[Effects of enhancer of zeste homolog (EZH2) downregulation on the proliferation and invasion of nasopharyngeal carcinoma cell and the possible mechanisms].

OBJECTIVE: To investigate the effects of the enhancer of zeste homolog 2 (EZH2) gene on cell growth and invasion of the nasopharyngeal carcinoma (NPC). METHODS: Recombinant lentivirus vector for shRNA delivery of EZH2 was constructed and transfected into 293FT cells. After collecting the viral particles, the NPC cell line 5-8F cells were transfected. The effects of EZH2 silence on cell proliferation and cell cycle were detected using MTT assay, plate colony formation assay and flow cytometry. The migration and invasion of 5-8F cells were determined by wound healing assay and matrigel invasion assay, respectively. The expressions of EZH2 and epithelial-mesenchymal transition (EMT)-related markers at mRNA and protein levels were examined by real-time PCR and Western blot respectively. RESULTS: The expressions of EZH2 mRNA and protein in the transfected 5-8F cells were obviously reduced. MTT assay showed that EZH2 downregulation significantly inhibited the growth of 5-8F/shEZH2 cells (P < 0.001). Colony formation rate (84.44%) of 5-8F/shEZH2 cells was lower than control (31.56%, P = 0.001). Cell cycle analysis showed that most 5-8F/shEZH2 cells were arrested in G0/G1 phase, with a very low ratio of cells in S phase. Wound healing assay indicated that the migration ability of cells silencing EZH2 decreased significantly, and the 48-hour relative migration distance of 5-8F/ShEZH2 cells and control cells was 0.58 ± 0.05, and 0.81 ± 0.02, respectively (P < 0.000). Matrigel invasion assay, showed the invasive capacity of cells silencing EZH2 was significantly inhibited, with less penetrating cells (72.23 ± 4.08) compared to control (150.95 ± 16.27), P < 0.000. The mRNA expressions of epithelial markers E-cadherin and Keratin 18 in the cells silencing EZH2 increased by 177% and 158% respectively, and the mRNA expressions of mesenchymal markers ß-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot analysis. CONCLUSION: EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro, which might be mediated by inducing EMT.

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

OBJECTIVE: To study the effect of Epstein-Barr virus nuclear antigen 1 (EBNA1) on cell proliferation and cell cycle in nasopharyngeal carcinoma (NPC) cells. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.