APE1通过NF-κB通路调节结直肠癌PD-L1表达和细胞增殖

APE1 Inhibits Colorectal Cancer Proliferation through Inhibition of PD-L1 and Regulation of NF-κB Signaling Pathway

目的探讨脱嘌呤/脱嘧啶核酸内切酶1(apurinic/aprimidinic endonuclease1,APE1)对结直肠癌组织及细胞程序性死亡配体-1(PD-L1)表达和细胞增殖的影响及其信号通路。方法收集67例结直肠癌及配对的癌旁组织,免疫组织化学染色检测组织APE1和PD-L1表达,分析APE1表达与结直肠癌患者临床资料的关系,并绘制不同APE1表达水平结直肠癌患者的生存曲线。利用siRNA技术沉默结直肠癌HT29、Ls174t中APE1表达,分别利用CCK-8和平板细胞克隆形成实验检测细胞增殖情况,蛋白质印迹法检测HT29、Ls174t细胞NF-κB信号通路相关蛋白表达。结果结直肠癌组织APE1、PD-L1阳性染色评分均明显高于癌旁组织,HT29、Ls174t细胞APE1、PD-L1蛋白相对表达量也明显高于结直肠上皮细胞HCEpi,差异具有统计学意义(均P<0.01)。相关性分析显示,结直肠癌组织APE1与PD-L1表达呈显著正相关(P<0.01)。APE1高表达与肿瘤直径(P=0.002)和病理学N分期(P=0.014)有关;APE1高表达组5年总生存率和无病生存率分别为49.7%和37.1%,明显低于APE1低表达组(75.1%和63.2%),差异均具有统计学意义(均P<0.05)。转染APE1 siRNA能下调HT29、Ls174t细胞APE1表达并导致PD-L1表达下调,肿瘤细胞增殖率和克隆形成数也明显降低(P<0.05,P<0.01)。同时,转染APE1 siRNA的HT29、Ls174t细胞磷酸化NF-κB表达水平明显低于NC-siRNA组和空白对照组(均P<0.01)。结论结直肠癌组织和细胞APE1、PD-L1表达上调,抑制APE1可能通过下调磷酸化NF-κB表达影响结直肠癌细胞的增殖。

Objective To investigate the effect of apurinic/aprimidinic endonuclease1(APE1)on the PD-L1 expression and proliferation in colorectal cancer cells and signal pathway mediated by it.Methods A total of 67 cases of colorectal cancer tissues and paired adenocarcinoma tissues were collected.The expression levels of APE1 and PD-L1 were measured by immunohistochemistry.Correlation between APE1 expression and colorectal cancer clinicopathologic features was also analyzed,survival curves were described by Kaplan-Meier method.Human colorectal cancer cell lines HT29 and Ls174 t were interfered with siRNAs for silencing APE1 expression.Cell proliferation was determined by CCK-8 assay and colony formation assay,respectively.The expression protein related to NF-κB pathway was detected by Western blotting.Results The staining scores of APE1 and PD-L1 were significantly higher in colorectal cancer tissues than in adenocarcinoma tissues.Besides,the expression of APE1 protein was significantly up-regulated in CRC cell lines HT29,Ls174 t compared with colorectal epithelial cells HCEpi(P<0.01).Correlation analysis showed that there was positive correlation between APE1 and PD-L1 expression in colorectal cancer tissues(P<0.01).APE1 expression level was strongly correlated with the tumor size(P=0.002)and N classification(P=0.014)in this cohort of 67 cases of CRC.Kaplan-Meier survival analysis confirmed that patients with high APE1 levels had poorer overall survival(P=0.020)and disease-free survival(P=0.029)than patients with low APE1 levels.APE1 siRNA effectively inhibited the expression of APE1 and PD-L1,which decreased the rates of cell apoptosis and colony number in HT29,Ls174 t cells(P<0.05).Meanwhile,in cells tranfected with APE1 siRNA,NF-κB phosphorylation expression was also inhibited when compared to the NC-siRNA group and blank control group(P<0.01).Conclusion APE1 and PD-L1 is up-regulated in colorectal cancer tissues and cells,down-regulation of APE1 is able to inhibit the proliferation of human CRC via the inhibition of NF-κB phosphorylation.