CCAAT增强子结合蛋白β对胃癌细胞功能的影响  

Effect of CCAAT enhancer-binding proteinβon function of gastric cancer cells

作　　者：钟海均[1] 吕汪霞[1] 凌哲南 凌志强[3] Zhong Haijun;Lyu Wangxia;Ling Zhenan;Ling Zhiqiang(Department of Oncology,Zhejiang Cancer Hospital,Hangzhou 310022,China;Department of General Surgery,First Affiliated Hospital of Zhejiang University,Hangzhou 310000,China;Department of Molecular Biology,Zhejiang Cancer Institute,Zhejiang Cancer Hospital,Hangzhou 310022,China)

机构地区：[1]浙江省肿瘤医院肿瘤内科,杭州310022 [2]浙江大学附属第一医院普外科,杭州310000 [3]浙江省肿瘤医院浙江省肿瘤研究所分子生物学实验室,杭州310022

出　　处：《中华实验外科杂志》2025年第1期65-69,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(32271238);国家卫生健康委科学研究基金-浙江省卫生健康重大科技计划重点项目(WKJ-ZJ-2117);浙江省自然科学基金面上项目(LY20H160005)。

摘　　要：目的探讨CCAAT增强子结合蛋白β(C/EBPβ)对胃癌细胞生物学行为的影响及其机制。方法人胃癌细胞系AGS购自中国科学院上海细胞库,实验分为AGS-C/EBPβshRNA组与AGS-Vector对照组:AGS-C/EBPβshRNA组采用小发夹状RNA技术干扰AGS细胞中C/EBPβ表达,AGS-Vector对照组转染空白对照病毒;通过实时荧光反转录聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测两组细胞中C/EBPβmRNA和蛋白表达以检查AGS-C/EBPβshRNA组中C/EBPβ敲减效果,采用细胞计数试剂盒(CCK-8)和Transwell侵袭实验分别检测两组细胞中细胞增殖和侵袭能力,采用流式细胞术分析两组细胞中细胞周期分布情况,采用Western blot检测两组细胞中细胞核增殖抗原(Ki-67)和TET2蛋白表达水平。组间比较采用单因素方差分析(One-way ANOVA)。结果AGS-C/EBPβshRNA组中C/EBPβmRNA和蛋白表达水平均显著低于AGS-Vector对照组(C/EBPβmRNA:0.210±0.038比1.120±0.083,t=14.817,P<0.01;C/EBPβ蛋白:0.120±0.016比0.890±0.027,t=8.136,P<0.01)。CCK-8实验结果显示,在培养24、48、72 h后,AGS-C/EBPβshRNA组细胞的增殖水平(0.391±0.023、0.618±0.038、0.886±0.049)显著低于AGS-Vector对照组(0.443±0.035、0.691±0.045、1.138±0.068),差异均有统计学意义(t=4.974、5.905、8.126,均P<0.01);Transwell侵袭实验结果显示,AGS-C/EBPβshRNA组侵袭穿过Transwell小室膜的细胞数[(486.0±30.5)个]明显低于AGS-Vector对照组[(1144.0±50.2)个],差异有统计学意义(t=13.836,P<0.01);细胞周期分析显示,AGS-C/EBPβshRNA组细胞周期阻滞于S期,其S期占51.12%,AGS-Vector对照组S期占29.58%(t=4.715,P<0.01);Western blot检测显示,与AGS-Vector对照组比较,AGS-C/EBPβshRNA组细胞中Ki-67蛋白表达显著减少(0.310±0.020比0.910±0.033,t=5.572,P<0.01)、TET2蛋白表达显著增加(0.810±0.021比0.170±0.016,t=12.338,P<0.01)。结论体外敲减C/EBPβ表达可抑制胃癌细胞增殖和侵袭能力,其机制可能是通过调控TET2通路来实现的Objective To investigate the influence of CCAAT enhancer-binding proteinβ(C/EBPβ)on the biological behavior of gastric cancer cells.Methods The human gastric cancer cell line AGS was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.The experiment was divided into C/EBPβknockout group and control group:AGS-C/EBPβshRNA group was given small-hairpin RNA(shRNA)technique to interfere with C/EBPβexpression in AGS cells;the AGS-Vector control group was transfected with the blank control virus.The C/EBPβmRNA and protein expression was detected by real-time fluorescence reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting respectively to check the effect of C/EBPβknockdown in the AGS-C/EBPβshRNA group.The cell proliferation and invasion capacity was measured by the cell counting kit-8(CCK-8)and the Transwell invasion assays respectively.The cell cycle distribution was analyzed in both cell groups by flow cytometry.The protein expression levels of proliferation cell nuclear antigen(Ki-67)and TET 2 in both cell groups were determined by Western blotting.One-way ANOVA was used for comparison between groups.Results The expression levels of C/EBPβmRNA and protein were significantly lower in AGS-C/EBPβshRNA group than in AGS-Vector control group(C/EBPβmRNA:0.210±0.038 vs.1.120±0.083,t=14.817,P<0.01;C/EBPβprotein:0.120±0.016 vs.0.890±0.027,t=8.136,P<0.01).The results of the CCK-8 experiment showed,after 24,48,and 72 h of culture,the proliferation level of cells in the AGS-C/EBPβshRNA group(0.391±0.023,0.618±0.038,0.886±0.049)were significantly lower than those in the AGS-Vector control group(0.443±0.035,0.691±0.045,1.138±0.068)(t=4.974,5.905,and 8.126,respectively,all cases:P<0.01).The results of the Transwell-invasion assay showed that,the number of cells[(486.0±30.5)cells]in the AGS-C/EBPβshRNA group was significantly less than that in the AGS-Vector control group[(1144.0±50.2)cells,t=13.836,P<0.01].Cell cycle analysis revealed the cell cycle arrest in S

关 键 词：CCAAT增强子结合蛋白β 增殖 侵袭 细胞周期 

分 类 号：R735.2[医药卫生—肿瘤]