RRM2通过调控CDK1对胃癌细胞恶性生物学行为及有氧糖酵解的影响  

Effects of RRM2 on malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating CDK1

作　　者：谭荣坚 欧雯婷 翟嘉伟 全祯豪 孙利君 周才进 Tan Rongjian;Ou Wenting;Zhai Jiawei;Quan Zhenhao;Sun Lijun;Zhou Caijin(Department of Gastrointestinal Surgery,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524000,China;Department of Digestive Oncology,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524000,China)

机构地区：[1]广东医科大学附属医院胃肠外科,湛江524000 [2]广东医科大学附属医院消化肿瘤内科,湛江524000

出　　处：《国际肿瘤学杂志》2025年第1期23-30,共8页Journal of International Oncology

基　　金：湛江市科技发展专项资金竞争性分配项目(2022A01174);湛江市非资助科技攻关计划(2022B01096)。

摘　　要：目的探究核糖核苷酸还原酶调节亚基M2(RRM2)通过调控周期蛋白依赖性激酶(CDK)1对胃癌细胞恶性生物学行为及有氧糖酵解的影响。方法将人胃癌MKN-45细胞分为si-NC组(转染空白片段)、CoCl_(2)+si-NC组(低氧环境转染空白片段)、CoCl_(2)+si-RRM2组(低氧环境沉默RRM2)、CoCl_(2)+si-RRM2+pcDNA3.1 NC组(低氧环境沉默RRM2+空白载体)、CoCl_(2)+si-RRM2+pcDNA3.1 CDK1组(低氧环境沉默RRM2+过表达CDK1)。实时荧光定量反转录PCR分析RRM2和CDK1 mRNA的相对表达量;免疫共沉淀分析RRM2和CDK1蛋白间的相互作用;MTT法检测细胞增殖活性;细胞划痕实验检测细胞迁移距离;流式细胞术检测细胞凋亡水平;三磷酸腺苷(ATP)和葡萄糖试剂盒检测ATP产生和葡萄糖消耗情况;蛋白质印迹法检测ENO1、RRM2、HK2、PKM2、GLUT1及p-CDK1/CDK1蛋白表达情况。结果实时荧光定量反转录PCR结果显示,si-NC组、CoCl_(2)+si-NC组和CoCl_(2)+si-RRM2组CDK1 mRNA相对表达量分别为1.01±0.15、1.30±0.06、0.51±0.18,RRM2 mRNA相对表达量分别为1.03±0.32、1.59±0.28、0.44±0.17,差异均有统计学意义(F=25.52,P=0.001;F=14.47,P=0.005);与si-NC组比较,CoCl_(2)+si-NC组细胞RRM2和CDK1 mRNA表达均较高;与si-NC组、CoCl_(2)+si-NC组比较,CoCl_(2)+si-RRM2组细胞RRM2和CDK1 mRNA表达均较低(均P<0.05)。免疫共沉淀结果显示,RRM2与CDK1之间存在相互作用。MTT法、细胞划痕实验和流式细胞术结果显示,si-NC组、CoCl_(2)+si-NC组、CoCl_(2)+si-RRM2组、CoCl_(2)+si-RRM2+pcDNA3.1 NC组和CoCl_(2)+si-RRM2+pcDNA3.1 CDK1组细胞增殖活性分别为1.04±0.01、1.18±0.04、0.84±0.03、0.81±0.03、0.93±0.05,细胞迁移距离分别为(301.83±2.75)、(369.67±0.76)、(176.50±6.38)、(175.83±3.69)、(254.17±1.61)μm,细胞凋亡率分别为8.05%±0.21%、5.75%±0.20%、28.28%±0.04%、30.18%±1.51%、17.79%±0.22%,差异均有统计学意义(F=73.82,P<0.001;F=1600.01,P<0.001;F=787.15,P<0.001);与si-NC组和CoCl_(2)+si-NC组比较,CoCl_(2)+siObjective To investigate the effect of ribonucleotide reductase regulatory subunit M2(RRM2)on the malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating cyclin-dependent kinase(CDK)1.Methods Human gastric cancer MKN-45 cells were divided into si-NC group(transfected with blank fragment),CoCl_(2)+si-NC group(hypoxia control transfected with blank fragment),CoCl_(2)+si-RRM2 group(hypoxia with RRM2 silencing),CoCl_(2)+si-RRM2+pcDNA3.1 NC group(hypoxia with RRM2 silencing and blank vector)and CoCl_(2)+si-RRM2+pcDNA3.1 CDK1 group(hypoxia with RRM2 silencing and CDK1 overexpression).The mRNA relative expression levels of RRM2 and CDK1 were analyzed by real time fluorescent quantitative reverse transcription PCR.Co-immunoprecipitation(CoIP)was used to analyze the interaction between RRM2 and CDK1 protein.MTT assay was used to analyze the proliferation activity of cells.The cell migration distance was detected by cell scratch assay.Cell apoptosis was detected by flow cytometry.Adenosine triphosphate(ATP)and glucose kit were used to detect ATP production and glucose consumption.The protein expressions of ENO1,RRM2,HK2,PKM2,GLUT1 and p-CDK1/CDK1 were detected by Western blotting.Results Real time fluorescent quantitative reverse transcription PCR results showed that the relative expression levels of CDK1 mRNA in si-NC group,CoCl_(2)+si-NC group and CoCl_(2)+si-RRM2 group were 1.01±0.15,1.30±0.06 and 0.51±0.18,and the relative expression levels of RRM2 mRNA were 1.03±0.32,1.59±0.28 and 0.44±0.17,respectively,and there were statistically significant differences(F=25.52,P=0.001;F=14.47,P=0.005).The mRNA expressions of RRM2 and CDK1 in CoCl_(2)+si-NC group were higher than those in si-NC group.Compared with the si-NC group and the CoCl_(2)+si-NC group,the mRNA expressions of RRM2 and CDK1 were lower in the CoCl_(2)+si-RRM2 group(all P<0.05).CoIP results showed that there was interaction between RRM2 and CDK1.MTT assay,cell scratch assay and flow cytometry showed that the cell prolifera

关 键 词：胃肿瘤 核苷酸还原酶类 细胞周期蛋白质依赖性激酶类 

分 类 号：R73[医药卫生—肿瘤]