LIANG Bi-jun, LI Xiang-ping, LU Juan, LIN Shao-xiong, LIU Xiong, LI Gang, ZHANG Bao, WANG Lu, LUO Hua-nan, WAN Ren-qiang, TIAN Wen-dong
Department of Otorhinolaryngology Head and Neck Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2012 Apr;47(4):298-304.
To investigate the effects of the enhancer of zeste homolog 2 (EZH2) gene on cell growth and invasion of the nasopharyngeal carcinoma (NPC).
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.
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 β-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot analysis.
EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro, which might be mediated by inducing EMT.
探讨zeste同源物2（EZH2）基因对鼻咽癌（NPC）细胞生长和侵袭的影响。
构建用于EZH2基因短发夹RNA（shRNA）传递的重组慢病毒载体，并转染至293FT细胞。收集病毒颗粒后，转染至NPC细胞系5-8F细胞。采用MTT法、平板克隆形成试验和流式细胞术检测EZH2基因沉默对细胞增殖和细胞周期的影响。分别采用划痕愈合试验和基质胶侵袭试验检测5-8F细胞的迁移和侵袭能力。分别通过实时荧光定量PCR和蛋白质免疫印迹法检测EZH2及上皮-间质转化（EMT）相关标志物在mRNA和蛋白质水平的表达。
转染后的5-8F细胞中EZH2 mRNA和蛋白质表达明显降低。MTT法显示，EZH2基因下调显著抑制5-8F/shEZH2细胞的生长（P<0.001）。5-8F/shEZH2细胞的克隆形成率（84.44%）低于对照组（31.56%，P=0.001）。细胞周期分析显示，大多数5-8F/shEZH2细胞停滞于G0/G1期，S期细胞比例极低。划痕愈合试验表明，沉默EZH2基因的细胞迁移能力显著降低，5-8F/ShEZH2细胞和对照细胞在48小时的相对迁移距离分别为0.58±0.05和0.81±0.02（P<0.000）。基质胶侵袭试验显示，沉默EZH2基因的细胞侵袭能力显著受到抑制，穿透细胞数（72.23±4.08）少于对照组（150.95±16.27），P<0.000。沉默EZH2基因的细胞中上皮标志物E-钙黏蛋白和角蛋白18的mRNA表达分别增加了177%和158%，间质标志物β-连环蛋白和N-钙黏蛋白的mRNA表达分别降低了18.04%和41.18%。蛋白质免疫印迹分析也得到了类似结果。
EZH2在体外显著增强了鼻咽癌细胞的增殖和侵袭能力，其机制可能与诱导EMT有关。
