EB病毒核抗原1对鼻咽癌细胞miR-34a/Notch1轴及增殖、凋亡的影响

Effects of Epstein-Barr virus EBNA1 on miR-34a/Notch1 axis,proliferation and apoptosis of nasopharyngeal carcinoma cells

目的探讨EB病毒核抗原1(Epstein-Barr virus nuclear antigen1,EBNA1)对鼻咽癌细胞miR-34a/Notch1轴及增殖、凋亡的影响。方法体外培养人EB病毒(Epstein-Barr virus,EBV)阴性和阳性鼻咽癌细胞系CNE2、C666-1,CNE2细胞设置对照组、空转染组、EBNA1过表达组、miR-34a对照组和双转染组,进行不同的转染处理。实时荧光定量PCR法检测EBNA1、Notch1 mRNA及miR-34a表达;CCK-8法检测细胞增殖;流式细胞仪检测细胞凋亡及细胞周期分布;蛋白免疫印迹法检测Notch1、细胞周期蛋白D1(cyclin D1)蛋白表达。结果与CNE2细胞相比,C666-1细胞中Notch1 mRNA表达水平升高(P<0.05),miR-34a表达水平降低(P<0.05);转染EBNA1后,CNE2细胞中EBNA1、Notch1 mRNA表达水平、细胞OD值、S、G2/M期CNE2细胞比例及Notch1、cyclin D1蛋白表达水平升高,miR-34a表达水平、细胞凋亡率、G0/G1期CNE2细胞比例降低(P<0.05);进一步转染miR-34a后,CNE2细胞中以上指标的变化均被逆转(P<0.05)。结论EBNA1可能通过降低miR-34a水平,上调Notch1表达,对鼻咽癌CNE2细胞发挥增殖促进、凋亡抑制作用。

Objective To investigate the effects of Epstein-Barr virus nuclear antigen 1(EBNA1)on microRNA-34a/Notch1 axis,proliferation and apoptosis of nasopharyngeal carcinoma cells.Methods Human Epstein-Barr virus(EBV)negative and positive nasopharyngeal carcinoma cell lines,CNE2 and C666-1 were cultured in vitro.CNE2 cells were divided into control group,empty transfection group,EBNA1 over-expression group,microRNA-34a(miR-34a)control group and double transfection group according to different transfection protocols.The expression levels of EBNA1,Notch1 mRNA and miR-34a were detected by real-time fluorescence quantitative PCR(qRT-PCR).The proliferation of the cells was detected by CCK-8 method.The apoptosis and cell cycle distribution were detected by flow cytometry.Western blot was used to detect the expressions of Notch1 and cyclin D1.Results Compared with those in CNE2 cells,the expression level of Notch1 mRNA in C666-1 cells was significantly higher(P<0.05),and the expression level of miR-34a was significantly lower(P<0.05).After transfected with EBNA1,the expression levels of EBNA1 and Notch1 mRNA,OD valueof the cell,the proportions of CNE2 cells in S and G2/M phases,and the protein expression levels of Notch1 and cyclin D1 were significantly higher in the EBNA1 overexpression group while the expression level of miR-34a,apoptosis rate and the proportion of CNE2 cells in G0/G1 phase were significantly lower(P<0.05).Further transfection of miR-34a resulted in all the changes in CNE2 cells to be reversed(P<0.05).Conclusions EBNA1 may promote proliferation and inhibit apoptosis of CNE2 cells by decreasing miR-34a level and up-regulating Notch1 expression.