of miRNAs, in colon epithelial cells. This interaction occurs through PLEKHA7, a novel partner of the E-cadherin cell-cell adhesion complex. PLEKHA7 loss results in compromised epithelial integrity, decreased levels and function of a set of miRNAs and in increased anchorage-independent growth, an indicator of epithelial transformation. Our preliminary data show extensive mis-localization or loss of PLEKHA7 in colon cancer cell lines and tumor patient samples. Interestingly, an RNA-CLIP experiment followed by RNA sequencing revealed association of PLEKHA7 with long non-coding RNAs (lncRNAs). LncRNAs can interact with miRNAs and the RNAi machinery in multiple ways and a number of them has been implicated in intestinal diseases. However, our knowledge on lncRNA regulation and function is still limited. Our preliminary data show that PLEKHA7 loss results in altered expression of a number of these lncRNAs, including upregulation of MIR17HG, a known promoter of cellular transformation. We hypothesize that the adherens junctions recruit and regulate the RNAi machinery and lncRNAs to maintain colon epithelial homeostasis. We will test our hypothesis in two Aims that will determine whether: 1) PLEKHA7 suppresses MIR17HG levels through RISC and miRNAs at adherens junctions; 2) PLEKHA7 maintains the normal colon epithelial phenotype through miRNAs and MIR17HG. This study is significant, since it provides a missing mechanistic link between epithelial architecture and cell behavior and a new unexpected localized regulation of RNAi and lncRNAs. The proposed work is innovative, because it tethers two previously unrelated fields, cell-cell adhesion and non-coding RNA biology. The impact of the study is that it will advance our understanding of the underlying mechanistic causes of intestinal diseases and will lay the foundation for the systematic interrogation of the newly discovered connection between the adherens junctions, the RNAi machinery and non-coding RNAs.