摘要
目的探讨IQ结构域GTP酶激活蛋白3(IQ domain GTPase-activating protein 3, IQGAP3)对人肝癌HepG2细胞顺铂耐药的影响及作用机制。方法冻存HepG2细胞株,其中对照组细胞正常培养,IQGAP3组细胞转染IQGAP3,shIQGAP3组细胞转染shIQGAP3,采用Western blot法检测3组IQGAP3蛋白相对表达量。3组细胞均加入顺铂进行培养,采用MTT法检测培养第1-5天细胞存活率,采用实时荧光定量PCR法检测细胞Nanog mRNA、Oct4 mRNA、Bmi1 mRNA、c-Myc mRNA、cyclinD1 mRNA相对表达量,基因集合富集分析GO、KEGG数据库肝细胞癌样本数据中IQGAP3表达与Wnt/β-catenin通路的关系,采用Top/Fop双荧光素酶报告基因实验检测细胞β-catenin激活程度(Top/Fop比值),采用Western blot法检测细胞β-catenin核浆比例。shIQGAP3组细胞再分为shIQGAP3-氯化锂(lithium chloride, LiCl)组和shIQGAP3对照组,其中shIQGAP3对照组加入顺铂进行培养,shIQGAP3-LiCl组加入顺铂+LiCl进行培养,采用MTT法检测2组培养第1-5天细胞存活率,采用Top/Fop双荧光素酶报告基因实验检测细胞β-catenin激活程度(Top/Fop比值)。结果 IQGAP3组细胞IQGAP3蛋白相对表达量(9.13±0.42)均高于对照组(1.08±0.25)和shIQGAP3组(0.42±0.07)(P<0.05),对照组高于shIQGAP3组(P<0.05)。培养第3-5天,IQGAP3组细胞存活率均高于对照组和shIQGAP3组(P<0.05),对照组高于shIQGAP3组(P<0.05);培养第1-5天,IQGAP3组、shIQGAP3组、对照组细胞存活率均依次降低(P<0.05)。IQGAP3组细胞Nanog mRNA、Oct4 mRNA、Bmi1 mRNA、c-Myc mRNA、cyclinD1 mRNA相对表达量均高于对照组和shIQGAP3组(P<0.05),对照组高于shIQGAP3组(P<0.05)。GO、KEGG数据库肝细胞癌样本数据分析结果显示,活化的Wnt/β-catenin信号通路在IQGAP3表达上调的样本中富集(富集分数=0.497,P<0.001;富集分数=0.527,P<0.001);IQGAP3组细胞β-catenin核浆比例(3.61±0.24)和Top/Fop比值(2.61±0.29)均高于对照组(0.98±0.14、1.13±0.08)和shIQGAP3组(0.25±0.03、0.57±0.13)(P<0.05),对照组高于shIQGAP3组(P<0.05)。shIQGAP3-LiCl组细胞Top/Fop比值(3.18±0.33)高于shIQGAP3对照组(1.02±0.06)(P<0.05);培养第4-5天,shIQGAP3-LiCl组细胞存活率高于shIQGAP3对照组(P<0.05);培养第0-5天,shIQGAP3-LiCl组、shIQGAP3对照组细胞存活率均依次降低(P<0.05)。结论 IQGAP3可能通过激活Wnt/β-catenin通路增加肿瘤干性基因表达,促进HepG2细胞对顺铂耐药。
Objective To investigate the effect of IQ domain GTPase-activating protein 3(IQGAP3)on cisplatin resistance in HepG2 cells and its mechanism.Methods The cryopreserved human HepG2 cells were divided into control group(normal cells),IQGAP3 group(transfected with IQGAP3)and shIQGAP3 group(transfected with shIQGAP3).The relative expression of IQGAP3 was detected by Western blot in three groups.The cells in three groups were cultured with cisplatin,and the survival rate by day 1 to 5 was detected by MTT assay.The mRNA relative expressions of Nanog,Oct4,Bmi1,c-Myc and cyclinD1 were detected by real-time fluorescence quantitative PCR.Gene set enrichment analysis software was used to analyze the correlation between IQGAP3 expression and Wnt/β-catenin pathway in hepatocellular carcinoma database of GO and KEGG database.The level ofβ-catenin activation(Top/Fop ratio)was detected by Top/Fop double luciferase assay.The nucleoplasma ratio ofβ-catenin was determined by Western blot.The cells in shIQGAP3 group were further divided into shIQGAP3-lithium chloride(LiCl)group and shIQGAP3 control group.shIQGAP3 control group was cultured with cisplatin,and shIQGAP3-LiCl group was cultured with cisplatin and LiCl.MTT assay was used to detect the survival rate of cells cultured for 1 to 5 days in two groups.Top/Fop double luciferase assay was used to detect the level ofβ-catenin activation(Top/Fop ratio).Results The relative expression of IQGAP3 protein was higher in IQGAP3 group(9.13±0.42)than that in control group(1.08±0.25)and shIQGAP3 group(0.42±0.07)(P<0.05),and higher in control group than that in shIQGAP3 group(P<0.05).By day 3 to 5 of culture,the survival rate was higher in IQGAP3 group than that in control group and shIQGAP3 group(P<0.05),and higher in control group than that in shIQGAP3 group(P<0.05).By day 1 to 5 of culture,the survival rate decreased gradually in turn in IQGAP3,shIQGAP3 and control groups(P<0.05).The mRNA relative expressions of Nanog,Oct4,Bmi1,c-Myc and cyclinD1 were higher in IQGAP3 group than those in control group and shIQGAP3 group(P<0.05),and higher in control group than those in shIQGAP3 group(P<0.05).The hepatocellular carcinoma samples from GO and KEGG database showed that the activated Wnt/β-catenin pathway was enriched in the samples with high IQGAP3 expression(enrichment score=0.497,P<0.001;enrichment score=0.527,P<0.001).The nucleoplasma ratio ofβ-catenin and Top/Fop ratio were higher in IQGAP3 group(3.61±0.24,2.61±0.29)than those in control group(0.98±0.14,1.13±0.08)and shIQGAP3 group(0.25±0.03,0.57±0.13)(P<0.05),and higher in control group than those in shIQGAP3 group(P<0.05).The Top/Fop ratio was higher in shIQGAP3-LiCl group(3.18±0.33)than that in shIQGAP3 control group(1.02±0.06)(P<0.05).By day 4 to 5 of culture,the cell survival rate was higher in shIQGAP3-LiCl group than that in shIQGAP3 control group(P<0.05).By day 0 to 5 of culture,the cell survival rate decreased gradually in shIQGAP3-LiCl and shIQGAP3 control groups(P<0.05).Conclusion IQGAP3 may promote cisplatin resistance of HepG2 cells by increasing tumor stemness genes expression through Wnt/β-catenin pathway activation.
作者
黄思聪
石永杰
周强
嘉红云
申刚
HUANG Si-cong;SHI Yong-jie;ZHOU Qiang;JIA Hong-yun;SHEN Gang(Department of Clinical Laboratory,the Second Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong 510260,China;Department of Hematiffioma Interventional Therapy,Children's Hospital of Capital Institute of Pediatrics,Beijing 100020,China)
出处
《中华实用诊断与治疗杂志》
2021年第7期666-671,共6页
Journal of Chinese Practical Diagnosis and Therapy
基金
广东省医学科学技术研究基金项目(A2019189)
广州市卫生健康科技项目(20191A010057,20211A011079)。
