ACSL6对肺癌细胞放射敏感性的调控作用及机制研究

Recently, the energetic metabolism remodeling of cancer cells induced by IR has attracted more and more attention all over the world, among which lipid metabolism alterations are most closely related to malignant transformation of cancer cells. Long-chain acyl-coenzyme A synthetase (ACSLs), the key enzyme in lipid metabolism, are reported to be unregulated among a great spectrum of clinical tumors and significantly correlated with poor prognosis in some patients. In our previous study, the ACSL6 was specifically up-regulated in the Lewis lung cancer cells after IR and its radiosensitivity was greatly increased after knockout the gene by CRISPR CAS9, suggesting the potential vital role of ACSL6 in radiosensitization of lung cancer and the possibility to become a new breakthrough to improve the radiotherapeutic effect of lung carcinoma. The regulatory role of ACSL6 in the radiosensitivity of human lung cancer cells will be studied at multiple levels in this project. Taking BEAS-2B, A549, NCL-H1299 as the research object, we plan to conduct the mechanism exploration in metabolomics, lipid metabolism analysis, aerobic glycolysis, mitochondrial function, DNA damage response, ROS, autophagy and senescence. Then, further studies such as protein interaction and functional analysis of molecular domain will be performed for focused molecules to investigate the exact mechanism. Finally, this study will try to elucidate the role and potential value of ACSL6 in regulating radiosensitivity of lung cancer cells and reveal the underlying mechanism, providing new ideas and strategies for the development of lung cancer radiosensitizers.

近来，IR引发的癌细胞能量代谢重塑受到了逐渐关注，而脂代谢变化与癌细胞恶性转化最为相关。已知长链酯酰辅酶A合成酶（ACSLs）是脂代谢关键酶，而ACSLs失调广泛存在于临床肿瘤组织且与患者预后显著相关。预实验结果显示，受照肺癌细胞特异性上调ACSL6，以CRISPR CAS9敲除该基因后其放射敏感性显著增高，有力提示了ACSL6在肺癌放射增敏方面的重要作用，极有可能成为提高肺癌放疗效果的新突破点。本项目将在多层次研究ACSL6对人肺癌细胞放射敏感性的调控作用；以BEAS-2B、A549、NCL-H1299等为研究对象，在代谢组学、脂代谢、有氧糖酵解、线粒体功能、DNA损伤应答、ROS、自噬、衰老等方面探索其作用机制；并针对关键分子开展蛋白互作、分子结构域功能分析等深入研究其作用机理。通过阐明ACSL6调控肺癌细胞放射敏感性的作用、机理及其潜在价值，为开发肺癌放疗增敏剂提供新思路、新策略。

研究背景：肺癌严重威胁人类健康，探寻肺癌放疗抵抗分子机制，寻找肺癌放射增敏新策略，对于提高肺癌放射敏感性（LCRS）具有重要价值。近年来，电离辐射（IR）改变肿瘤细胞能量代谢引起了国际上的广泛关注。其中，脂质代谢与肿瘤发生发展关系最为密切。我们的前期研究发现，长链脂酰辅酶 A 合成酶6（ACSL6）具有调控LCRS的潜在价值。本课题即系统研究LCRS的调控效应及机制，为增强LCRS提供新思路。.主要内容：利用细胞系，荷瘤和基因工程小鼠及临床样本，从体内外多个层面证实了ACSL6的LCRS调控作用和规律。经由转录组、代谢组、生物信息学分析，甄别ACSL6对LCRS的调控主要依赖于何种方式，然后以分子生物学手段对其中的关键分子机理作了进一步锁定，深入和阐明。.重要结果及关键数据：①通过R语言等对TCGA数据库等进行挖掘，发现ACSL6在肺癌中高表达，且该现象进一步被临床相关性分析等验证。②成功构建了不同ACSL6表达水平的H460、BEAS 2B细胞系。③通过克隆形成、细胞活力、凋亡、DNA损伤修复、ROS、荷瘤增长、组织病理学及小动物肺功能等多方面检测，发现ACSL6 敲除后显著提高了LCRS。④通过转录组、代谢组及其RT-PCR和WB等验证，我们提出了以下机制假设：ACSL6 敲除后上调FLI1抑制COLs，从而引起H460 KO细胞株IR反应性的自噬水平下降，最终提高了LCRS。⑤进一步通过构建FLI1和COLs不同表达水平的细胞株并进行WB、IP等检测发现，0 Gy处理的si-COL2A1和si-COL4A3的LC3-II/LC3-I水平下降；但是在0 Gy处理的si-FLI1的LC3-II/LC3-I水平并未出现明显变化，反而在8 Gy处理后si-FLI1自噬水平上升，且siCOLs和si-FLI1自噬水平变化与前述ACSL6 敲除后FLI1显著上调而COLs显著抑制，从而引起H460 KO细胞株IR诱发的自噬水平下降这一趋势完全相符，从而印证了上述机制假设。.科学意义：本课题原始发现了ACSL6对于调控LCRS的重要作用，且阐明了该作用所依赖的ACSL6-FLI1-COLs-Autophagy分子机制，为进一步增强LCRS提供了诸多新靶点、新策略，对于提高肺癌疗效具有重要价值。其应用前景乐观、学术借鉴意义重要，有望成为提高LCRS的新增长极、新突破点。