Table_3_QSOX2 Is an E2F1 Target Gene and a Novel Serum Biomarker for Monitoring Tumor Growth and Predicting Survival in Advanced NSCLC.DOC

BackgroundQuiescin Q6 sulfhydryl oxidase 2 (QSOX2), an enzyme that can be directly secreted into the extracellular space, is known to be associated with oxidative protein folding. However, whether QSOX2 is abnormally expressed in non-small cell lung cancer (NSCLC) and its role in tumor growth remains unclear.
MethodsReal-time quantitative PCR (qPCR), immunohistochemistry (IHC), bioinformatics analyses were applied to analyze the expression pattern and prognostic significance of QSOX2 in NSCLC. Xenografts model, enzyme-linked immunosorbent assays (ELISA), western blot analysis (WB), and IHC were preformed to examine in vivo tumor suppression and intracellular and extracellular expression of QSOX2. Flow cytometry, WB and qPCR analyses were used to elucidate the role of QSOX2 in cell cycle regulation. Chromatin immunoprecipitation assay (ChIP) assay and Dual-Luciferase reporter assay were employed to investigate transcriptional regulation of QSOX2 by E2F Transcription Factor 1 (E2F1).
ResultsQuiescin sulfhydryl oxidase 2 was significantly overexpressed in NSCLC and associated with poor survival in advanced-stage patients. The intracellular and extracellular expression of QSOX2 by tumor cells markedly decreased after anti-cancer therapy in vitro, in vivo and in the clinic. Moreover, QSOX2 silencing in NSCLC cell lines resulted in inhibition of cancer cell proliferation, induction of apoptosis, and decreased expression of cell division-related genes (CENPF and NUSAP1) and Wnt pathway activators (PRRX2 and Nuc-β-catenin). Mechanistically, QSOX2 was expressed periodically during cell cycle and directly regulated by E2F1.
ConclusionsOur findings demonstrate that QSOX2 is directly regulated by E2F1 in the cell cycle, which is essential for the proliferation of NSCLC cells. Furthermore, QSOX2 is a prognostic indicator for NSCLC and may be developed into a biomarker for monitoring tumor burden and therapeutic progress.

**背景**：静止素Q6巯基氧化酶2（Quiescin Q6 sulfhydryl oxidase 2, QSOX2）是一种可直接分泌至细胞外间隙的酶，已知其与氧化蛋白质折叠密切相关。然而，QSOX2在非小细胞肺癌（non-small cell lung cancer, NSCLC）中是否存在异常表达，及其在肿瘤生长中的具体作用仍不明确。

**方法**：本研究采用实时定量PCR（real-time quantitative PCR, qPCR）、免疫组织化学（immunohistochemistry, IHC）及生物信息学分析，探究QSOX2在非小细胞肺癌中的表达模式及其预后意义。通过异种移植模型、酶联免疫吸附试验（enzyme-linked immunosorbent assays, ELISA）、蛋白质印迹分析（western blot analysis, WB）及IHC，检测QSOX2的体内抗肿瘤活性及细胞内、外表达水平。采用流式细胞术、WB及qPCR分析，阐明QSOX2在细胞周期调控中的作用。通过染色质免疫沉淀试验（chromatin immunoprecipitation assay, ChIP）与双荧光素酶报告基因试验（Dual-Luciferase reporter assay），探究E2F转录因子1（E2F Transcription Factor 1, E2F1）对QSOX2的转录调控作用。

**结果**：QSOX2在非小细胞肺癌组织中显著高表达，且与晚期患者的不良生存预后密切相关。在体外、体内及临床样本中，肿瘤细胞的QSOX2细胞内与细胞外表达水平均在抗肿瘤治疗后显著降低。此外，在非小细胞肺癌细胞系中沉默QSOX2，可抑制癌细胞增殖、诱导细胞凋亡，并降低细胞分裂相关基因（CENPF与NUSAP1）及Wnt通路激活因子（PRRX2与核β-连环蛋白）的表达。机制研究表明，QSOX2在细胞周期中呈周期性表达，且直接受E2F1调控。

**结论**：本研究结果证实，QSOX2在细胞周期中受E2F1的直接调控，这一机制对非小细胞肺癌细胞的增殖至关重要。此外，QSOX2可作为非小细胞肺癌的预后指标，有望成为监测肿瘤负荷与治疗进展的生物标志物。