解旋酶DDX46基因对食管鳞癌Eca-109细胞增殖及凋亡的影响

doi:10.3969/j.issn.1004-616x.2017.03.011

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (3): 216-221,229.doi: 10.3969/j.issn.1004-616x.2017.03.011

解旋酶DDX46基因对食管鳞癌Eca-109细胞增殖及凋亡的影响

高华¹, 宋铁牛², 李斌^2,3, 杨建宝², 朱多杰², 郭权威²

1. 兰州大学第二医院门诊部, 兰州大学第二临床医学院, 甘肃兰州 730030;
2. 兰州大学第二医院胸外科, 兰州大学第二临床医学院, 甘肃兰州 730030;
3. 甘肃省消化系肿瘤重点实验室, 甘肃兰州 730030

收稿日期:2016-09-01 修回日期:2016-12-15 出版日期:2017-05-31 发布日期:2017-05-31
通讯作者: 李斌,E-mail:dr.leebin@outlook.com E-mail:dr.leebin@outlook.com
作者简介:高华,E-mail:gao.hua@outlook.com。
基金资助:
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）

Effect of RNA helicase gene DDX46 knockdown on proliferation and apoptosis in esophageal squamous cell carcinoma Eca-109 cells

GAO Hua¹, SONG Tieniu², LI Bin^2,3, YANG Jianbao², ZHU Duojie², GUO Quanwei²

1. Outpatient Department, Lanzhou University Second Hospital, Lanzhou University Second Clinical Medical College, Lanzhou 730030;
2. Thoracic Surgery Department, Lanzhou University Second Hospital, Lanzhou University Second Clinical Medical College, Lanzhou 730030;
3. Gansu Provincial Key Laboratory of Digestive System Tumors, Lanzhou 730030, Gansu, China

Received:2016-09-01 Revised:2016-12-15 Online:2017-05-31 Published:2017-05-31

摘要/Abstract

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shRNA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-shRNA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：DDX46 mRNA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。

关键词: 食管鳞癌, RNA解旋酶, DDX46, 增殖, 凋亡

Abstract: OBJECTIVE:To explore the effect of DDX46 knockdown by RNA interference on proliferation and apoptosis in esophageal squamous cell carcinoma Eca-109 cells. METHODS:DDX46 mRNA expression in Eca-109 and normal esophageal epithelium cell Het-1A were quantified using quantitative real-time PCR (qPCR). Then,DDX46 -shRNA-mediated RNA interference was applied to silence DDX46 in 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:DDX46 mRNA was significantly up-regulated in Eca-109 cells compared with that in Het-1A cells (P < 0.01). DDX46 knockdown in Eca-109 led 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 DDX46 and up-regulated the expression of cleaved Caspase-3 and cleaved PARP-1 (P < 0.01). CONCLUSION:DDX46 mRNA was over-expressed in Eca-109 cells. Targeted silencing of the DDX46 gene inhibited cell proliferation,arrested cell cycle in the G0/G1 phase,and induced cell apoptosis. The DDX46 silencing effect could be mediated by positive regulation of the apoptosis signaling pathway which played the roles of inhibiting Eca-109 cells growth and inducing apoptosis.

Key words: esophageal squamous cell carcinoma, RNA helicase, DDX46, proliferation, apoptosis

中图分类号:

R730.2

高华, 宋铁牛, 李斌, 杨建宝, 朱多杰, 郭权威. 解旋酶DDX46基因对食管鳞癌Eca-109细胞增殖及凋亡的影响[J]. 癌变·畸变·突变, 2017, 29(3): 216-221,229.

GAO Hua, SONG Tieniu, LI Bin, YANG Jianbao, ZHU Duojie, GUO Quanwei. Effect of RNA helicase gene DDX46 knockdown on proliferation and apoptosis in esophageal squamous cell carcinoma Eca-109 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(3): 216-221,229.

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解旋酶DDX46基因对食管鳞癌Eca-109细胞增殖及凋亡的影响

Effect of RNA helicase gene DDX46 knockdown on proliferation and apoptosis in esophageal squamous cell carcinoma Eca-109 cells

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