Objective: To explore the correlation of CPNE3 expression with long-term prognosis of patients with gastric cancer (GC) and the possible mechanism.
Methods: We retrospectively collected the data of 104 GC patients undergoing radical surgery in our hospital from February, 2013 to October, 2017. TCGA database and immunohistochemistry were used to analyze CPNE3 expression level in GC tissues and its effects on tumor progression and long-term prognosis of the patients. GO analysis was performed to predict the biological role of CPNE3 in GC. We also conducted cell experiments with MGC803 cells and observed the effects of CPNE3 knockdown, CPNE3 overexpression and LY294002 (a PI3K/AKT inhibitor) treatment on cell apoptosis and cellular expressions of apoptotic proteins using flow cytometry and Western blotting.
Results: TCGA analysis and immunohistochemistry both showed high expressions of CPNE3 in GC (P < 0.05). The patients with high CPNE3 expressions had a reduced 5-year survival (P < 0.01), and a high CPNE3 expression, CEA level≥5 μg/L, CA19-9 level ≥37 kU/L, T3-T4 stage, and N2-N3 stage were all independent risk factors for a lowered 5-year survival rate after surgery. The sensitivity and specificity of CPNE3 for predicting 5-year mortality was 79.59% and 74.55%, respectively (P < 0.05). GO analysis predicted that CPNE3 negatively regulated GC cell apoptosis. In MGC803 cells, CPNE3 knockdown significantly increased cell apoptosis, enhanced Bax and Cleaved Caspase-3 expressions and decreased Bcl-2 expression, while CPNE3 overexpression produced the opposite results (P < 0.05). The cellular expressions of p-PI3K and p-AKT were significantly decreased following CPNE3 knockdown and increased following CPNE3 overexpression (P < 0.05). Treatment with LY294002 obviously attenuated the inhibitory effect of CPNE3 overexpression on apoptosis of MGC803 cells (P < 0.05).
Conclusion: CPNE3 is highly expressed in GC tissues and affects the long-term prognosis of the patients possibly by inhibiting GC cell apoptosis through activation of PI3K/AKT signaling.
目的: 明确CPNE3在胃癌组织中表达情况与患者远期预后的关系，探究其调控胃癌细胞凋亡的作用和分子机制。
方法: 纳入2013年2月~2017年10月104例于我院行胃癌根治术的患者，通过癌症和肿瘤基因图谱（TCGA)的公共数据和免疫组织化学染色分析CPNE3在胃癌中的表达和对患者肿瘤进展与远期预后的影响。GO富集分析预测CPNE3在胃癌中的生物学功能。采用干扰和过表达CPNE3以及对照慢病毒载体转染MGC803细胞，获取对照组（NC）、敲低组（si-CPNE3）、过表达组（LV-CPNE3）细胞，采用PI3K/AKT抑制剂（LY294002）干预LV-CPNE3组细胞获取LY294002组。流式细胞术分析细胞凋亡，Western blot检测Bax、Cleaved Caspase-3、Bcl-2、p-PI3K和p-AKT表达。
结果: TCGA预测分析和免疫组化结果显示CPNE3在胃癌中高表达（P＜0.05）。Kaplan-Meier生存分析显示CPNE3高表达组患者5年生存率显著降低（P＜0.01）。单因素及Cox回归分析显示CPNE3高表达、CEA≥5 μg/L、CA19-9≥37 kU/L、T3-T4期和N2-N3期是影响胃癌根治术后5年生存率的独立危险因素。ROC曲线分析显示CPNE3预判术后5年死亡的敏感度为79.59%，特异性为74.55%（P＜0.05）。GO富集分析预测显示CPNE3可能与负向调控胃癌细胞凋亡有关。体外实验显示，si-CPNE3组的细胞凋亡率以及Bax、Cleaved Caspase-3表达的增多，而Bcl-2表达减少；LV-CPNE3组则结果相反（P＜0.05）。机制分析显示，si-CPNE3组p-PI3K、p-AKT表达降低，LV-CPNE3组则增多（P＜0.05）；另外，LY294002减弱了CPNE3过表达对胃癌细胞凋亡的抑制作用（P＜0.05）。
结论: CPNE3在胃癌组织中高表达且对患者的远期预后具有预测价值，可能与其通过激活PI3K/AKT信号通路抑制胃癌细胞的凋亡有关。
Keywords: CPNE3; PI3K/AKT; cell apoptosis; gastric cancer; prognosis.