摘要
目的:探讨RNA解旋酶DDX46基因对食管鳞癌细胞株Eca-109增殖和凋亡的影响及可能的作用机制。方法:以人永生化食管鳞状上皮细胞Het-1A为对照,实时荧光定量PCR(qPCR)检测DDX46 mRNA在Eca-109细胞中的相对表达水平。应用sh RNA干扰技术沉默Eca-109细胞DDX46基因后,分别采用MTT实验、克隆形成实验及流式细胞术检测细胞增殖情况、克隆形成能力以及细胞周期和细胞凋亡情况。并用Western blot检测DDX46基因沉默后Eca-109细胞DDX46蛋白和凋亡信号转导通路关键分子Caspase-3和PARP-1蛋白表达的变化。结果:与Het-1A细胞相比,Eca-109细胞DDX46 mRNA的表达水平显著升高(P<0.01)。与对照组相比,靶向沉默DDX46基因后MTT实验显示Eca-109细胞活力明显减弱,增殖能力被显著抑制(P<0.01);克隆形成实验显示Eca-109细胞克隆形成能力被显著抑制(P<0.01);流式细胞术检测显示处于G1期的细胞增加,而处于S期的细胞减少(P<0.05),细胞周期停滞于G0/G1期;凋亡实验显示细胞凋亡率显著增加(P<0.01)。Western blot检测显示,与对照组比较,DDX46-sh RNA干扰使Eca-109细胞DDX46蛋白表达水平显著下降(P<0.01),而cleaved Caspase-3和cleaved PARP-1蛋白表达水平显著上升(P<0.01)。结论:DDX46mRNA在食管鳞癌Eca-109细胞中高表达,DDX46基因沉默可能通过激活细胞凋亡信号转导通路而发挥抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡的作用。
OBJECTIVE:To explore the effect of DDX46knockdown by RNA interference on proliferation and apoptosis in esophageal squamous cell carcinoma Eca-109cells.METHODS:DDX46mRNA expression in Eca-109and normal esophageal epithelium cell Het-1A were quantified using quantitative real-time PCR(qPCR).Then,DDX46-shRNA-mediated RNA interference was applied to silence DDX46in Eca-109.Subsequently,cell viability was measured by MTT assay,cell colony-forming capacity was measured by colony formation assay,cell cycle progression and apoptosis were determined by flow cytometry.Western blot analysis was used to detect changes of apoptosis signaling molecules.RESULTS:DDX46mRNA was significantly up-regulated in Eca-109cells compared with that in Het-1A cells(P<0.01).DDX46knockdown in Eca-109led to reduced cell proliferation(P<0.01),reduced number and size of cell colonies(P<0.01),increased percentage of G1-phase cells,decreased percentage of S-phase cells(P<0.05)and increased percentage of apoptotic cells(P<0.01).Western blot analysis showed that the knockdown effectively suppressed the expression of DDX46and up-regulated the expression of cleaved Caspase-3and cleaved PARP-1(P<0.01).CONCLUSION:DDX46mRNA was over-expressed in Eca-109cells.Targeted silencing of the DDX46gene inhibited cell proliferation,arrested cell cycle in the G0/G1phase,and induced c ell apoptosis.T he D DX46silencing effect could be ediated by positive regulation of the apoptosis signaling pathway which played t he roles of inhibiting E ca-109c ells growth and inducing apoptosis.
作者
高华
宋铁牛
李斌
杨建宝
朱多杰
郭权威
GAO Hua;SONG Tieniu;LI Bin;YANG Jianbao;ZHU Duojie;GUO Quanwei
出处
《癌变．畸变．突变》
CAS
CSCD
2017年第3期216-221,229,共7页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
甘肃省自然科学基金(1606RJZA040)
兰州大学中央高校基本科研业务费专项资金(lzujbky-2013-140)
关键词
食管鳞癌
RNA解旋酶
DDX46
增殖
凋亡
esophageal squamous cell carcinoma
RNA helicase
DDX46
proliferation
apoptosis
