环状RNAcirc_0071036在胰腺癌发生发展中的作用及其机制研究

Pancreatic ductal adenocarcinoma (PDAC), generally regarded as pancreatic cancer, is a highly aggressive and one of the most lethal malignancies with a 5-year overall survival rate of less than 5%. Circular RNAs (circRNAs), comprise a novel class of widespread non-coding RNAs that may regulate gene expression, are broadly expressed in eukaryotic cells. However, the characterization and function of circRNAs in tumorigenesis and metabolism of PDAC remains obscure. Here we identified at least 13617 distinct circRNAs candidates and a series of circRNAs that are differentially expressed in PDAC tissues compared with matched normal tissues. We further characterized one circRNAs derived from the INPP4B gene and termed it as circ_0071036. The circ_0071036 was remarkably upregulated in PDAC serve as diagnostic biomarker and fluorescence in situ hybridization showed that overexpressed circ_0071036 was mainly located in cytoplasm. In vitro, gene silence of circ_0071036 suppressed migration and proliferation of PANC-1 cells. In this study, we plan 1) To analyze whether or not in vitro silencing endogenous circ_0071036 or specific overexpression of circ_0071036 can significantly affect cell functions of PDAC, such as proliferation, invasion, metastasis, apoptosis and cell cycle; 2) Via luciferase screening assay, to determine whether or not circ_0071036 can sponge to miR-489 with potential binding sites and directly inhibits miR-489 activity, and to screen and validate their potential downstream targets; 3) In vivo, to clarify the relationship between expressions of circ_0071036 and malignant clinical features and survival outcomes of PDAC patients and mice. In summary, the regulatory role of circ_0071036 in tumorigenesis and progression and their clinical implications in malignant behaviors and outcomes are expectable from this project, which will definitely explode our understading of complex mechanisms of PDAC and contribute to the clinical screening of targeted therapy.

环状RNA(circRNAs)是一种以闭合环状结构为特征的内源性非编码RNA，作为竞争性内源RNA(ceRNA)靶向调控miRNA影响基因表达。大部分circRNAs在胰腺癌中的具体作用尚不清楚。前期研究发现circ_0071036在胰腺癌组织中呈高表达；RNA-FISH表明circ_0071036定位于肿瘤细胞的细胞质；高表达circ_0071036与胰腺癌局部淋巴结转移等恶性临床病理学特征存在相关性；沉默circ_0071036能抑制胰腺癌细胞的增殖和迁徙。本研究将进一步揭示circ_0071036的生物学特征，从体外层面通过沉默或上调circ_0071036的表达进行细胞功能学实验，明确其在胰腺癌增殖、侵袭、凋亡和细胞周期中的作用；探索circ_0071036是否能作为miR-489的“海绵”靶向调控miR-489并影响下游靶基因，从体内层面验证其在胰腺癌发生发展及侵袭转移中的作用。

环状RNA(circRNAs)是一种以闭合环状结构为特征的内源性非编码RNA，主要通过竞争性内源RNA靶向调控miRNA影响肿瘤的发生发展。胰腺癌恶性程度高，预后极差，是真正的癌中之王。大部分circRNAs在胰腺癌中的具体作用尚不清楚。申请人首先建立了胰腺癌circRNA芯片，通过显著性分析方法进行筛选，明确hsa_circ_0071036在胰腺癌中表达显著上调。采用荧光定量PCR和RNA荧光原位杂交的方法证实hsa_circ_0071036在胰腺癌组织和细胞中表达显著上调，并且可以作为诊断胰腺癌的标志物，其敏感度与特异性均较好；此外，hsa_circ_0071036的表达水平与胰腺癌患者的恶性生物学行为和生存预后密切相关。同时，胰腺癌组织中hsa_circ_0071036的表达水平与miR-489呈显著负相关。体外细胞实验证实沉默hsa_circ_0071036能够促进胰腺癌细胞的凋亡，抑制胰腺癌细胞的迁徙、克隆形成和增殖。通过体内裸鼠成瘤实验进一步表明敲减hsa_circ_0071036可以抑制胰腺癌细胞的增殖和皮下成瘤能力。在机制研究中，通过Luciferase荧光素酶结合实验和RNA pull-down实验表明hsa_circ_0071036可直接与胰腺癌细胞中的miR-489相结合，hsa_circ_0071036可以作为miR-489的“海绵”靶向调控miR-489并影响下游基因。本项目紧密结合临床，明确了hsa_circ_0071036与miR-489构成的ceRNA网络在胰腺癌发生发展及侵袭转移中的作用，为胰腺癌的靶向治疗提供新思路和理论依据。