长非编码RNA CILA1促进舌鳞癌细胞干性与化疗耐药的机制研究

Long non-coding RNA (lncRNA) plays an important role in regulating chemoresistance of diverse cancer cells. Recent studies demonstrate that the main cause of chemoresistance is the existence of cancer stem cells (CSCs). However, the biological characters and molecular mechanisms of chemotherapy-induced CSCs in tongue squamous cell carcinoma (TSCC) remain largely unknown. Our previous study (Mol Ther., 2018) has identified a novel lncRNA termed chemotherapy-induced long noncoding RNA 1(CILA1), which is closely associated with the chemoresistance of TSCC. Furthermore, the pre-experiment results show that CILA1 regulates the cancer stem-like properties in TSCC and is expressed both in the cytoplasm and the nucleus. In addition, CILA1 could regulate the classical CSCs-related signaling (Wnt/β-catenin pathway) via miR-1-3p and miR-33a-5p in the cytoplasm, and inhibit the expression of CSCs-related factors (PTEN and AXIN2) in the nucleus through binding to EZH2. In the following project, we will elucidate the underlying mechanism of CILA1 upregulation through CHIP assays and detecting the histone modification and upstream transcription factors. And we will confirm the downstream mechanism of CILA1 regulating the cancer stem-like properties in TSCC through luciferase assays, RNA pulldown, RIP assays and biological function experiments. Our project will provide a novel target for inhibiting cancer stem-like properties and chemoresistance in TSCC.

长非编码RNA（lncRNA）在肿瘤化疗耐药过程中发挥重要作用，最新研究显示化疗诱导的肿瘤细胞干性是导致化疗耐药的主要因素，但lncRNA是否影响化疗诱导的舌鳞癌细胞干性并不清楚。本课题组前期研究（Mol Ther., 2018）已鉴定出lncRNA CILA1与舌鳞癌化疗耐药密切相关，预实验发现CILA1调控舌鳞癌细胞干性，CILA1在胞核胞浆均有表达，在胞浆内它通过miR-1-3p和miR-33a-5p影响干性相关的Wnt/β-catenin通路，在胞核内它结合EZH2影响干性因子PTEN和AXIN2表达。本项目将通过组蛋白修饰和上游转录调控因子检测、CHIP等阐明CILA1上调机制，并通过荧光素酶报告分析、RNA pull down、RIP和生物功能实验等明确CILA1调控舌鳞癌干性的下游机制，为抑制舌鳞癌细胞干性和化疗耐药提供新靶点。

化疗是舌鳞癌综合序列治疗的重要组成部分，化疗导致的肿瘤干细胞（CSC）富集、增殖分化，是肿瘤复发的重要因素。长非编码RNA（lncRNA）在调控肿瘤干细胞样特性中发挥重要作用，针对肿瘤干细胞的特殊lncRNA靶点开发特异性药物, 将为开发及筛选新药靶标带来诸多新的方向。申请人在前期筛选出全新的舌鳞癌化疗耐药特异性lncRNA CILA1，但其调控机制不明确。为了研究lncRNA CILA1对舌鳞癌干性和化疗耐药性的影响，课题组构建了敲减/过表达lncRNA CILA1的舌鳞癌细胞系稳转株，通过细胞球囊形成实验、流式细胞凋亡实验、TUNEL细胞实验，从细胞水平验证了lncRNA CILA1可以增强舌鳞癌细胞自我更新能力，化疗耐药性。我们通过PROMO及TCGA数据库筛选出调控CILA1的关键转录因子FOXM1，证实了FOXM1对CILA1的直接转录调控作用。课题组进一步检测了lncRNA CILA1在细胞中的定位以探索其影响舌鳞癌进展的内在机制，发现lncRNA CILA1主要定位于舌鳞癌细胞系的细胞质中，提示其可能发挥ceRNA的作用。课题组通过检索miRDB和AnnoLnc数据库，发现miR-425-5p与lncRNA CILA1及FGF2 mRNA的3'UTR片段存在结合位点，而FGF2的表达被发现有助于维持舌鳞癌细胞的干性。为此，我们构建了相应的野生型和突变型质粒，通过双荧光素酶报告基因实验验证了miR-425-5p可以分别与IncRNA CILA1和FGF2 nRNA的3’UTR结合，通过RT-q PCR实验验证了IncRNA CILA1的表达与miR-425-5p的表达呈负相关，而FGF2 mRNA的表达亦与miR-425-5p呈负相关，提示lncRNA CILA1可能通过miR-425-5p/FGF2轴促进舌鳞癌进展。我们在敲减了lncRNA CILA1的舌鳞癌细胞系中转染了miR-425-5p inhibitor发现lncRNA CILA1可以通过miR-425-5p/FGF2增强Wnt通路，从而促进舌鳞癌的进展。在临床样本中，我们发现舌鳞癌化疗耐药组织中CILA1、FOXM1及FGF2显著高表达，及三者直接具有正相关性。相关研究成果已经编撰成文并投稿。