DDX46基因调控TE-1食管鳞状细胞癌细胞侵袭转移行为的相关性研究

Relationship between DDX46 gene and invasion and metastasis behaviors of TE-1 esophageal squamous cell carcinoma cells

目的 探索DDX46基因与TE-1食管鳞状细胞癌(鳞癌)细胞侵袭和转移行为的相关性及可能的分子机制。方法 荧光标记shRNA慢病毒转染TE-1食管鳞癌细胞敲减DDX46基因为实验组(shDDX46组),空载体转染为对照组(shCtrl组)。镜下绿色荧光蛋白表达率评价细胞转染效率,实时荧光定量聚合酶链式反应和蛋白质印迹法(Western blotting,WB)分别检测DDX46基因在mRNA和蛋白表达水平的敲减效率。采用划痕实验、侵袭实验、Transwell小室实验检测DDX46基因敲减前后TE-1食管鳞癌细胞侵袭和转移能力的变化。采用生物信息学方法进行经典通路分析探讨信号通路的变化,进一步通过WB检测纤连蛋白表达,探讨DDX46在食管鳞癌细胞侵袭和转移过程中可能存在的作用机制。结果 shRNA慢病毒转染TE-1食管鳞癌细胞后DDX46基因在mRNA和蛋白水平上的表达受到显著抑制。划痕实验提示shDDX46组TE-1食管鳞癌细胞在划痕后8 h细胞迁移率相比shCtrl组明显降低(P=0.001)。侵袭实验表明shDDX46组TE-1食管鳞癌细胞在培养24 h后侵袭细胞数量低于shCtrl组(P<0.001)。Transwell实验结果显示shDDX46组在24 h观察点的转移细胞数少于shCtrl组(P<0.001)。经典通路分析结果表明整合素信号通路活性被抑制,进一步探究其作用机制发现,敲减DDX46基因后与细胞黏附相关的纤连蛋白表达下降11%。结论 DDX46基因与TE-1食管鳞癌细胞的侵袭和转移行为相关,敲减DDX46基因可能是通过下调整合素通路信号,降低细胞黏附性及细胞骨架重构,从而抑制食管鳞癌细胞侵袭和转移过程。

Objective To investigate the relationship between DDX46 genes and invasion and migration of esophageal squamous cell carcinoma cells.Methods Human esophageal squamous cell carcinoma cells TE-1 were transfected by fluorescent marker shRNA lentivirus(shDDX46 group),and an empty vector was transfected as a control(shCtrl group).The expression rate of green fluorescent protein under the microscope was used to evaluate the cell transfection efficiency.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blotting(WB)detected the knockdown efficiency of the target gene at the mRNA and protein expression levels.Wound healing,invasion assay and migration assay detected the changes of invasion and metastasis ability.Classical pathway analysis was used to explore signaling pathway changes and the possible mechanism of DDX46 in the invasion and metastasis was explored by detecting fibronectin expression.Results DDX46 gene at mRNA and protein levels was significantly inhibited after lentiviral transfection.Wound healing showed that after 8 h the cell mobility of TE-1 cells decreased significantly(P<0.001).Invasion assay showed that after 24 h the average cell metastasis rate of TE-1 cells was lower in the shDDX46 group than that in the shCtrl group(P<0.001).The cell metastasis rate in the shDDX46 group corresponding to observation points in the transwell assay was lower than that in the shCtrl group(P<0.001)after 24h culture.The results of the classical pathway analysis showed that the integrin signaling pathway activity was inhibited,further exploration of the mechanism of action found that the expression of fibronectin associated with cell adhesion was decreased.Conclusion DDX46 gene is related to the invasion and migration ability of esophageal squamous cell carcinoma cells.Knock-down DDX46 genes may reduce cell adhesion by downregulating the integrin pathway signaling.