摘要
背景与目的:DDX46是一种三磷酸腺苷依赖的RNA解旋酶,在mRNA前体剪接和核糖体组装中发挥核心作用,其在人类肿瘤中的表达及与肿瘤的相关性少见报道。该研究旨在探讨DDX46与食管鳞癌细胞恶性生物学行为之间的关系及可能的调控机制。方法:构建慢病毒载体和shDDX46质粒,转染食管鳞癌TE-11细胞,建立沉默DDX46表达的稳转细胞株,采用反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)和蛋白质印迹法(Western blot)鉴定转染效果;采用细胞计数和克隆形成实验检测细胞增殖能力;采用Annexin V-APC单染法流式细胞术检测细胞凋亡情况;采用Western blot检测PI3K-Akt-mTOR信号通路相关分子及自噬相关蛋白的表达水平。结果:慢病毒介导的RNAi有效抑制DDX46在TE-11细胞中的表达(P<0.001)。与对照组相比,靶向沉默DDX46基因后细胞计数显示,TE-11细胞生长被显著抑制(P<0.01);克隆形成实验显示,细胞克隆形成能力被显著抑制(P<0.01);流式细胞术凋亡检测显示,细胞凋亡率显著增加(P<0.01);Western blot检测发现,PIK3CG、Akt、P-Akt和mTOR表达水平显著下降(P<0.01),PTEN表达水平无显著变化(P>0.05),而Beclin 1和LC3表达显著上调(P<0.01)。结论:DDX46与食管鳞癌细胞增殖和凋亡相关;沉默DDX46可能通过PI3K-Akt-mTOR信号转导通路,同时激活了细胞凋亡和自噬,进而影响食管鳞癌细胞生长增殖。
Background and purpose： DDX46 plays critical roles in cellular metabolism, and in many cases it has been implicated in cellular proliferation. The expression pattern of DDX46 in tumor tissues and its biologic role in neoplastic transformation have rarely been reported previously. This study aimed to elucidate the role of DDX46 in regulating the malignant process of esophageal squamous cell carcinoma. Methods： Lentivirus-mediated RNA interference was applied to silencing DDX46 in esophageal squamous carcinoma cell line TE-11. Cell growth was monitored using high-content screening（HCS）. Cell colony-forming capacity was measured by colony formation assay. Cell apoptosis was determined by flow cytometry. Furthermore, the changes of proteins related to PI3 K-Akt-mTOR and autophagy were measured by Western blot. Results： Lentivirus-mediated RNAi efficiently decreased DDX46 expression in TE-11 cells（P〈0.001）. DDX46 silencing led to decreased cell growth（P〈0.01）, reduced the number and size of cell colonies（P〈0.01）, and increased the percentage of apoptotic cells in TE-11（P〈0.01）. Moreover, DDX46 silencinginduced the apparent down-regulation of PIK3 CG, Akt, P-Akt and mTOR（P〈0.01）, and significantly increased expression of Beclin 1 and LC3（P〈0.01）, without change in expression level of PTEN（P〉0.05）. Conclusion： DDX46 plays an essential role in the progression of esophageal squamous cell carcinoma, and DDX46-mediated regulation of autophagy and apoptosis probably affects tumorigenesis in esophageal squamous cell carcinoma via PI3 K-Akt-mTOR signaling pathway.
作者
李斌
蔺军平
冯海明
杨建宝
宋铁牛
敬涛
孟于琪
LI Bin;LIN Junping;FENG Haiming;YANG Jianbao;SONG Tieniu;JING Tao;MENG Yuqi(Department of Thoracic Surgery,Lanzhou University Second Hospital,Lanzhou University Second Clinical Medical College,Lanzhou 730030,Gansu Provence,China)
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2018年第9期671-678,共8页
China Oncology
基金
甘肃省卫生行业科研计划项目(GSWSKY2017-04)
甘肃省自然科学基金项目(1606RJZA040)
兰州大学第二医院"萃英科技创新"计划项目(CY2017-MS02)
关键词
DDX46
食管鳞癌
增殖
凋亡
自噬
DDX46
Esophageal squamous cell carcinoma
Growth
Apoptosis
Autophagy
