Twist1竞争性内源RNAs调控网络促进肺腺癌恶性进展研究

The competitive endogenous RNAs network (ceRNET),which was composed of the mRNA of novel oncogene and its competitive endogenous RNAs (ceRNAs), has recently been shown to play significant roles in tumor progression. According to the published studies and our previous investigation, Twist1 was characterized as an essential oncogene which played crucial roles during lung adenocarcinoma progression. However, the function of the Twist1 competitive endogenous RNAs network (Twist1-ceRNET) in lung adenocarcinoma remains largely unknown. An integrated Twist1-ceRNAs regulation network was firstly predicted by bioinformatic infomation, and then the top 20 ceRNAs were validated by TCGA database and real-time RT-PCR of clinical lung adenocarcinoma tissues. The mRNAs of SLC12A5, MSRB3, AKAP2 and ZFHX4 were considered as candidate ceRNAs of Twist1 among the predicted top 20 ceRNAs . SLC12A5 provided a perfect introduction of our further preliminary study as recent published papers concluded that SLC12A5 was a promising oncogene. Further investigation revealed that ectopic expression of 3’UTR of SLC12A5 mRNA resulted in eleveted expression of Twist1, as well as remarkable proliferation and invasion capacities of A549 cell line, which were consistent with malignant phenotypes caused by overexpression of Twist1 in A549 cell line. The mRNA of SLC12A5 therefore was supposed as the functional ceRNA of Twist1. miR-32-5p and miR-92b-3p were speculated to be mechanically involved in the cross-regulation of Twist1 and SLC12A5 by our preliminary investigation. Collectively, these findings promoted us to hypothesize that the Twist1-ceRNET might play collaborative roles in promoting lung adenocarcinoma progression. In this proposal we firstly design to screen out functional ceRNAs of Twist1 and verify the composition of the Twist1-ceRNET according to the luciferase reporter assays and the expression of target genes by using silencing and overexpressing strategy. We next plan to explore the functional roles of Twist1-ceRNET during the development and progression of lung adenocarcinoma both in vitro and in vivo. Finally we will investigate the mechanisms in the Twist1-ceRNAs cross-regulating network, and demonstrate the key microRNAs involved in their cross-talk by using MS2-RNA immunoprecipitation (RIP) and TALEN-mediated precise genome modification strategy. This study might provide a new idea for lung adenocarcinoma treatment and monitoring in the future.

癌基因竞争性内源RNAs（ceRNAs）调控网络对肿瘤进展具有重要作用。Twist1是促进肺腺癌恶性进展关键癌基因。肺腺癌中Twist1竞争性内源RNAs调控网络（Twist1-ceRNET）存在与否、功能如何尚属未知。前期经预测和验证，确定SLC12A5等四个癌基因转录本为Twist1候选ceRNAs；体外干预及功能实验提示SLC12A5转录本为Twist1功能性ceRNA，其3’UTR促进肺腺癌恶性进展；miR-32-5p等可能参与其交互调节。因此提出“Twist1-ceRNET促进肺腺癌恶性进展”科学假说。本研究拟利用过表达/沉默策略筛选Twist1功能性ceRNAs，明确Twist1-ceRNET构成；应用体外、体内实验研究Twist1-ceRNET生物学功能；应用MS2-RIP和TALEN基因敲除技术阐明Twist1-ceRNET交互调节分子机制。本研究可为肺腺癌防治提供新思路。

目的 在肺腺癌中构建mRNA-mRNA相关ceRNA网络，寻找在肺腺癌中发挥重要作用的潜在ceRNA组合并研究其相互调节的分子机制。方法 通过Perl语言对miRBASE、Targetscan以及starbase这三个靶基因预测网站进行全面数据搜索，利用The Cancer Genome Atlas (TCGA)数据库RNAseq数据，对所有潜在mRNA-mRNA以及mRNA-miRNA的表达相关性进行全面筛选并挑选潜在ceRNA组合。通过RNA沉默、过表达技术分别上调或敲降TWIST1/SLC12A5/ZFHX4以及miR-194-3p、miR-514a-3p的表达，观察潜在靶基因RNA及蛋白层面的表达变化。用RNA免疫共沉淀（RNA immunoprecipitation, RIP）、生物素标记的RNA pull down （biotin-pull down）、双荧光素酶报告基因等技术探索miR-194-3p以及miR-514a-3p对TWIST1/SLC12A5/ZFHX4进行调控的分子机制。结果 通过建立起的ceRNA调控网络，我们挑选出了以TWIST1为核心，包含SLC12A5以及ZFHX4的ceRNA组合。A549及H1299这两株代表性肺腺癌细胞中，在mRNA及蛋白层面，敲降SLC12A5/ZFHX4后TWIST1明显下调，过表达SLC12A5/ZFHX43’UTR区域后TWIST1明显上调，且这种调节作用可以分别被miR-194-3p及miR-514a-3p的同向表达变化所稀释。在A549及H1299细胞中过表达或敲降miR-194-3p及miR-514a-3p，发现miR-194-3p可负向调节SLC12A5/TWIST1的表达，而miR-514a-3p可负向调节ZFHX4/TWIST1的表达。在Ago2 RIP中，TWIST1/SLC12A5/ZFHX4以及miR-194-3p/miR-514a-3p都与AGO2蛋白充分结合。在biotin-pulldown中，miR-194-3p能富集到较多的TWIST1/SLC12A5，miR-514a-3p能富集到较多的TWIST1/ZFHX4。结论 本研究建立的肺腺癌mRNA-mRNA ceRNA网络能够卓有成效的寻找潜在ceRNA组合。SLC12A5和ZFHX4能够在肺腺癌细胞中正向调节TWIST1的表