磷酸果糖激酶-2/果糖-2,6-二磷酸酶3对肝细胞癌细胞生物学行为的影响  

Influence of PFKFB3 on the biological behaviors of hepatocellular carcinoma cells

作　　者：师文楷[1] 张予川 赵永福[1] SHI Wen-kai;ZHANG Yu-chuan;ZHAO Yong-fu(Department of hepatopancreatobiliary surgery,the first affiliated hospital of zhengzhou university,zhengzhou,henan 450052,china;Department of ophthalmology,henan provincial people's hospital,zhengzhou university people's hospital,zhengzhou,henan 450003,china)

机构地区：[1]郑州大学第一附属医院肝胆胰外科,河南郑州450052 [2]河南省人民医院郑州大学人民医院眼科,河南郑州450003

出　　处：《中华实用诊断与治疗杂志》2022年第8期769-773,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20190041);河南省自然科学基金(222300420340)。

摘　　要：目的探讨磷酸果糖激酶-2/果糖-2,6-二磷酸酶3(phosphofructokinase-2/fructose-2,6-bisphosphatase 3,PFKFB3)表达对肝细胞癌细胞增殖、侵袭和迁移的影响及调控机制。方法对数生长期SMMC-7721细胞分为PFKFB3敲低组(转染shRNA-PFKFB3慢病毒)和对照组(转染shRNA-NC慢病毒)。转染后采用实时荧光定量PCR法检测细胞PFKFB3 mRNA相对表达量,采用Western blot法检测细胞PFKFB3蛋白相对表达量,采用CCK-8实验检测0、24、48、72 h细胞增殖吸光度(optical density,OD)值,采用Transwell小室实验检测侵袭细胞数和迁移细胞数;采用基因芯片技术检测并分析2组细胞基因表达谱差异,GO和KEGG富集分析差异表达基因参与的生物学过程及信号通路;采用实时荧光定量PCR法检测受PFKFB3敲低影响较大的FoxO信号通路相关基因FoxO1、FoxO4、CCNB1、CCND1、CDKN1B、BCL6、GADD45A mRNA相对表达量,筛选出表达差异最明显的基因FoxO4,采用免疫共沉淀法验证PFKFB3与FoxO4的相互作用。结果PFKFB3敲低组PFKFB3 mRNA(0.23±0.02)及蛋白(0.40±0.03)相对表达量均低于对照组(1.01±0.07、1.12±0.06)(t=34.862,P<0.001;t=18.591,P<0.001)。PFKFB3敲低组转染后培养24、48、72 h时细胞增殖OD值(1.11±0.09、1.74±0.03、3.05±0.09)均低于对照组(1.55±0.02、2.44±0.06、4.00±0.00)(P<0.05),0 h时细胞增殖OD值与对照组比较差异无统计学意义(P>0.05)。PFKFB3敲低组侵袭细胞数[(56.00±6.08)个]、迁移细胞数[(120.67±7.57)个]均少于对照组[(432.00±11.00)、(474.67±6.11)个](t=51.811,P<0.001;t=63.108,P<0.001)。2组共检测出2178个差异表达基因(1473个上调基因和705个下调基因),差异表达基因主要参与细胞周期、DNA转录调控的生物学过程,FoxO信号通路受PFKFB3敲低的影响较大。PFKFB3敲低组细胞FoxO1、FoxO4、GADD45A mRNA相对表达量均高于对照组(P<0.05),CCNB1、CCND1、CDKN1B、BCL6 mRNA相对表达量均低于对照组(P<0.05),其中FoxO4表达与对照组差异最明显。Objective To investigate the influence of phosphofructokinase-2/fructose-2,6-bisphosphatase 3(PFKFB3)on the proliferation,invasion and migration of hepatocellular carcinoma,and its regulation mechanism.Methods The SMMC-7721 cells in logarithmic growth phase were divided into PFKFB3 knockdown group(transfected with shPFKFB3 lentivirus)and control group(transfected with SHRNA-NC lentivirus).The relative expressions of PFKFB3 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The optical density(OD)values were detected by CCK-8 assay at 0,24,48 and 72 h after transfection,and numbers of invaded and migrated cells were detected by Transwell assay.Gene microarray technology was used to detect and analyze the differences in gene expression profiles between two groups.GO and KEGG enrichment was used to analyze the biological processes and signaling pathways involved in the differentially expressed genes.The relative expressions of FoxO signaling pathway related genes greatly affected by PFKFB3 knockdown as FoxO1,FoxO4,CCNB1,CCND1,CDKN1 B,BCL6 and GADD45 A mRNAs were detected by real-time fluorescence quantitative PCR,and FoxO4 gene with the most significant expression difference was screened out.The interaction between PFKFB3and FoxO4was verified by immunoprecipitation method.Results The relative expressions of PFKFB3 mRNA and protein were lower in PFKFB3knockdown group(0.23±0.02,0.40±0.03)than those in control group(1.01±0.07,1.12±0.06)(t=34.862,P<0.001;t=18.591,P<0.001).The OD values at 24,48and 72hafter transfection were lower in PFKFB3knockdown group(1.11±0.09,1.74±0.03,3.05±0.09)than those in control group(1.55±0.02,2.44±0.06,4.00±0.00)(P<0.05),and there was no significant difference in OD value at 0hbetween two groups(P>0.05).The numbers of invaded and migrated cells were smaller in PFKFB3 knockdown group(56.00±6.08,120.67±7.57)than those in control group(432.00±11.00,474.67±6.11)(t=51.811,P<0.001;t=63.108,P<0.001).Totally 2178differentially expres

关 键 词：肝细胞癌 磷酸果糖激酶-2/果糖-2 6-二磷酸酶3 FoxO4 增殖 侵袭 迁移 SMMC-7721细胞 

分 类 号：R735.7[医药卫生—肿瘤]