Effect of Epstein-Barr virus nuclear antigen 1 on cell proliferation and cell cycle in nasopharyngeal carcinoma cells

Abstract

To study the effect of Epstein-Barr virus nuclear antigen 1 (EBNA1) on cell proliferation and cell cycle in nasopharyngeal carcinoma (NPC) cells. Recombinant lentivirus that encoded EBNA1 short hairpin RNA (shRNA) was prepared. The C666-EBNA1 (CE) cells were transduced with lentivirus and selected by fluorescence activated cell sorting (FACS) to repress EBNA1 expression. The protein expression levels of EBNA1 were examined by Western blot. The effect of EBNA1 silence on cell proliferation was analyzed by MTT assay and cell growth assay, respectively. Cell cycle was assessed by flow cytometry. The mRNA and protein levels of cell cycle regulators were examined by real-time PCR and Western blot. Recombinant lentivirus encoded EBNA1 shRNA was successfully constructed. The EBNA1 expression in CE cells was significantly reduced by lentivirus-mediated RNA interference. The results of cell counting and MTT assay showed that EBNA1 down-regulation significantly inhibited cell growth in CE-shRNA EBNA1 cells (P < 0.05). Compared with the control group, the percentage of cells in G0-G1 phase was increased from (62.43 ± 6.62)% to (89.66 ± 0.64)% (t = -7.091, P = 0.002), and that in S phase was decreased from (34.93 ± 7.36)% to (7.82 ± 2.44)% (t = 6.095, P = 0.004). The mRNA expressions of c-myc, CDK4, CDK6 and pRb were decreased by 65.60%, 34.06%, 41.05% and 55.29% respectively with the similar results in protein expression levels. Suppression of EBNA1 may inhibit the growth of nasopharyngeal carcinoma cells in vitro and induce a G1-phase cell cycle arrest, which might be mediated by down-regulation of c-myc, CDK4, CDK6 and pRb.