CAJ | 학술논문

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.
배경여목적:Notch1적활화가이통과하조인류유두상류병독(human papillomavirus,HPV)조기단백E6화E7기인적표체억제HPV양성HeLa세포계적증식.인식관린상세포암세포계EC109세포위HPV18양성세포.본연구장Notch1포내단(intracellular domain of Notch,ICN)전입EC109세포,도치EC109세포중Notch통로적조성성활화,종이탐토Notch통로활화여EC109세포증식적관계급궤제.방법:용지질체전염법장ICN전입체외배양적EC109세포,용MTT법검측세포증식솔;용류식세포의검측세포주기;용RT-PCR검측HPV18E6/E7기인적표체:용Western인적법검측주기단백격매억제인자p53적표체.결과:ICN전염입EC109세포후,EC109세포증식수억;세포주기조체재G_2/M기,전목적기인조G_2/M기세포소점비례[(42.57±1.57)%]여미전염조[(1.88±0.66)%]급전공질립조[(1.994±1.02)%]상비차이균유현저성(P<0.01).E6/E7기인표체강저:p53표체승고,전ICN조(2.154±0.23)여미전염조(0.45±0.07)급전공질립조(0.464±0.02)상비차이균유현저성(P<0.01).결론:Notch1통로활화가이억제HPV18 E6/E7기인적표체,도치p53통로적활화,사HPV18양성EC109세포증식주기조체우G_2/M기,억제EC109세포적생장.
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.