IDH1基因在肝内胆管癌细胞HuCCT1增殖与迁移中的作用及其初步机制  

The biological function and mechanism of IDH1 gene in intrahepatic cholangiocarcinoma cell HuCCT1

作　　者：林美佳 雷宇清 叶洲杰 朱丽萍 王心睿 黄雄飞[1,8] Lin Mei-Jia;Lei Yu-Qing;Ye Zhou-Jie;Zhu Li-Ping;Wang Xin-Rui;Huang Xiong-Fei(Department of Pathology,School of Basic Medical Sciences,Fujian Medical University,Fuzhou,Fujian 350004,China;NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-Human Primate/Fujian Maternity and Child Health Hospital,Fuzhou,Fujian 350000,China;College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics,Fujian Medical University,Fuzhou,Fujian 350004,China;Department of Cardiac Surgery,Fuzhou,Fujian 350011,China;Fujian Maternity and Child Health Hospital,Fuzhou,Fujian 350001,China;Fujian Children's Hospital,Fuzhou,Fujian 350011,China;Medical Research Center,Fujian Maternity and Child Health Hospital,Affiliated Hospital of Fujian Medical University,Fuzhou,Fujian 350001,China;Key Laboratory of Gastrointestinal Malignancy of Ministry of Education,Fujian Medical University,Fuzhou,Fujian 350108,China)

机构地区：[1]福建医科大学基础医学院病理学系,福建福州350004 [2]国家卫健委非人灵长类生育调节技术评价重点实验室/福建省妇幼保健院,福建福州350000 [3]福建医科大学妇儿临床医学院,福建福州350004 [4]福建省儿童医院心脏外科,福建福州350011 [5]福建省妇幼保健院,福建福州350001 [6]福建省儿童医院,福建福州350011 [7]福建医科大学附属医院福建省妇幼保健院医学研究中心,福建福州350001 [8]福建医科大学消化道恶性肿瘤教育部重点实验室,福建福州350108

出　　处：《解放军医学杂志》2024年第2期194-203,共10页Medical Journal of Chinese People's Liberation Army

基　　金：福建省自然科学基金(2022J01662)。

摘　　要：目的探讨异柠檬酸脱氢酶1(IDH1)在肝内胆管癌(iCCA)细胞HuCCT1增殖与迁移中的作用及其可能的分子机制。方法采用CRISPR/Cas9基因编辑技术构建IDH1基因敲除的HuCCT1细胞(HuCCT1^(IDH1-/-));CCK-8法和克隆形成实验检测IDH1野生型HuCCT1(HuCCT1^(WT))细胞和HuCCT1^(IDH1-/-)细胞的增殖能力;细胞划痕和Transwell实验检测细胞的迁移和侵袭能力;Western blotting检测细胞上皮间质转化(EMT)相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-9(MMP-9)、Wnt3a、β-连环蛋白(β-catenin)的表达水平。生物信息学方法分析上述两种HuCCT1细胞的转录组测序结果,Western blotting验证转录组信息通路相关蛋白的表达。结果与HuCCT1细胞比较,HuCCT1^(IDH1-/-)细胞增殖和克隆形成数目明显减少(P<0.05),阻滞在G_(2)/M期细胞的比例明显增加(P<0.01),划痕愈合率明显降低(P<0.01),迁移细胞数目(P<0.001)和侵袭细胞数目(P<0.05)明显减少;q RT-PCR检测结果显示,HuCCT1^(IDH1-/-)细胞IDH1、Vimentin、MMP-9和调控G_(2)/M期增殖相关基因Cyclin A2、Cyclin B1及CDK1 mRNA表达水平降低(P<0.05),编码E-cadherin的CDH1 mRNA表达水平升高(P<0.01);Western blotting检测结果显示,HuCCT1^(IDH1-/-)细胞中E-cadherin表达水平升高(P<0.05),N-cadherin、Vimentin及MMP-9蛋白表达水平降低(P<0.05)。转录组测序结果显示,HuCCT1^(WT)与HuCCT1^(IDH1-/-)存在1476个差异表达基因(DEGs);基因本体论(GO)分析显示上述DEGs显著富集在炎症反应、细胞信号转导和细胞代谢等生物学过程;KEGG通路分析显示上述DEGs显著富集在Wnt、MAPK、Rap1、Hippo、TNF等与肿瘤细胞增殖和侵袭转移密切相关的信号通路。Western blotting验证结果显示,与HuCCT1^(WT)比较,HuCCT1^(IDH1-/-)细胞Wnt信号通路的Wnt3a和β-catenin蛋白表达水平降低(P<0.05)。结论IDH1基因参与调控iCCA细胞HuCCT1的迁移、侵袭及EMT过程,其机制可能与激活Wnt/�Objective To explore the role and possible molecular mechanism of Isocitrate dehydrogenase 1(IDH1)gene in proliferation and migration of intrahepatic cholangiocarcinoma(iCCA)cell HuCCT1.Methods HuCCT1 cells with IDH1 gene knockout(HuCCT1^(IDH1-/-))were constructed by CRISPR/Cas9 gene editing technology.To investigate the capacities of proliferation,migration and invasion of HuCCT1^(WT)(HuCCT1 cells with wild-type IDH1 gene)and HuCCT1^(IDH1-/-)cells,assays of CCK-8,clone formation,scratch and transwell were performed.Western blotting was used to detect the expression levels of epithelial-mesenchymal transition(EMT)associated proteins E-cadherin,N-cadherin,Vimentin,MMP-9,Wnt3a andβ-catenin in two groups of cells.The transcriptome sequencing data of HuCCT1^(WT) and HuCCT1^(IDH1-/-)cells were analyzed by bioinformatics methods,Western blotting was used to verify the expression of signaling pathway-related proteins.Results Compared with HuCCT1^(WT) cells,HuCCT1^(IDH1-/-)cells showed the number of proliferation and clone formation significantly reduced(P<0.05),the proportion of cells blocked in G_(2)/M phase was significantly increased(P<0.01),the rate of scratch healing was significantly decreased(P<0.01),and the number of migrated cells(P<0.001)and invaded cells(P<0.05)was significantly reduced.qRT-PCR assay showed that the expression levels of IDH1,Vimentin,MMP-9 and genes related to the regulation of G_(2)/M cycle proliferation,Cyclin A2,Cyclin B1 and CDK1 mRNA were down-regulated in HuCCT1^(IDH1-/-)cells(P<0.05),and the expression of CDH1 mRNA encoding E-cadherin was up-regulated(P<0.01);Western blotting assay showed that the expression level of E-cadherin in HuCCT1^(IDH1-/-)cells was significantly increased(P<0.05),and the expression level of N-cadherin,Vimentin and MMP-9 protein was significantly decreased(P<0.05)than that in HuCCT1^(WT) cells.Data of transcriptome sequencing revealed 1476 differentially expressed genes(DEGs)between two groups of HuCCT1 cells.Go enrichment analysis showed the DEGs were signific

关 键 词：肝内胆管癌 异柠檬酸脱氢酶1 细胞迁移 细胞侵袭 转录组 

分 类 号：R735.8[医药卫生—肿瘤]