胃癌中PCMT1表达的预后价值及其对纺锤体组装检查点的调控作用

Prognostic Value of PCMT1 Expression in Gastric Cancer and Its Regulatory Effect on Spindle Assembly Checkpoints

目的 探讨蛋白质-L-异天冬氨酸(D-天冬氨酸)-甲基转移酶(PCMT1)在胃癌中的表达及对预后的影响,并分析其潜在的作用机制。方法 UALCAN数据库在线分析PCMT1在胃癌组织的表达情况,通过DAVID数据库进行基因本体注释(GO)及京都基因和基因组百科全书(KEGG)信号通路富集分析其可能的功能与信号通路。纳入2014年1月–2017年12月于我院接受胃癌根治术的120例患者,免疫组织化学染色检测PCMT1、Ki67在胃癌组织中的表达;Cox回归、Kaplan-Meier曲线和受试者工作特征(ROC)曲线用于胃癌患者术后5年生存率的预后分析。采用慢病毒构建干扰或过表达PCMT1载体,转染人胃癌细胞系MGC-803与HGC-27细胞,设置干扰空载体(sh-NC)组与干扰PCMT1载体(sh-PCMT1)组,过表达空载体(LV-Vec)组与过表达PCMT1载体(LV-PCMT1)组;Western blot检测各组细胞中PCMT1、CyclinB1、CDC20蛋白表达,CCK-8法检测胃癌细胞增殖能力,流式细胞术检测细胞周期。注射4组MGC-803细胞构建裸鼠皮下移植瘤模型,每组3只,测量体质量,14 d后处死裸鼠,测量肿瘤体积,Western blot法检测肿瘤组织中CyclinB1与CDC20蛋白表达水平。结果 UALCAN数据库分析示PCMT1在胃癌组织中高表达且在不同病理分期、分级与淋巴结转移的胃癌组织中均表达升高(P<0.05)。GO与KEGG富集示PCMT1主要参与有丝分裂、纺锤体组装检查点与细胞周期等信号。免疫组化结果显示PCMT1与Ki67在患者胃癌组织中呈高表达且二者呈正相关关系(P<0.05)。Cox多因素分析示PCMT1高表达[风险比(HR)=2.921,95%置信区间(CI):1.628~5.239]是影响胃癌患者术后5年生存率的独立危险因素之一。Kaplan-Meier曲线分析示高表达PCMT1的患者术后5年生存率(16.7%,HR=4.651,95%CI=2.846~7.601)低于低表达PCMT1患者(70.0%,HR=0.215,95%CI=0.132~0.351)。ROC曲线表明,PCMT1预测患者术后5年生存率的曲线下面积为0.764(95%CI:0.674~0.854)。Western blot对PCMT1的检测结果表明慢病毒干扰或过表达PCMT1细胞系构建成功。CCK-8结果表明下调PCMT1表达MGC-803与HGC-27细胞增殖能力减弱,过表达PCMT1则促进细胞增殖(P<0.05)。干扰PCMT1后CDC20蛋白表达下调,CyclinB1蛋白表达上升,细胞周期阻滞于G2/M期,而过表达则呈现相反趋势(P<0.05)。sh-PCMT1组裸鼠肿瘤的体积与质量减小,肿瘤组织CDC20蛋白表达降低,CyclinB1蛋白表达升高(P<0.05,与sh-NC组相比),LV-PCMT1组则呈相反趋势(P<0.05,与LV-Vec组相比)。结论 PCMT1在胃癌组织中呈高表达,与患者不良预后相关,可能通过调控细胞有丝分裂进程中纺锤体检查点影响肿瘤细胞恶性增殖。

