摘要
目的探讨胞裂蛋白9(SEPTIN9)基因甲基化对食管癌细胞增殖、凋亡、迁移和侵袭的影响。方法采用亚硫酸盐测序聚合酶链式反应(PCR)法检测正常食管上皮细胞HEEC与食管癌ECA109、KYSE150细胞中SEPTIN9基因甲基化水平。分别应用甲基化抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dc)(实验组)和二甲基亚砜(DMSO)(对照组)与细胞共培养72 h,采用实时荧光定量聚合酶链反应及蛋白质印迹法(Western blot)检测SEPTIN9 mRNA及其蛋白,采用噻唑蓝(MTT)法及膜联蛋白V-异硫氰酸荧光素标记磷脂结合蛋白(Annexin V-FITC)凋亡实验检测增殖与凋亡,Transwell小室法检测迁移和侵袭。组间比较采用独立样本t检验。结果食管癌细胞ECA109、KYSE150的SEPTIN9基因甲基化程度高于正常食管上皮细胞HEEC[(83.69±7.15)%比(21.36±4.58)%;(84.26±6.97)%比(21.36±4.58)%,t=12.714、13.063,P<0.01]。实验组食管癌细胞ECA109、KYSE150 SEPTIN9 mRNA及SEPTIN9蛋白相对表达量高于对照组(1.66±0.17比1.23±0.12、1.68±0.20比1.25±0.13;1.76±0.18比1.35±0.15、1.85±0.21比1.38±0.16,t=3.579、3.122、3.031、3.083,P<0.05)。实验组食管癌细胞ECA109、KYSE150增殖能力低于对照组(0.38±0.06比0.91±0.17、0.40±0.05比0.97±0.16,t=5.092、5.890,P<0.01)。实验组食管癌细胞ECA109、KYSE150凋亡率高于对照组[(35.69±4.37)%比(17.89±2.26)%、(41.18±5.49)%比(18.96±3.05)%,t=6.267、6.128,P<0.01]。实验组食管癌细胞ECA109、KYSE150迁移细胞数目、侵袭细胞数目少于对照组[(116.75±12.59)个比(187.24±19.26)个、(120.24±13.68)个比(193.25±20.34)个;(61.34±7.52)个比(108.35±12.56)个、(63.58±6.69)个比(112.24±15.06)个,t=5.905、5.159、5.562、5.114,P<0.01]。结论抑制SEPTIN9基因甲基化可使食管癌细胞中SEPTIN9基因重新表达,从而抑制食管癌细胞增殖、迁移和侵袭,促进细胞凋亡。
Objective To investigate the effects of cytosolic protein 9(SEPTIN9)gene methylation on oesophageal cancer cell proliferation,apoptosis,migration and invasion.Methods The methylation level of SEPTIN9 gene in normal oesophageal epithelial cells HEEC and oesophageal cancer ECA109 and KYSE150 cells was detected by sulfite sequencing polymerase chain reaction(PCR)method.The methylation inhibitor 5-aza-2′-deoxycytidine(5-Aza-dc)(experimental group)and dimethylsulfoxide(DMSO)(control group)were applied to co-culture with the cells for 72 h,respectively,and the real-time fluorescence quantitative polymerase chain reaction(RTFQPCR)and protein blotting(Western blotting)were used to detect SEPTIN9 mRNA and its proteins by using the diphenyltetrazolium bromide(MTT)method and fluorescein isothiocyanate labelled phospholipid binding protein(Annexin V-FITC)apoptosis assay to detect proliferation and apoptosis,and Transwell method to detect migration and invasion.Independent samples t-tests was used for comparison between groups.Results The methylation of SEPTIN9 gene in esophageal cancer cells ECA109 and KYSE150 was higher than that in normal esophageal epithelial cells HEEC[(83.69±7.15)%vs.(21.36±4.58)%;(84.26±6.97)%vs.(21.36±4.58)%,t=12.714,13.063,P<0.01].The relative expression of ECA109 and KYSE150 SEPTIN9 mRNA and SEPTIN9 protein in the experimental group was higher than that in the control group(SEPTIN9 mRNA:1.66±0.17 vs.1.23±0.12,1.68±0.20 vs.1.25±0.13;SEPTIN9 protein:1.76±0.18 vs.1.35±0.15,1.85±0.21 vs.1.38±0.16,t=3.579,3.122,3.031,3.083,P<0.05).The proliferation ability of esophageal cancer cells ECA109 and KYSE150 in the experimental group was lower than that of the control group[(0.38±0.06)vs.(0.91±0.17),(0.40±0.05)vs.(0.97±0.16),t=5.092,5.890,P<0.01].The apoptosis rate of esophageal cancer cells ECA109 and KYSE150 in the experimental group was higher than that in the control group[(35.69±4.37)%vs.(17.89±2.26)%,(41.18±5.49)%vs.(18.96±3.05)%,t=6.267,6.128,P<0.01].The number of migrating and inva
作者
吴恺
乔镇东
薛文华
陈铖鑫
刘东雷
李金豪
胡亚梅
吴彬
Wu Kai;Qiao Zhendong;Xue Wenhua;Chen Chengxing;Liu Donglei;Li Jinhao;Hu Yamei;Wu Bin(Department of Thoracic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Pharmacy,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Outpatient Office,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
2024年第1期62-65,共4页
Chinese Journal of Experimental Surgery
基金
河南省中青年卫生健康科技创新优秀青年人才培养项目(YXCC2020055)
河南省医学科技攻关项目(SBGJ202002077)
河南省自然科学基金项目(202300410460)
河南省高等学校重点科研项目(23A320061)
河南省中青年卫生健康科技创新优秀青年人才培养项目(YXKC2022060)
河南省医学科技攻关计划联合共建项目(LHGJ20200300)。
关键词
食管癌
增殖
凋亡
迁移
侵袭
Esophageal cancer
Proliferation
Apoptosis
Migration
Invasion
