CircRNA-6957作为ceRNA调控MondoA在肺腺癌糖代谢重编程中的作用及机制研究

Glucose metabolic reprogramming plays important roles in the biological processes of lung adenocarcinoma cells, such as proliferation, apoptosis, migration and invasion. Recently, we found that the expression of circRNA-6957 was significantly down-regulated in human lung adenocarcinoma tissues, overexpression of circRNA-6957 in adenocarcinoma cells can significantly inhibit its’ glucose uptake and lactate secretion rate. MondoA plays a vital role in the process of glucose metabolic reprogramming in many types of cancer cells, and the results of the pre-experiment showed that the expression of MondoA was significantly down-regulated in human adenocarcinoma tissues.Bioinformatics analysis revealed that miRNA-93-5p can regulate the expression of MondoA, while there are multiple binding sites of miRNA-93-5p on circRNA-6957, the results of the following experiments showed that over-expression of circRNA-6957 could lead to the down regulation of miRNA-93-5p, and the expression of MondoA was up-regulated. The above evidences strongly suggest that circRNA-6957 is likely to regulate the expression of miRNA-93-5p and further affects the expression of MondoA, which will affect the glucose reprogramming process as well as the progression of lung adenocarcinoma. This study hopes to elucidate the progression mechanism of adenocarcinoma from the point of view that the regulation of glucose metabolic reprogramming of circRNA, to develop a theoretical basis for the prevention and treatment of lung adenocarcinoma.

糖代谢重编程在肺腺癌细胞的增殖、凋亡、迁移及侵袭等生物学进程中起重要作用。近期我们发现circRNA-6957在肺腺癌组织中显著下调，过表达circRNA-6957可显著抑制肺腺癌细胞葡萄糖摄取及乳酸分泌速率。MondoA在肿瘤细胞糖代谢重编程中发挥着重要的作用，预实验表明其在人肺腺癌组织中显著下调。生物信息学分析表明miRNA-93-5p可调控MondoA的表达，而circRNA-6957上存在多个miRNA-93-5p结合位点，后续细胞水平研究表明过表达circRNA-6957的同时伴随miRNA-93-5p下调，MondoA上调，以上证据提示：circRNA-6957可能是通过调控miRNA-93-5p影响下游MondoA表达参与肺腺癌糖代谢重编程及其进展过程。本研究希望能从circRNA对糖代谢重编程调控的角度阐明肺腺癌进展的相关机制，为开发防治肺腺癌的的方法或药物奠定理论基础。

背景：肺癌是肿瘤中发病率以及死亡率最高的疾病，对于肺癌的发生、发展的机制仍有待于进一步研究。环状RNA和m6A甲基化修饰广泛参与非小细胞肺癌的发生与发展过程，且环状RNA和m6A阅读蛋白主要位于细胞质中。所以本文拟探讨在非小细胞肺癌中circRNA能否和m6A阅读蛋白相互作用从而对下游甲基化RNA产生影响。.方法及结果：首先通过收集三对非小细胞肺癌(NSCLC)样本并进行环状RNA测序，筛选到hsa_circ_0001523(circZNF608)，通过qPCR验证，circZNF608在NSCLC及多种细胞系中显著低表达，同时与肿瘤的T分期显著相关。在体外实验中，过表达circZNF608显著抑制A549及H1299细胞增殖、迁移及侵袭能力，敲减circZNF608，显著增强细胞增殖、迁移及侵袭能力。通过RNA pull-down及RIP，我们发现circZNF608能够与IGF2BP3结合。且FISH及免疫荧光结果也提示circZNF608、IGF2BP3共定位于细胞质中。通过敲减及过表达IGF2BP3，我们发现IGF2BP3在NSCLC扮演着重要的促癌作用。通过拯救实验，我们发现过表达circZNF608能够抑制igf2bp3过表达细胞的增殖、迁移及侵袭能力，而敲减circZNF608能够显著增加igf2bp3敲减组的增殖、迁移及侵袭能力。通过蛋白截短实验我们发现circZNF608主要与IGF2BP3的KH3-4结构域相结合。MYC是IGF2BP3下游的重要靶基因，与KH3-4结构域结合。我们通过WB验证发现，过表达及敲减circZNF608，细胞中MYC的表达量分别下降或上升，过表达igf2bp3时细胞中myc表达量上升，敲减igf2bp3时显著下降。通过拯救实验，我们发现过表达circZNF608能够显著抑制igf2bp3过表达所导致的myc含量的上升，而敲减circZNF608能够显著增加igf2bp3敲减组细胞内myc的表达。.结论：本研究发现外显子环状circZNF608是一种新的肿瘤抑制因子，通过内源性阻断IGF2BP3对MYC的识别，从而抑制肿瘤进展，提示circZNF608可能成为NSCLC患者潜在的治疗靶点