Objective The study was conducted to investigate the expression of protein-L-isoaspartate(D-aspartate)O-methyltransferase(PCMT1)in gastric cancer and its effect on the prognosis,and to analyze its potential mechanism.Methods UALCAN,a cancer data analysis platform,was used to conduct online analysis of the expression of PCMT1 in gastric cancer tissues.Through the Database for Annotation,Visualization and Integrated Discovery(DAVID),Gene Ontology(GO)annotation and signaling pathway enrichment by Kyoto Encyclopedia of Genes and Genomes(KEGG)were performed to analyze the possible functions and signaling pathways.A total of 120 patients who underwent radical gastrectomy for gastric cancer between January 2014 and December 2017 in our hospital were enrolled for the study.Immunohistochemical staining was performed to determine the expression of PCMT1 and Ki67 in gastric cancer tissues.Cox regression,Kaplan-Meier curve,and receiver operating characteristic(ROC)curves were used for prognostic analysis of 5-year survival in gastric cancer patients after surgery.Lentivirus was used to construct PCMT1-interfering or PCMT1-overexpressing vectors,which were then used to transfect human gastric cancer cell lines of MGC-803 and HGC-27 cells.The interfering empty vector(sh-NC)group,the interfering PCMT1 vector(sh-PCMT1)group,the overexpressing empty vector(LV-Vec)group,and the overexpressing PCMT1 vector(LV-PCMT1)group were set up.Western blot was performed to determine the protein expression levels of PCMT1,CyclinB1,and CDC20.CCK-8 assay was performed to measure the proliferation of gastric cancer cells.Flow cytometry was performed to determine the cell cycle.MGC-803 cells were injected in four groups of nude mice to construct a subcutaneous xenograft tumor model,with three nude mice in each group.The body mass of the nude mice was measured.The nude mice were sacrificed after 14 days and the tumor volume was monitored.The expression levels of CyclinB1 and CDC20 proteins in the tumor tissues were determined by Western blot assay.Results Analysis with UALCAN showed that PCMT1 was highly expressed in gastric cancer tissues.Moreover,elevated expression was found in gastric tumor tissues of different pathological stages and grades and those with lymph node metastasis(P<0.05).GO and KEGG enrichment analyses showed that PCMT1 was mainly involved in the signal regulation of mitosis,spindle assembly checkpoints,and cell cycle.The immunohistochemical results showed that PCMT1 and Ki67 were highly expressed in gastric cancer tissues and that they were positively correlated with each other(P<0.05).Cox multivariate analysis showed that high PCMT1 expression(hazard ratio[HR]=2.921,95%confidence interval[CI]:1.628-5.239)was one of the independent risk factors affecting the 5-year survival rate of gastric cancer patients after surgery.Kaplan-Meier curve showed that patients with high PCMT1 expression had a lower 5-year survival after surgery(16.7%,HR=4.651,95%CI:2.846-7.601)than patients with low PCMT1 expression(70.0%,HR=0.215,95%CI:0.132-0.351)did.The ROC curve showed that PCMT1 had an area under the curve(AUC)of 0.764(95%CI:0.674-0.854)for predicting 5-year patient survival after surgery.Western blot results showed that lentiviral interference or overexpression of PCMT1 cell lines was successfully constructed.The results of CCK-8 showed that the proliferative ability of MGC-803 and HGC-27 cells was weakened with the downregulation of PCMT1,and the overexpression of PCMT1 promoted cell proliferation(P<0.05).With the interference of PCMT1,the expression of CDC20 protein was decreased,the expression of CyclinB1 protein was increased,and the cell cycle was arrested in the G2/M phase.In contrast,the overexpression of PCMT1 led to the opposite trends(P<0.05).In the sh-PCMT1 group,the tumor volume and mass were decreased and the expression of CDC20 protein was decreased and the expression of CyclinB1 protein was increased in the tumor tissues of the nude mice(P<0.05),compared with those of the sh-NC group.In contrast,the LV-PCMT1 group showed the opposite trends(P<0.05,compared with those of the LV-Vec group).Conclusion The high expression of PCMT1 in gastric cancer tissues is associated with poor prognosis in patients and may affect tumor cell malignant proliferation via regulating spindle checkpoints in the process of mitosis.