摘要
目的 :探讨通过转染短发夹RNA(short hairpin RNA,shRNA)下调Notch2表达的方法阻滞Notch2信号通路对食管鳞癌Eca109细胞增殖的影响。方法 :首先应用实时荧光定量-PCR法检测包括食管鳞癌Eca109细胞在内的多种肿瘤细胞中Notch受体的表达水平。然后将无效shRNA(Control-shRNA)和针对Notch2基因的shRNA(Notch2-shRNA)分别转染高表达Notch2的Eca109细胞。实时荧光定量-PCR法检测转染后Notch2及其下游靶基因Hes1的mRNA表达,蛋白质印迹法检测转染后Notch2蛋白的表达,CCK-8(cell counting kit-8)法检测Eca109细胞增殖情况,FCM法检测细胞周期变化。结果:食管鳞癌Eca109细胞中Notch2 mRNA高表达。与Control-shRNA转染组相比,Notch2-shRNA转染后Eca109细胞中Notch2 mRNA及蛋白表达明显下调(P<0.01),下游靶基因Hes1的mRNA表达水平明显降低(P<0.01)。Notch2-shRNA转染72 h后,Eca109细胞增殖率明显降低(P<0.05);G0/G1期细胞所占百分率为(70.66±6.25)%,明显高于Control-shRNA组的(45.34±1.88)%和未转染对照组的(47.10±1.70)%(P<0.01)。结论 :Notch2在体外培养的食管鳞癌Eca109细胞中高表达,下调Notch2表达可以通过诱导G0/G1期细胞阻滞而减缓Eca109细胞的体外增殖。
Objective： To investigate the effect of blocking Notch2 signaling pathway on the proliferation of esophageal squamous cell carcinoma cell line Eca109 through down-regulating the expression level of Notch2 by transfection with short-hairpin RNA （shRNA）. Methods： The expression of Notch receptor in multiple tumor cell lines including esophageal squamous cell carcinoma Eca109 cells was detected by real-time fluorescence quantitative-PCR （RFQ-PCR）. Then Eca109 cells with high expression of Notch2 were transfected with Control-shRNA and specific shRNA of Notch2 （Notch2-shRNA）, respectively. The mRNA expressions of Notch2 and its downstream gene Hes-1 in Eca109 cells after transfection with shRNA were detected by RFQ-PCR. The Notch2 protein expression was detected by Western blotting. The proliferation of Eca109 cells was detected by cell counting kit-8 （CCK-8）. In additon, flow cytometry was performed to assess the cell cycle distribution of Eca109 cells. Results： Notch2 gene highly expressed in Eca109 cell line. Compared with the control group, the expression levels of Notch2 mRNA and protein and its downstream gene Hes-1 were significantly decreased after transfection with Notch2-shRNA （P 〈 0.01）. Moreover, the proliferation of Eca109 cells transfected with Notch2-shRNA for 72 h was inhibited （P 〈 0.05）, and the percentage of Eca109 cells in Go/G1 phase in Notch2-shRNA transfection group （70.66±6.25）% was significantly higher as compared with Control-shRNA transfection group [（45.34±1.88）%] and the untransfection group [（47.10±1.70）%] （P 〈 0.01）. Conclusion： Notch2 is significantly highly expressed in Eca109 cells in vitro. The down-regulation of Notch2 expression can suppress the proliferation of Eca109 cells through inducing cell cycle arrest in the G0/G1 phase.
出处
《肿瘤》
CAS
CSCD
北大核心
2014年第6期501-506,540,共7页
Tumor
基金
国家自然科学基金资助项目(编号:81172871)
