死亡相关蛋白激酶1及结节性硬化复合物蛋白2基因在胰腺癌组织中的表达及与预后的关系  

Expression of death-associated protein kinase 1 and tuberous sclerosis complex 2 gene in pancreatic carcinoma tissues and its relationship with prognosis

作　　者：孔祥泉[1] 杜开新 马礼钦[2] 李金銮[2] 廖雪洪 洪俊强 罗水英[1] 林小艺 吴倩 李永恒[4] Kong Xiangquan;Du Kaixin;Ma Liqin;Li Jinluan;Liao Xuehong;Hong Junqiang;Luo Shuiying;Lin Xiaoyi;Wu Qian;Li Yongheng(Department of Radiotherapy,Xiamen Humanity Hospital,Fujian Medical University,Fujian Province,Xiamen 361000,China;Department of Radiotherapy,Fujian Cancer Hospital,Cancer Hospital,Fujian Medical University,Fuzhou 350014,China;Department of Pathology,Zhortgshan Hospital,Xiamen University,Fujian Province,Xiamen 361000,China;Department of Radiotherapy,Beijing Cancer Hospital,Beijing Institute for Cancer Research,Key Laboratory of Carcinogenesis and Translational Research(Ministry of Education),Beijing 100142,China)

机构地区：[1]福建医科大学附属厦门弘爱医院放疗科,361000 [2]福建省肿瘤医院福建医科大学附属肿瘤医院放疗科,福州350014 [3]厦门大学附属中山医院病理科,361000 [4]北京大学肿瘤医院暨北京市肿瘤防治研究所放疗科恶性肿瘤发病机制及转化研究教育部重点实验室,100142

出　　处：《中国医药》2021年第4期570-574,共5页China Medicine

基　　金：福建省自然科学基金(2018J01266);首都卫生发展科研专项(首发2020-2-1027)。

摘　　要：目的通过生物信息学方法研究死亡相关蛋白激酶1(DAPK1)和结节性硬化复合物蛋白2(TSC2)基因在胰腺癌中的表达情况,并探讨分析其与预后的关系。方法利用来自癌症和肿瘤基因图谱(TCGA)数据库的胰腺癌患者数据进行Kaplan-Meier分析,进而深层次探讨DAPK1和TSC2基因表达水平与患者总生存期或无进展生存期(PFS)的关系,同时,对影响预后的因素进行Cox多因素分析,以及分析胰腺癌中DAPK1和TSC2基因启动子的甲基化水平。此外,通过双荧光素酶报告基因检测系统,验证靶向DAPK1的微小RNA(miR),并应用定量逆转录聚合酶链反应和蛋白质免疫印迹法验证在胰腺癌细胞中miR-324-5p负调控DAPK1的表达。结果多因素分析显示,肿瘤分级(P=0.005)、R0切除(P<0.001)及TSC2表达情况(P=0.005)是影响胰腺癌患者PFS的独立危险因素;肿瘤部位(P=0.006)、病理类型(P=0.002)及分子靶向治疗(P<0.001)是影响胰腺癌患者总生存期的独立危险因素。DAPK1基因低表达且TSC2基因高表达的胰腺癌患者具有更好的总生存期(P=0.024)。DAPK1和TSC2甲基化水平与相应的mRNA表达水平均呈负相关(r=-0.69、-0.37,均P<0.05)。体外实验结果证实,过表达的miR-324-5p显著抑制了胰腺癌Pa Ca-2和PANC-1细胞DAPK1 mRNA和蛋白的表达[mRNA:(0.37±0.02)比(1.00±0.02)、(0.53±0.01)比(1.00±0.06);蛋白:(0.54±0.06)比(1.04±0.12)、(0.63±0.11)比(1.01±0.11)](均P<0.05)。结论DAPK1基因联合TSC2基因的表达情况可以预测胰腺癌患者的预后;在胰腺癌组织中,DAPK1和TSC2启动子的低甲基化水平是DAPK1和TSC2高表达的重要因素;miR-324-5p负调控DAPK1的表达,有望成为胰腺癌靶向治疗的新靶点。Objective To investigate the expression of death-associated protein kinase 1(DAPK1)and tuberous sclerosis complex 2(TSC2)gene in pancreatic carcinoma tissues and its relationship with prognosis through bioinformatics method.Methods The data of patients with pancreatic cancer were obtained from The Cancer Genome Atlas(TCGA)database.Kaplan-Meier analysis was performed to investigate the association between DAPK1 and TSC2 gene expression levels and overall survival(OS)or progression-free survival(PFS)of patients.Cox multivariate analysis was performed for the factors influencing prognosis.The levels of DAPK1 and TSC2 gene promoter methylation in pancreatic cancer were assessed.Subsequently,the microRNA(miR)targeting DAPK1 was verified by the dual-luciferase reporter gene assay system,and the negative regulation of DAPK1 expression by miR-324-5p in pancreatic cancer cells was verified by quantitative reverse transcription polymerase chain reaction and western blotting.Results Multivariate analysis showed that tumor grade(P=0.005),R0 excision(P<0.001)and TSC2 expression(P=0.005)were independent risk factors affecting PFS in pancreatic cancer patients;tumor location(P=0.006),pathological type(P=0.002)and molecular targeted therapy(P<0.001)were independent risk factors affecting OS in pancreatic cancer patients.Pancreatic cancer patients with low expression of the DAPK1 gene and high expression of the TSC2 gene had better OS(P=0.024).DAPK1 and TSC2 methylation levels were negatively associated with corresponding mRNA expression levels(r=-0.69,-0.37,both P<0.05).In vitro experiments showed that overexpression of miR-324-5p significantly inhibited the expression of DAPK1 mRNA and protein in PaC a-2 and PANC-1 cells[mRNA:(0.37±0.02)vs(1.00±0.02),(0.53±0.01)vs(1.00±0.06);protein:(0.54±0.06)vs(1.04±0.12),(0.63±0.11)vs(1.01±0.11)](all P<0.05).Conclusions The expression of DAPK1 gene combined with TSC2 gene can predict the prognosis of patients with pancreatic cancer.The hypomethylation of DAPK1 and TSC2 promoters is a

关 键 词：胰腺癌 死亡相关蛋白激酶1 结节性硬化复合物蛋白2 微小RNA-324-5p 

分 类 号：R735.9[医药卫生—肿瘤]