UCA1/KRAS/YAP1环路促进胰腺癌细胞上皮间质转化的作用及其机制

It is the challenge of clinical treatment for pancreatic cancer due to its invasiveness and metastasis. Recent evidence indicates that KRAS/YAP1 is a key factor involved in epithelial-mesenchymal transition (EMT), invasiveness and metastasis in pancreatic cancer. Furthermore, YAP1/TAZ upregulates long non-coding RNA (lncRNA) UCA1. However, the underlying mechanism remains to be determined yet. Our preliminary studies combined with bioinformatics analysis suggest that UCA1 plays a critical role in maintaining the high level as well as the high activity of KRAS and YAP1 possibly through 1) interaction with the key factors in the Hippo pathway, promoting nuclear translocation of YAP1, and interaction with hnRNPA2B1, enhancing the KRAS activity; 2) interaction with microRNAs targeting KRAS and YAP1 by ceRNA mechanism, leading to a high level of KRAS and YAP1. Finally, KRAS and YAP1 in turn upregulates UCA1 by a positive feedback mechanism, thus, forming a vicious loop. Therefore, we propose to perform a series of experiments to dissect how the UCA1/KRAS/YAP1 axis promotes EMT and metastasis in pancreatic cancer. The success of this study will help to develop novel molecular targeted therapy for pancreatic cancer.

胰腺癌的侵袭和转移是临床治疗的难题。研究发现KRAS/YAP1通路驱动了胰腺癌细胞的上皮间质转化（EMT）和转移，且YAP1/TAZ可上调长链非编码RNA UCA1的表达。然而，UCA1与KRAS、YAP1间相互作用机制尚需进一步阐明。基于我们预研结果和生物信息学分析，推测UCA1维持了KRAS和YAP1持续激活和高表达:（1）通过结合Hippo通路关键分子形成“屏蔽复合物”而促进YAP1的核转位；UCA1通过结合hnRNPA2B1增强KRAS的活性；（2）UCA1可结合靶向KRAS和YAP1的microRNAs而维持KRAS和YAP1的高表达；（3）持续激活、高水平的KRAS和YAP1又促进UCA1表达，从而形成正反馈环路促进胰腺癌EMT和转移。本项目拟通过体内、外实验验证上述假说，旨在揭示UCA1/KRAS/YAP1途径调控的分子机制及其在EMT和转移中的作用。

胰腺癌是预后极差的消化道恶性肿瘤之一。最近研究发现 KRAS/YAP1 通路驱动了胰腺癌细胞的上皮间质转化（Epithelial-mesenchymal transition, EMT）和转移，且YAP1/TAZ可上调长链非编码RNA UCA1的表达。然而，UCA1与KRAS、YAP1间相互作用机制尚需进一步阐明。本研究在理论上将阐明 KRAS/UCA1/YAP1 途径发挥作用的分子机制，及其在促进胰腺癌细胞恶性行为中的调控作用，为进一步胰腺癌的靶向治疗提供理论依据。主要研究内容包括：①探究UCA1在胰腺癌细胞恶性行为中的调控作用；②分析 UCA1 调控 YAP1 磷酸化水平的作用；③ 分析 UCA1 调控 KRAS 磷酸化的作用；④分析 UCA1 竞争性结合靶向 YAP1 和 KRAS microRNAs 的作用；⑤体内致瘤试验验证上述的研究结果。.主要结果如下：（1）通过CCK8（Cell Counting Kit-8）法、 平板细胞克隆形成实验、划痕实验、 Transwell迁移实验和成球实验分别表明UCA1能够促进胰腺癌细胞增殖能力、 克隆形成能力、迁移能力和干细胞成球能力；（2）应用荧光定量 PCR、 Western blotting、免疫荧光及核浆分离实验等探究UCA1 调控 YAP1 磷酸化水平的作用及机制；（3）应用蛋白免疫沉淀法、RNA结合蛋白免疫沉淀技术、phos-tag system 实验以及点突变实验等探究UCA1 调控 KRAS 磷酸化的作用机制；（4）应用双荧光素酶报告基因、点突变技术及RNA结合蛋白免疫沉淀技术分析 UCA1 竞争性结合靶向 YAP1 和 KRAS microRNAs 的作用；（5）用 Western blotting、基因转染等手段分析 UCA1/KRAS/YAP1 作用环路中上下游分子的变化，探明 UCA1 调控胰腺癌上皮间质转化的作用机制。