P16INK4A调控Wnt/β-catenin信号通路对结肠腺癌细胞生物学行为的影响  

Effect of P16~(INK4A)regulating Wnt/β-catenin signaling pathway on biological behaviours of colon adenocarcinoma cells

作　　者：郭伟 魏亚哲 李鑫 GUO Wei;WEI Yazhe;LI Xin(Department of General Surgery,the First Affiliated Hospital of Henan University,Kaifeng,Henan 475000,China)

机构地区：[1]河南大学第一附属医院普通外科,河南开封475000

出　　处：《中华实用诊断与治疗杂志》2024年第10期1006-1012,共7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(LHGJ20220649)。

摘　　要：目的观察结肠腺癌组织和HT29细胞P16^(INK4A)表达变化,探讨上调P16^(INK4A)表达对HT29细胞Wnt/β-catenin信号通路及生物学行为的影响。方法2023年1-12月河南大学第一附属医院诊治结肠腺癌患者23例,收集手术切除的结肠腺癌组织和癌旁组织标本,采用实时荧光定量PCR法检测P16^(INK4A)mRNA相对表达量。取对数生长期HT29细胞,分为对照组(正常培养)、pcDNA3.0组(转染pcDNA3.0质粒)、pcDNA3.0-P16^(INK4A)组(转染pcDNA3.0-P16^(INK4A)质粒),转染后培养48 h取3组细胞,采用CCK-8法检测细胞增殖吸光度(OD)值,采用流式细胞术检测细胞凋亡率,采用细胞划痕实验检测细胞迁移率,采用Transwell小室实验检测侵袭细胞数,采用实时荧光定量PCR法检测P16^(INK4A)、β-catenin、c-Myc、细胞周期蛋白D1(cyclin D1)、T细胞因子-4(TCF-4)mRNA相对表达量,采用Western blot法检测P16^(INK4A)、β-catenin、c-Myc、cyclin D1、TCF-4蛋白相对表达量。结果结肠腺癌组织P16^(INK4A)mRNA相对表达量(0.53±0.08)低于癌旁组织(1.00±0.10)(t=17.604,P<0.001)。转染后培养48 h,3组细胞增殖OD值、细胞凋亡率、细胞迁移率、侵袭细胞数比较差异均有统计学意义(F=39.000、241.449、44.816、24.052,P均<0.05);pcDNA3.0-P16^(INK4A)组细胞增殖OD值(0.47±0.05)、细胞迁移率[(21.38±2.85)%]均低于pcDNA3.0组[0.85±0.10、(40.79±4.53)%]、对照组[0.82±0.09、(41.85±4.97)%](P<0.05),细胞凋亡率[(35.28±3.74)%]均高于pcDNA3.0组[(10.09±1.13)%]、对照组[(9.26±1.02)%](P<0.05),侵袭细胞数[(102.33±12.27)个]均少于pcDNA3.0组[(158.33±16.45)个]、对照组[(155.33±17.93)个](P<0.05)。3组细胞P16^(INK4A)、β-catenin、c-Myc、cyclin D1、TCF-4 mRNA及蛋白相对表达量比较差异均有统计学意义(F=31.484~83.046,P均<0.05);pcDNA3.0-P16^(INK4A)组P16^(INK4A)mRNA及蛋白相对表达量均高于pcDNA3.0组、对照组(P<0.05),β-catenin、c-Myc、cyclin D1、TCF-4 mRNA及蛋白相对表达量均低于pcDNA3.0组、对Objective To observe the changes of P16^(INK4A)expression in colon adenocarcinoma tissues and HT29 cells,and to explore the effects of upregulating P16^(INK4A)expression on the Wnt/β-catenin signaling pathway and the biological behaviours of HT29 cells.Methods The samples of surgically resected colon adenocarcinoma tissues and paracancerous tissues were harvested from 23 colon adenocarcinoma patients in the First Affiliated Hospital of Henan University from January to December 2023.The relative expression of P16^(INK4A)mRNA was detected by real-time fluorescence quantitative PCR.The HT29 cells in logarithmic growth phase were divided into control group(normal culture),pcDNA3.0 group(transfected with pcDNA3.0 plasrid),and pcDNA3.0-P16^(INK4A)group(transfected with pcDNA3.0-P16^(INK4A)plasmid).After 48-h culture,the optical density(OD)value of cell proliferation in three groups was detected by CCK-8 assay,the apoptosis rate was detected by flow cytometry,the migration rate was detected by cell scratch assay,and the number of invasive cells was detected by Trans well chamber assay.The relative expressions of P16^(INK4A),β-catenin,c-Myc,cyclin D1 and T cell factor-4(TCF-4)mRNAs were detected by real-time fluorescence quantitative PCR,and the relative expressions of P16^(INK4A),β-catenin,c-Myc,cyclin D1 and TCF-4 proteins were detected by Western blot.Results The relative expression of P16^(INK4A)mRNA was lower in colon adenocarcinoma tissues(0.53±0.08)than that in paracancerous tissues(1.00±0.10)(t=17.604,P<0.001).After 48-h culture,the OD value,apoptosis rate,migration rate,and the number of invasive cells showed significant differences among three groups(F=39,000,241.449,44.816,24.052;all P values<0.05),the OD value and migration rate were lower in pcDNA3.0-P16^(INK4A)group[0.47±0.05,(21.38±2.85)%]than those in pcDNA3.0 group[0.85±0.10,(40.79±4.53)%]and control group[0.82±0.09,(41.85±4.97)%](P<0.05),the apoptosis rate was higher in pcDNA3.0-P16^(INK4A)group[(35.28±3.74)%]than that in pcDNA3.0 group[(10.

关 键 词：结肠腺癌 HT29细胞 P16INK4A WNT/Β-CATENIN信号通路 

分 类 号：R735.35[医药卫生—肿瘤]