外泌体传递的circ-0040809参与调控肺腺癌侵袭转移的功能及机制研究

Circ-0040809 was identified as a novel circular RNA (circRNA) which was highly expressed and functionally unknown in lung adenocarcinoma tissues using microarray technology. And this circRNA was enriched in lung adenocarcinoma cell lines and their exosomes through quantitative real-time PCR. Our previous studies have shown that exosomes could deliver circ-0040809 to the recipient cells, functionally promoting the invasion and metastasis of lung adenocarcinoma. Based on database and dual luciferase reporter assay, we found that circ-0040809 could act as a competitive endogenous RNA (ceRNA) via absorbing the potential tumor suppressor gene miR-138-5p. Therefore we proposed the scientific hypothesis that circ-0040809 delivered by exosomes could promote the invasion and metastasis of lung adenocarcinoma through ceRNA mechanism by absorbing miR-138-5p. We plan to validate that circ-0040809 delivered by exosomes could regulate the invasion and metastasis of lung adenocarcinoma with overexpression/silencing strategy both in vitro and in vivo experiments. Further, to screen and verify the downstream target of circ-0040809, we carry out microarray analysis, bioinformatic software analysis and multiple experiments. Cross-rescue experiments were designed to elucidate the ceRNA moluclular mechanism by which circ-0040809 regulates the invasion and metastasis of lung adenocarcinoma. The role of circRNA delivered by exosomes in the regulation of invasion and metastasis of lung adenocarcinoma has been rarely reported. Therefore our study may provide a new target for the treatment of lung adenocarcinoma.

circ-0040809是应用表达谱芯片从肺腺癌组织获得的一条差异高表达的circRNA，并且在肺腺癌细胞株及其外泌体中获得了验证。前期研究证明外泌体可传递circ-0040809至受体细胞，功能上促进肺腺癌的侵袭转移。通过数据库、双荧光素酶报告基因初步确定circ-0040809能吸附潜在的抑癌基因miR-138-5p发挥竞争性内源RNA（ceRNA）机制。故提出“外泌体传递的circ-0040809可吸附miR-138-5p通过ceRNA机制促进肺腺癌的侵袭转移”的科学假说。本课题拟体内外系统论证外泌体传递的circ-0040809调控肺腺癌侵袭转移的功能。采用表达谱芯片联合生物信息学筛选并多实验验证下游靶基因，设计交叉拯救实验，阐明circ-0040809调控肺腺癌侵袭转移的ceRNA分子机制。外泌体传递的环状RNA调控肺腺癌侵袭转移的功能机制鲜有报道，本课题可为其治疗提供新靶标。

背景：肺腺癌是肺癌的一种常见亚型，其复发率和死亡率都很高。Circ_0001361 已被公认为在多种恶性肿瘤中发挥调控作用，但其在肺腺癌中的作用仍然不明确。方法：通过RT-qPCR评估Circ_0001361、miR-525-5p 和 VMA21的表达水平。 肺腺癌细胞的生长和转移分别通过 MTT、克隆形成、划痕和 Transwell 法分别检测。circ_0001361/VMA21 和 miR-525-5p 之间的交互作用通过双荧光素酶、RNA免疫沉淀和 RNA pull-down 试验检测。通过Western blotting检测VMA21的蛋白水平。建立裸鼠异种移植模型以确定circ_0001361在体内肿瘤生长中的作用。结果：在肺腺癌组织和细胞中，Circ_0001361上调，而miR-525-5p下调。功能实验表明，circ_0001361 促进了肺腺癌细胞的生长和转移。从机制上讲，circ_0001361充当 miR-525-5p的海绵来上调下游靶基因VMA21 的表达水平。 MiR-525-5p/VMA21轴参与 circ_0001361 介导的肺腺癌细胞恶性表型。体内实验表明抑制circ_0001361的表达可通过调节 miR-525-5p/VMA21 轴来抑制小鼠异种移植肿瘤的生长。结论：我们的研究结果阐明 circ_0001361通过 miR-525-5p/VMA21 轴促进肺腺癌的肿瘤发生和发展，为 circ_0001361作为肺腺癌临床治疗的潜在预后生物标志物和治疗靶点提供了证据。