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

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。PFKFB3与FoxO4存在相互作用。结论下调PFKFB3表达可抑制肝细胞癌细胞增殖、侵袭和迁移,可能与PFKFB3调控FoxO4信号通路有关。

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 expressed genes including 1473up-regulated genes and 705down-regulated genes in two group were mainly involved in the biological process of cell cycle and DNA transcription regulation,and FoxO signaling pathway was significantly affected by PFKFB3knockdown.The relative expressions of FoxO1,FoxO4and GADD45A mRNAs were higher in PFKFB3knockdown group than those in control group(P<0.05),and the relative expressions of CCNB1,CCND1,CDKN1Band BCL6mRNAs were lower in PFKFB3knockdown group than those in control group(P<0.05).FoxO4expression showed the most significant difference among FoxO signaling pathway related genes between two groups.PFKFB3and FoxO4were interacted.Conclusion Down-regulating PFKFB3can suppress the proliferation,invasion and migration of SMMC-7721 cells,probably by regulating FoxO4 signaling pathway.