uMtCK在胃癌细胞线粒体损伤及转移中的作用及机制研究

Previously, we discovered that ubiquitous mitochondrial creatine kinase (uMtCK) promoted the metastasis of gastric cancer (GC) cells and mitochondrial DNA (mtDNA) damage. Moreover, high uMtCK expression was significantly correlated with high p-Smad2/3 expression, which were the key transcriptional factors of TGF-β/Smads signaling pathway. uMtCK wide spreads in mitochondrion, working as the key enzyme in the process of oxidative phosphorylation. mtDNA is vulnerable to harmful element, regarded as an important factor in the carcinogenesis and development of cancers. Therefore, we suppose that, via activating the TGF-β/Smads signalling pathway, uMtCK may induce mtDNA damage resulted in GC cells metastasis. In this project, gene transfection, nude mice models et al will be used to verify the role of uMtCK on cell biological behaviors of GC in vitro and in vivo; CO-IP-MS/MS, GST-pull down and other related assays will be applied to identify the molecular mechanism of uMtCK activated TGF-β/Smads signaling pathway; we would further confirm the clinical significance of aberrant expression of uMtCK and key markers of TGF-β/Smads signalling in large sample of GC tissue, then established combinations of multi-genes predicting the metastasis of GC cells. This project would verify the molecular mechanism of uMtCK in metastasis of GC, and may be helpful to guide précising treatment and develop novel anti-metastasis drugs.

前期实验发现，广泛型线粒体肌酸激酶（uMtCK）能促进胃癌细胞转移和线粒体DNA（mtDNA）损伤，且uMtCK高表达与TGF-β/Smads信号通路中p-Smad2/3升高相关。uMtCK是细胞内氧化磷酸化过程中关键酶，广泛存在于线粒体中。mtDNA易受不良因素刺激而损伤，是肿瘤发生发展的重要因素。据此推测， uMtCK可能通过活化TGF-β/Smads信号通路，诱导胃癌细胞线粒体DNA损伤，促进肿瘤转移。本研究拟运用基因转染、裸鼠实验等方法，体内外检测uMtCK对胃癌细胞生物学行为的影响；运用蛋白质免疫共沉淀联合质谱分析、GST-pull down等技术阐明uMtCK活化TGF-β/Smads信号通路的分子机制；综合临床大样本验证确定其临床意义，并构建预测肿瘤转移的多基因组合模型。本项目将明确uMtCK促进胃癌转移的分子机制，有利于制定精准治疗策略，甚至指引抗肿瘤转移的酶抑制新药研发。

普遍存在型线粒体肌酸激酶(uMtCK)将线粒体产生的ATP中的高能磷酸盐转移到肌酸中，在能量需求波动较大的组织中生成磷酸肌酸。据报道uMtCK在一些恶性肿瘤中过表达，往往提示预后不良。但uMtCK在胃癌(GC)中的临床意义及影响尚未得到全面的研究。我们的初步结果显示，uMtCK在胃癌组织中的表达明显高于相邻的正常粘膜组织，且其表达的升高与胃癌患者的不良预后显著相关，这与两个独立公开胃癌数据集的结果相一致。有趣的是，独立于UICC(国际癌症控制联盟)分期和肿瘤复发的Kaplan-Meier生存分析提示，uMtCK阳性表达胃癌患者组的总生存期明显缩短。在单因素和多因素分析中，uMtCK可作为无病生存期和总生存期的独立预后因素。细胞体外功能实验中，胃癌细胞中过表达uMtCK可促进GC细胞增殖，诱导细胞周期转化，促进细胞迁移和侵袭，而敲除uMtCK可抑制胃癌的相关恶性进展。此外，我们还发现，uMtCK的高表达促进了肿瘤对5-氟尿嘧啶化疗的耐药性。在体内细胞功能实验中，过表达uMtCK可促进裸鼠皮下移植瘤的形成和肿瘤的转移。我们进一步验证了uMtCK通过激活转化生长因子-β信号通路，进而增加pSmad2和pSmad3的水平，从而增强了GC细胞的恶性程度。综上所述，uMtCK可以作为GC的一种新的独立预后生物标志物，特别是对于UICC晚期和肿瘤复发的GC患者。