NDUFS1通过线粒体ROS/HIF-1α/FBLN5途径调控胃癌进展的研究

Gastric cancer is a common malignant tumor with high morbidity and mortality. It is urgent to clarify the mechanisms and find the key targets of its carcinogenesis and development, thus obtaining new breakthroughs in gastric cancer prevention and treatment. We previously found that the expression of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) was decreased, whereas the expression of fibulin-5 (FBLN5) was increased in gastric cancer clinical tissue specimens by isobaric tags for relative and absolute quantification (iTRAQ) analysis. However, the role and correlation of these two proteins in gastric cancer and its underlying mechanism is poorly unclear. Based upon the clue from previous reports on NDUFS1 and FBLN5, we performed preliminary studies and further demonstrated that NDUFS1 deficiency promoted the growth and metastasis in gastric cancer. NDUFS1 deficiency also increased mitochondrial reactive oxygen species (ROS), activated hypoxia-inducible factor-1α (HIF-1α) and upregulated HIF-1α downstream target FBLN5 in gastric cancer cells. Thus, we hypothesize that NDUFS1 deficiency would be involved in gastric cancer progression very possibly via activation of mitochondrial ROS/HIF-1α/FBLN5 pathway. For validation of the above hypothesis, the project will focus on regulatory effects of NDUFS1 in gastric cancer progression by establishing lentivirus-mediated NDUFS1-overexpressed or NDUFS1-knocked down gastric cancer cell line and then elucidating critical roles of NDUFS1/mitochondrial ROS/HIF-1α/FBLN5 pathway in gastric cancer at different levels including cell model, animal model and clinical samples, in order to explore and establish NDUFS1 as a novel target for gastric cancer treatment.

胃癌是一种发病率和死亡率高的消化道恶性肿瘤，亟需阐明其发生发展机制、寻找关键调控靶点，以期获得防治突破。本课题组利用iTRAQ技术研究发现，胃癌组织中NDUFS1表达下降、FBLN5表达升高。然而两者在胃癌中的作用、相互关系及其机制仍知之甚少。基于以往NDUFS1、FBLN5报道的提示，我们进一步预实验发现，NDUFS1缺失能促进胃癌生长、转移；并能促使胃癌细胞线粒体ROS升高，活化HIF-1α及上调靶基因FBLN5。因此我们推测：NDUFS1缺失可能通过激活线粒体ROS/HIF-1α/FBLN5途径参与胃癌的进展。为此，本项目拟以NDUFS1调控胃癌进展为研究主线，建立慢病毒介导的NDUFS1过表达、沉默胃癌细胞株，在细胞模型、动物模型及临床组织标本水平明晰NDUFS1/线粒体ROS/HIF-1α/FBLN5途径在胃癌中扮演的关键角色，探索和建立以NDUFS1为靶点治疗胃癌的新手段。

胃癌是一种发病率和死亡率较高的消化道恶性肿瘤，亟需阐明其发生发展机制、寻找关键调控靶点，以期获得防治突破。我们前期通过iTRAQ技术研究发现，胃癌组织中NDUFS1表达下降、FBLN5表达升高，而这两个分子在胃癌中尚未见报道，因此我们拟研究：NDUFS1在胃癌组织和细胞中的表达，以及NDUFS1的表达水平和胃癌病人预后的关系；NDUFS1蛋白的亚细胞定位；NDUFS1的表达水平对胃癌细胞在体内外的增殖、迁移、侵袭和转移的影响；NDUFS1表达下调对线粒体ROS生成及其下游信号分子表达的影响；NDUFS1表达下调促进胃癌进展的分子机制；FBLN5的表达对下调的NDUFS1促进胃癌进展的影响。针对上述研究内容，我们验证得出胃癌组织、胃癌细胞系中NDUFS1的蛋白表达均下调。同时，我们通过生信分析得出：相对于正常胃黏膜上皮组织，NDUFS1的mRNA水平在胃癌组织中明显减少，并且NDUFS1的mRNA表达与胃癌病人的预后呈正相关。利用免疫荧光定位到NDUFS1主要存在于细胞的线粒体，并且通过线粒体分离试剂盒提取得到线粒体组分，Western bolt实验结果也进一步证实了上述结果。通过转染慢病毒构建稳定细胞株，利用CCK8、平板克隆形成、Transwell等细胞功能学实验证明NDUFS1表达下调可以在体外促进胃癌细胞的增殖、迁移和侵袭的能力，利用裸鼠皮下移植瘤和尾静脉注射肺转移模型证明NDUFS1表达下调可以促进胃癌细胞在动物体内的生长和转移。下调NDUFS1可以升高线粒体内ROS，并进一步激活HIF-1α信号通路。同时发现FBLN5与NDUFS1的表达呈负相关，并且FBLN5作为HIF-1α转录激活信号通路的下游分子可以被下调的NDUFS1诱导升高。通过回复实验发现FBLN5的敲减可以逆转下调NDUFS1引起的胃癌进展，结果证明下调的NDUFS1最终通过FBLN5的表达升高促进胃癌细胞的恶性进展。以上结果表明NDUFS1通过ROS-HIF1α-FBLN5信号通路调控胃癌进展，为深入阐明胃癌进展的分子机制提供科学依据，也为胃癌的靶向治疗提供新的视角和方案。