SIRT7抑制胰腺癌细胞上皮-间充质转化的作用和机制研究  

Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism

作　　者：王梦迪 高天杨 黄蔚 杨云凯 王艳 Wang Mengdi;Gao Tianyang;Huang Wei;Yang Yunkai;Wang Yan(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Collaborative Innovation Center of Tianjin for Medical Epigenetics,Tianjin Key Laboratory of Medical Epigenetics,Ministry of Education,Key Laboratory of Immune Microenvironment and Disease,Ministry of Education,Tianjin 300070,China;Department of Biochemistry and Molecular Biology,School of Basic Medicine,Capital Medical University,Beijing Key Laboratory of Cancer Invasion and Metastasis Research,Beijing 100069,China;Key Laboratory of Cancer and Microbiome,State Key Laboratory of Molecular Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Science and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]天津医科大学基础医学院生物化学与分子生物学系,天津市医学表观遗传学协同创新中心,天津市医学表观遗传学重点实验室,免疫微环境与疾病教育部重点实验室,天津300070 [2]首都医科大学基础医学院生物化学与分子生物学系,肿瘤侵袭和转移机制研究北京市重点实验室,北京100069 [3]国家癌症中心,国家肿瘤临床医学研究中心,中国医学科学院北京协和医学院肿瘤医院恶性肿瘤与微生物组学重点实验室,分子肿瘤学国家重点实验室,北京100021

出　　处：《中华肿瘤杂志》2024年第6期566-582,共17页Chinese Journal of Oncology

基　　金：国家自然科学基金(41931291、82273403、82203820);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2019PT310027、2021-RC310-018);中国博士后科学基金(2022M710454)。

摘　　要：目的探讨SIRT7在胰腺癌细胞上皮-间充质转化(EMT)中的作用和机制。方法使用siRNA或质粒转染将胰腺癌细胞分为siControl组、siSIRT7组、过表达SIRT7组、siSIRT7+siCOL4A1组和siSIRT7+siSLUG组。EdU实验、细胞划痕实验和Transwell实验分别检测细胞的增殖、迁移和侵袭能力,实时荧光聚合酶链反应(qRT-PCR)和Western blot法检测EMT标志物和肿瘤干细胞标志物的表达水平。对敲低SIRT7的胰腺癌细胞进行转录组测序(RNA-seq),探究SIRT7调控的信号通路和靶基因,采用qRT-PCR验证靶基因的转录水平。定量染色质免疫共沉淀实验(q-ChIP)和染色质免疫共沉淀聚合酶链反应(ChIP-PCR)鉴定SIRT7直接调控的靶基因。免疫组织化学法检测人胰腺癌组织(2013-2016年购自武汉塞维尔生物科技有限公司)中SIRT7及靶基因的表达。癌症基因组图谱(TCGA)数据库数据分析SIRT7及其靶基因的表达相关性。KM-Plotter网站用以分析SIRT7和靶基因在胰腺癌中的生存相关性。GeneMANIA、STRING和ENCORI在线工具用以分析SIRT7相关蛋白及miRNAs等。结果EdU实验结果显示,过表达SIRT7组PANC-1和BxPC-3细胞增殖率分别为(19.33±0.35)%和(17.00±1.89)%,均低于对照组[分别为(31.60±1.37)%和(24.33±0.78)%,均P<0.05];而敲低SIRT7表达组PANC-1和BxPC-3细胞增殖率[分别为(23.94±1.00)%、(27.08±0.97)%]和[(22.00±1.86)%、(25.96±1.61)%]均高于siControl组[分别为(11.80±1.86)%和(13.42±1.39)%,均P<0.05]。在PANC-1细胞中,细胞划痕实验结果表明,过表达SIRT7组细胞相对迁移率为(76.67±2.74)%,低于对照组细胞[(100.00±2.13)%,P<0.05];而抑制SIRT7表达后细胞相对迁移率[分别为(134.22±4.08)%和(199.82±9.20)%]均高于siControl组细胞[(102.24±3.13)%,均P<0.05]。迁移实验中(BxPC-3细胞),过表达SIRT7组细胞迁移数为(28.33±2.62)个,低于于对照组[(45.66±1.69)个,P<0.05];敲低SIRT7表达组细胞迁移数[分别为(65.66±2.86)个、(82.00±2.94)个]均高于siControlObjectiveTo investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation(EMT)of pancreatic cancer cells.MethodsThe pancreatic cancer cells were divided into siControl,siSIRT7,over-expression SIRT7,siSIRT7+siCOL4A1,and siSIRT7+siSLUG groups using siRNA or plasmid transfection.The proliferation,migration and invasion of pancreatic cancer cells were detected by EdU,wound healing assay and Transwell experiments,respectively.The expression of EMT and cancer stem cell(CSC)markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay(qRT-PCR)and western blot.RNA sequencing(RNA-seq)in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7.Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment(q-ChIP)and chromatin immunoprecipitation polymerase chain reaction(ChIP-PCR).The expressions of SIRT7 and target genes were detected by immunohistochemical(IHC)in pancreatic cancer tissues,and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset.The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website.Finally,GeneMANIA,STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs.ResultsEdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1[(19.33±0.35)%]and BxPC-3 cells[(17.00±1.89)%]were lower than those in the control group[(31.60±1.37)%and(24.33±0.78)%,respectively,P<0.05].The proliferation rates of SIRT7-knockdown PANC-1[(23.94±1.00)%and(27.08±0.97)%]and BxPC-3 cells[(22.00±1.86)%and(25.96±1.61)%]were higher than those of the siControl group[(11.80±1.86)%and(13.42±1.39)%,respectively,P<0.05].In PANC-1 cells,the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells[(76.67±2.74)%]was lower than that of control cells[(100.00±2.13)%,P<0.05];the relative migration rate of c

关 键 词：胰腺肿瘤 SIRT7 上皮-间充质转化 

分 类 号：R735.9[医药卫生—肿瘤]