Notch1通路活化抑制EC109细胞的增殖及机制探讨

摘要

Background and purpose: It has been reported that activation of Notch1 could strongly inhibit proliferation of HPV (human papilloma virus)-positive HeLa cells by down-regulation of the E6 and E7 genes. The aim of this paper was to investigate the role of the Notch signaling pathway in growth arrest of EC109 cells in vitro and the molecular mechanism. Methods: EC109 cell lines, a well differentiated human ESCC (esophageal squamous cell carcinoma) cell line with HPV18-positive, was used in the study. Exogenous intracellular domain of Notch1(ICN) was transfected into cultured EC109 cells by lipofectamine transfection, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papilloma virus type 18 (HPV18) E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot.Results: Activation of Notchl signaling resulted in inhibition of EC109 cell proliferation with the induction of G_2/ M arrest. There was a significant difference in terms of the percentage of G_2/M phase cells among the ICN-transfected group (42.57±1.57)% and the non-transfected group (1.88±0.66)% or the empty plasmid transfected group (1.99±1.02)% (P<0.01). Down modulation of HPV18 E6/E7 gene expression and upregulation of p53 expression was (2.15±0.23) in ICN-transfected group higher than non- transfected group (0.45±0.07) and empty plasmid transfected group (0.46±0.02) (P<0.01). Conclusion: Repression of HPV18 E6/E7 expression by Notch1 signaling results in growth suppression of HPV18-positive EC109 cells with concomitant activation of p53-mediated pathways.%背景与目的:Notch1的活化可以通过下调人类乳头状瘤病毒(human papillomavirus,HPV)早期蛋白E6和E7基因的表达抑制HPV阳性HeLa细胞系的增殖.人食管鳞状细胞癌细胞系EC109细胞为HPV18阳性细胞.本研究将Notch1胞内段(intracellular domain of Notch,ICN)转入EC109细胞,导致EC109细胞中Notch通路的组成性活化,从而探讨Notch通路活化与EC109细胞增殖的关系及机制.方法:用脂质体转染法将ICN转入体外培养的EC109细胞,用MTT法检测细胞增殖率;用流式细胞仪检测细胞周期;用RT-PCR检测HPV18E6/E7基因的表达:用Western印迹法检测周期蛋白激酶抑制因子p53的表达.结果:ICN转染入EC109细胞后,EC109细胞增殖受抑;细胞周期阻滞在G_2/M期,转目的基因组G_2/M期细胞所占比例[(42.57±1.57)%]与未转染组[(1.88±0.66)%]及转空质粒组[(1.994±1.02)%]相比差异均有显著性(P<0.01).E6/E7基因表达降低:p53表达升高,转ICN组(2.154±0.23)与未转染组(0.45±0.07)及转空质粒组(0.464±0.02)相比差异均有显著性(P<0.01).结论:Notch1通路活化可以抑制HPV18 E6/E7基因的表达,导致p53通路的活化,使HPV18阳性EC109细胞增殖周期阻滞于G_2/M期,抑制EC109细胞的生长.