环状RNA circ-0109315 靶向ZEB1调控胰腺癌细胞EMT的分子机制研究

Circular RNAs is a hotspot in the field of non-coding RNA research which has a close connection with the initiation, progression and prognosis of various malignancies. Circular RNAs act as a “microRNA sponges” — they bind with miRNA competitively, thus they can impact the targets of multiple microRNAs. However, the study of circular RNAs in pancreatic cancer is still in its infancy. Our preliminary study demonstrated that circular RNA circ-0109315 which was upregulated in pancreatic cancer tissues was correlated with tumor size, vascular invasion and lymphatic metastasis. In-vitro studies confirmed the loop characteristic of circ-0109315, and found that circ-0109315 activated epithelial-mesenchymal transition (EMT) and increased the expression of ZEB1 which is the key transcription factors of EMT. Moreover, bioinformatics software predicted that circ-0109315 harbors twenty-four miRNAs response elements (MREs) of miR-23 family which is the direct upstream of ZEB1. Hence, based on “miRNA sponge” theory, we hypothesized that circ-0109315 could up-regulate the expression of ZEB1 by competitively binding with miR-23 family and further promote EMT of pancreatic cancer cells. In this project, we intend to illustrate the functional roles of circ-0109315 in the tumorigenesis of pancreatic cancer, as well as the regulation mechanism of ZEB1 and EMT through clinical data and in vitro and vivo experiments. Altogether, it is hopeful to promote the development of circular RNA study in pancreatic cancer, and provide new ideas for the diagnosis and treatment of pancreatic cancer.

环状RNA是非编码RNA研究中的前沿热点，其与肿瘤发生发展密切相关。但是，胰腺癌中环状RNA的研究还处于起步阶段。我们发现环状RNA circ-0109315在胰腺癌组织中高表达，并且与临床病理参数相关。细胞实验明确了circ-0109315的环状特性，并证明其正向调控上皮间质转化（EMT）以及EMT关键转录因子ZEB1。生物信息学软件分析提示circ-0109315包含24个miR-23家族结合位点，而ZEB1是miR-23家族的下游靶点。依据“miRNA海绵”理论，我们推测circ-0109315通过竞争结合miR-23家族使ZEB1表达上调，进而促进EMT。本项目通过环状RNA芯片筛选出胰腺癌中高表达的circ-0109315，从临床数据、细胞实验和动物实验三方面探索circ-0109315的作用及其分子机制。本项目有助于推动胰腺癌中环状RNA的研究进展，为胰腺癌的诊治提供新的思路。

缺氧是胰腺癌化疗耐药的重要原因，但其确切机制尚未完全阐明。在本研究中，我们发现低氧诱导胰腺癌细胞产生的的外泌体中的环状RNA可加强胰腺癌细胞对化疗药物耐药。环状RNA芯片结果显示，与常氧下胰腺癌细胞产生的外泌体相比，低氧所诱导的外泌体中circZNF91显著增加。过表达circZNF91可明显刺激胰腺癌细胞的耐药性，而敲低circZNF91可削弱低氧诱导的外泌体所导致的胰腺癌细胞耐药。进一步研究其机制，我们发现低氧诱导的外泌体circZNF91传递到常氧下的胰腺癌细胞后，可以竞争性地结合miR-23b-3p，这剥夺了miR-23b-3p对去乙酰化酶Sirtuin1 (SIRT1)表达的抑制作用，进而上调的SIRT1通过去乙酰化作用增强了HIF-1α蛋白的稳定性，促进胰腺癌细胞的糖酵解和对吉西他滨的耐药性。此外，我们发现低氧外泌体中circZNF91的增加与HIF-1α的转录调控有关。HIF-1α通过结合circZNF91线性序列的上游序列而增加circZNF91的表达。在动物实验中，在裸鼠皮下种植瘤中注射低氧外泌体可以明显增加种植瘤的化疗耐药性，但是敲低circZNF91或者过表达miR-23b-3p可以逆转这种耐药性。此外，临床数据显示，circZNF91在胰腺癌组织中显著上调，并与糖酵解酶相关蛋白和总生存时间相关连。总的来说，外泌体中circZNF91可以作为一种信号分子介导低氧和常氧肿瘤细胞之间的信号传输，以促进胰腺癌细胞的化疗耐药，这种机制可以作为一种潜在的治疗靶点。