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/β-catenin信号通路有关。
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 significantly enriched in cell biological processes associated with inflammatory response,cell signaling and cell metabolism.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis suggested that the DEGs may be involved in some signaling pathways such as Wnt,MAPK,Rap1,Hippo and TNF,which are closely related to the regulation of proliferation and invasion of tumor cells.Western blotting verification results showed that compared with HuCCT1^(WT) cells,the relative expression of Wnt3a andβ‐catenin proteins of HuCCT1^(IDH1-/-)cells was significantly decreased(P<0.05).Conclusions IDH1 gene may participate in the control of biological functions of HuCCT1 cells,including cell proliferation,migration,invasion and epithelial mesenchymal transition.The mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.
作者
林美佳
雷宇清
叶洲杰
朱丽萍
王心睿
黄雄飞
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)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
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)。
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
intrahepatic cholangiocarcinoma
isocitrate dehydrogenase 1
cell migration
cell invasion
transcriptome sequencing
