背景:胃癌(GC)是世界范围内最恶性的癌症之一。代谢紊乱是与肿瘤进展和转移相关的恶性肿瘤的关键特征。然而,苹果酸酶3(ME3)在GC中的表达及其分子机制的报道很少。在这项研究中,我们旨在研究ME3在GC发展中的分子机制,并探讨其作为GC预后和治疗靶点的潜在价值。
方法:使用RT-qPCR评估GC患者的ME3mRNA和蛋白表达。WB,和免疫组织化学,以及它们与临床病理指标的相关性。使用细胞计数试剂盒-8(CCK-8)评估ME3对增殖和转移的影响,5-乙炔基-20-脱氧尿苷(EdU)测定,transwell分析,伤口愈合试验,和皮下注射或尾静脉注射小鼠模型中的肿瘤细胞。测定ME3敲低对GC细胞代谢产物和缺氧诱导因子-1α(HIF-1α)蛋白水平的影响。测量氧化磷酸化以评估三磷酸腺苷(ATP)的产生。
结果:ME3在人GC组织中表达下调(P<0.001)。ME3mRNA表达下降与年龄增长有关(P=0.02),病理分期(P=0.049),淋巴结转移(P=0.001),ME3低表达与肿瘤大小相关(P=0.048),肿瘤浸润深度(P<0.001),淋巴结转移(P=0.018),TNM分期(P<0.001),预后不良(OS,P=0.0206;PFSP=0.0453)。ME3敲低促进GC细胞恶性表型。此外,在常氧下,ME3敲低组的α-酮戊二酸(α-KG)和NADPH/NADP比率降低,而苹果酸增加。当细胞在缺氧条件下培养时,NADPH/NADP比值和α-KG降低,而细胞内活性氧(ROS)明显增加。ME3敲低组显示ATP产生增加,而ME3过表达组显示相反。我们发现ME3和HIF-1α在GC细胞和组织中的表达呈负相关。并提出了以下假设:ME3的下调通过调节细胞内氧化应激和HIF-1α促进GC的进展。
结论:我们提供了ME3下调与GC患者预后不良相关的证据,并提出了关于GC进展中ME3调节机制的假设。本研究对探索GC的预后和治疗靶点具有重要的科学意义和临床价值。评估和提高患者的临床疗效,减少复发和转移,改善患者的预后和生活质量。
BACKGROUND: Gastric cancer (GC) is one of the most malignant cancers worldwide. Metabolism disorder is a critical characteristic of malignant tumors related to tumor progression and metastasis. However, the expression and molecular mechanism of malic enzyme 3 (ME3) in GC are rarely reported. In this study, we aim to investigate the molecular mechanism of ME3 in the development of GC and to explore its potential value as a prognostic and therapeutic target in GC.
METHODS: ME3 mRNA and protein expression were evaluated in patients with GC using RT-qPCR, WB, and immunohistochemistry, as well as their correlation with clinicopathological indicators. The effect of ME3 on proliferation and metastasis was evaluated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU) assay, transwell assay, wound healing assay, and subcutaneous injection or tail vein injection of tumor cells in mice model. The effects of ME3 knockdown on the level of metabolites and hypoxia-inducible factor-1α (HIF-1α) protein were determined in GC cells. Oxidative phosphorylation was measured to evaluate adenosine triphosphate (ATP) production.
RESULTS: ME3 was downregulated in human GC tissues (P < 0.001). The decreased ME3 mRNA expression was associated with younger age (P = 0.02), pathological staging (P = 0.049), and lymph node metastasis (P = 0.001), while low ME3 expression was associated with tumor size (P = 0.048), tumor invasion depth (P < 0.001), lymph node metastasis (P = 0.018), TNM staging (P < 0.001), and poor prognosis (OS, P = 0.0206; PFS P = 0.0453). ME3 knockdown promoted GC cell malignancy phenotypes. Moreover, α-ketoglutarate (α-KG) and NADPH/NADP+ ratios were reduced while malate was increased in the ME3 knockdown group under normoxia. When cells were incubated under hypoxia, the NADPH/NADP+ ratio and α-KG decreased while intracellular reactive oxygen species (ROS) increased significantly. The ME3 knockdown group exhibited an increase in ATP production and while ME3 overexpression group exhibited oppositely. We discovered that ME3 and HIF-1α expression were negatively correlated in GC cells and tissues, and proposed the hypothesis: downregulation of ME3 promotes GC progression via regulating intracellular oxidative stress and HIF-1α.
CONCLUSIONS: We provide evidence that ME3 downregulation is associated with poor prognosis in GC patients and propose a hypothesis for the ME3 regulatory mechanism in GC progression. The present study is of great scientific significance and clinical value for exploring the prognostic and therapeutic targets of GC, evaluating and improving the clinical efficacy of patients, reducing recurrence and metastasis, and improving the prognosis and quality of life of patients.