鼻咽癌CNE2细胞生物钟基因Per1表达及其对生物学行为影响的研究  

Expression of circadian clock gene Per1in nasopharyngeal carcinoma CNE2cell and its effect on biological behavior

作　　者：周湾 罗盼 曾艳 陈宇 唐红 龙金华 罗秀玲 金风 吴伟莉 ZHOU Wan;LUO Pan;ZENG Yan;CHEN Yu;TANG Hong;LONG Jin-hua;LUO Xiu-ling;JIN Feng;WU Wei-li(Department of Oncology,Af filiated Tumor Hospital of Guizhou Medical University,Guiyang 550004,China;School of Clinical Medicine,Guizhou Medical University,Guiyang 550004,China;Department of Oncology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学附属肿瘤医院肿瘤科,贵州贵阳550004 [2]贵州医科大学临床医学院,贵州贵阳550004 [3]贵州医科大学附属医院肿瘤科,贵州贵阳550004

出　　处：《中华肿瘤防治杂志》2022年第11期800-808,共9页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(82060555);贵州医科大学国家自然科学基金培育项目(19NSP047);贵州省肿瘤医院院级科技计划(YJ2019)。

摘　　要：目的 研究Per1对鼻咽癌CNE2细胞增殖、凋亡、侵袭及细胞周期分布的影响,初步探讨Per1在鼻咽癌中可能扮演的角色。方法 通过实时荧光定量PCR法及蛋白质印迹法检测低分化鼻咽癌细胞CNE2及正常鼻咽上皮细胞NP69中核心生物钟基因Per1 mRNA及蛋白的表达;采用基因过表达及干扰技术将实验分为过表达组(Per1-oe)、过表达对照组(Per1-oeNC)、干扰组(Per1-shRNA-646)、干扰对照组(CNE2)和空载体组(control-shRNA);采用CCK8测定Per1-shRNA、control-shRNA和CNE2组细胞增殖能力;采用流式细胞仪分析Per1对CNE2细胞周期分布的影响;采用Transwell小室及细胞划痕实验验证过表达和低表达各组细胞的迁移及侵袭能力。结果 CNE2细胞Per1 mRNA表达量为0.002±0.001,低于NP69细胞的1.000±0.001,t=4771.56,P<0.05;CNE2细胞Per1蛋白表达为0.60±0.15,低于NP69细胞的0.90±0.26,t=4.267,P<0.05。流式细胞分析显示,CNE2、control-shRNA和Per1-shRNA组S期比例分别为(36.54±0.19)%、(36.18±0.69)%和(44.31±0.36)%(F=323.423,P<0.001),G期比例分别为(52.88±1.27)%、(53.72±0.51)%和(37.68±0.62)%(F=55.356,P<0.001),G/M期比例分别为(10.58±1.21)%、(10.10±0.88)%和(18.00±0.98)%,F=296.47,P<0.001。CCK8实验检测0~5 d的光密度值,CNE2、control-shRNA和Per1-shRNA组差异有统计学意义,F=10.63,P<0.001;两两比较显示,Per1-shRNA组高于CNE2和control-shRNA组,均P<0.05。细胞划痕实验显示,CNE2、control-shRNA和Per1-shRNA组24 h迁移率分别为(15.02±0.57)%、(14.46±1.08)%和(21.55±0.52)%,F=66.241,P<0.001;干扰Per1使细胞迁移率增高,Per1-oe组24 h迁移率(7.00±1.59)%低于Per1-oeNC组(14.00±2.36)%(t=3.394,P<0.05),Per1-oe组48 h迁移率(18.00±2.01)%低于Per1-oeNC组(25.00±3.04)%,t=4.292,P<0.05。Transwell实验显示,CNE-2、control-shRNA和Per1-shRNA组穿膜细胞数分别为64.00±1.73、62.00±2.08和90.00±1.00,F=256.447,P<0.001;Per1-oe组细胞数(109.00±3.10)低于Per1-oeNC组(168.00±2.00),t=6.804,P<0.05。结论 核心�Objective To study the effect of Per1on the proliferation,apoptosis,invasion and cell cycle distribution of nasopharyngeal carcinoma CNE2cells,and to preliminarily explore the possible role of Per1in nasopharyngeal carcinoma.Methods The expression of core biological clock gene Per1mRNA and protein in poorly differentiated nasopharyngeal carcinoma cells CNE2and normal nasopharyngeal epithelial cells NP69were detected by real-time quantitative PCR and Western blotting.The experiments were divided into overexpression groups by gene overexpression and interference techniques(Per1-oe),overexpression control group(Per1-oeNC),low expression group Per1-shRNA-646,low expression control group(CNE2)and empty vector group(control-shRNA).CCK8was used to determine Per1-shRNA,control-shRNA and cell proliferation ability of CNE2group.Flow cytometry was used to analyze the effect of Per1on CNE2cell cycle distribution in CNE2,Per1-shRNA and control-shRNA groups.Transwell chamber and cell scratch assay were used to verify the overexpression and low expression of cells in each group migration and invasion ability.Results Per1mRNA expression in CNE2cells(0.002±0.001)was lower than that in NP69cells(1.000±0.001),t=4771.56,P<0.05;Per1protein expression in CNE2group(0.60±0.15)was lower than that in NP69cells(0.90±0.26),t=4.267,P<0.05.Flow cytometry analysis of CNE2,control-shRNA,Per1-shRNA group cell cycle distribution:the proportions of S phase ratios were(36.54±0.19)%,(36.18±0.69)%,(44.31±0.36)%,F=323.423,P<0.001;Gphase were(52.88±1.27)%,(53.72±0.51)%,(37.68±0.62)%,F=55.356,P<0.001;G/M phase were(10.58±1.21)%,(10.10±0.88)%,(18.00±0.98)%,F=296.47,P<0.001.The optical density values of CNE2,control-shRNA and Per1-shRNA groups on0-5days were detected by CCK8experiment,and there were differences among CNE2,control-shRNA and Per1-shRNA groups(F=10.63,P<0.001);pairwise comparison showed that,Per1-shRNA group was higher than CNE2and controlshRNA group,the difference was statistically significant,all P<0.05.Cell scratch experiment

关 键 词：鼻咽癌 CNE2细胞 Per1基因 增殖 凋亡 侵袭 

分 类 号：R739.6[医药卫生—肿瘤]