慢病毒介导的Nestin基因沉默抑制人食管癌ECA109细胞增殖

Silencing Nestin expression mediated by lentivirus inhibits human esophageal cancer ECA109 cell proliferation

目的通过体外实验探讨沉默巢蛋白(Nestin)基因对人食管癌ECA109细胞增殖能力的影响及可能机制。方法合成Lenti-Nestin慢病毒载体,实验设blank组、scrambled组和Lent-Nestin组,转染ECA109细胞,建立稳定沉默Nestin基因的细胞系Lenti-Nestin;用qRT-PCR和Western blot检测Nestin、c-myc和cyclin D1 mRNA和蛋白表达;CCK-8法检测细胞增殖能力;平板集落实验检测细胞的集落形成。结果稳定沉默Nestin的细胞系构建成功;与blank组和scrambled组相比,Lent-Nestin组中Nestin mRNA(P<0.01)和蛋白(P<0.05)水平明显降低;细胞增殖能力明显降低(P<0.01),集落形成能力显著下降(P<0.05);沉默Nestin表达后,c-myc、cyclin D1表达明显受到抑制(P<0.05)。结论沉默Nestin基因表达可抑制人食管癌ECA109细胞的增殖能力,其可能通过调控c-myc、cyclin D1表达参与其中。

Objective To investigate the effect of Nestin gene cancer ECA109 cells and possible mechanism. Methods silencing on the proliferation of human esophageal Lenti-Nestin was constructed and transfected into ECA109 cells to establish a stable Nestin-silencing cell line Lenti-Nestin. Blank group, scrambled group, Lenti- Nestin group were set up. The expressions of Nestin, c-myc and cyclin D1 mRNA and protein levels were detected by qRT-PCR and Western blot. The cell proliferation was analyzed by CCK8 assay. Results The stable Nestin-si- lencing cell line was successfully established. The expression of Nestin mRNA （ P 〈 0. 01 ） and protein （P 〈 0.05 ） levels were reduced significantly and the downregulation evidently suppressed cell proliferation （ P 〈 0.01 ） and col- ony forming capacity （P 〈 0. 05 ） compare with the scrambled group and blank group. However, The c-myc and cy- clin D1 expression levels in ECA109 cells in Lenti-Nestin group were significantly lower than that of scrambled group and blank group （P 〈 0. 05）. Conclusions Knockdown Nestin expression significantly inhibits the level of esophageal cancer ECA109 ceils proliferation,which may act via influencing the expression of c-myc, cyclin D1.