中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2012年
4期
298-304
,共7页
梁碧君%李湘平%鲁娟%林少雄%刘雄%李刚%张宝%王路%罗花南%万仁强%田文栋
樑碧君%李湘平%魯娟%林少雄%劉雄%李剛%張寶%王路%囉花南%萬仁彊%田文棟
량벽군%리상평%로연%림소웅%류웅%리강%장보%왕로%라화남%만인강%전문동
鼻咽肿瘤%DNA结合蛋白质类%转录因子%基因沉默%细胞增殖%肿瘤浸润%上皮间质转化
鼻嚥腫瘤%DNA結閤蛋白質類%轉錄因子%基因沉默%細胞增殖%腫瘤浸潤%上皮間質轉化
비인종류%DNA결합단백질류%전록인자%기인침묵%세포증식%종류침윤%상피간질전화
Nasopharyngeal neoplasms%DNA-binding proteins%Transcription factors%Gene silencing%Cell proliferation%Neoplasm invasiveness%Epithelial-mesenchy mal transition
目的 探讨果蝇zeste基因增强子人类同源物(enhancer of zeste homolog 2,EZH2)对鼻咽癌细胞增殖及侵袭能力的影响及其分子机制.方法 构建特异性的短发夹状(short hairpin RNA,shRNA)EZH2慢病毒干扰载体,转染293FT细胞包装成慢病毒后,感染鼻咽癌细胞株5-8F.采用四甲基偶氮唑蓝法、平板克隆形成实验和流式细胞术检测细胞增殖状态及细胞周期的改变,利用细胞划痕实验及Invasion Chamber侵袭小室测定法检测迁移和侵袭能力变化,荧光定量PCR及蛋白免疫印迹法检测转染EZH2基因后的mRNA和蛋白表达水平改变及上皮间质转化相关标志物的变化.结果 成功构建EZH2 shRNA慢病毒干扰载体,包装的慢病毒感染鼻咽癌细胞株5-8F后,EZH2的mRNA和蛋白表达水平显著下调.四甲基偶氮唑蓝法显示,细胞接种96 h后,5-8F/shEZH2组的生长速度较空载组显著下降(P＜0.001),平均(x±s,下同)细胞克隆形成率为(31.56±7.49)％明显小于空载组的(84.44±7.12)％,差异有统计学意义(P=0.001)；细胞周期结果表明,沉默EZH2表达的细胞增殖受抑,主要通过诱导G0/G1期阻滞,减少S期细胞的比例；基因转染沉默EZH2后,鼻咽癌细胞的运动迁移能力显著下降,5-8 F/shEZH2组细胞划痕48 h的相对迁移率为(0.58±0.05)明显低于空载组的(0.81±0.02),差异有统计学意义(P ＜0.000)；体外侵袭能力明显降低,5-8F/shEZH2组穿膜细胞数(72.23 ±4.08)个,与其相对应空载组(150.95 ±16.27)个比较,明显减少(P＜0.000)；5-8F/shEZH2细胞的上皮标志物上皮性钙黏附蛋白(E-cadherin)、细胞角蛋白18( Keratin 18)的mRNA水平分别上调1.77倍、1.58倍,间质标志物p-连环蛋白(β-catenin)、神经性钙黏附蛋白(N-cadherin)分别下调18.04％、41.18％；蛋白免疫印迹证实此四者的蛋白水平表达亦出现相同趋势.结论 EZH2可显著促进鼻咽癌细胞的体外增殖和侵袭能力,其机制可能是通过诱导上皮间质转化促进鼻咽癌转移.
目的 探討果蠅zeste基因增彊子人類同源物(enhancer of zeste homolog 2,EZH2)對鼻嚥癌細胞增殖及侵襲能力的影響及其分子機製.方法 構建特異性的短髮夾狀(short hairpin RNA,shRNA)EZH2慢病毒榦擾載體,轉染293FT細胞包裝成慢病毒後,感染鼻嚥癌細胞株5-8F.採用四甲基偶氮唑藍法、平闆剋隆形成實驗和流式細胞術檢測細胞增殖狀態及細胞週期的改變,利用細胞劃痕實驗及Invasion Chamber侵襲小室測定法檢測遷移和侵襲能力變化,熒光定量PCR及蛋白免疫印跡法檢測轉染EZH2基因後的mRNA和蛋白錶達水平改變及上皮間質轉化相關標誌物的變化.結果 成功構建EZH2 shRNA慢病毒榦擾載體,包裝的慢病毒感染鼻嚥癌細胞株5-8F後,EZH2的mRNA和蛋白錶達水平顯著下調.四甲基偶氮唑藍法顯示,細胞接種96 h後,5-8F/shEZH2組的生長速度較空載組顯著下降(P＜0.001),平均(x±s,下同)細胞剋隆形成率為(31.56±7.49)％明顯小于空載組的(84.44±7.12)％,差異有統計學意義(P=0.001)；細胞週期結果錶明,沉默EZH2錶達的細胞增殖受抑,主要通過誘導G0/G1期阻滯,減少S期細胞的比例；基因轉染沉默EZH2後,鼻嚥癌細胞的運動遷移能力顯著下降,5-8 F/shEZH2組細胞劃痕48 h的相對遷移率為(0.58±0.05)明顯低于空載組的(0.81±0.02),差異有統計學意義(P ＜0.000)；體外侵襲能力明顯降低,5-8F/shEZH2組穿膜細胞數(72.23 ±4.08)箇,與其相對應空載組(150.95 ±16.27)箇比較,明顯減少(P＜0.000)；5-8F/shEZH2細胞的上皮標誌物上皮性鈣黏附蛋白(E-cadherin)、細胞角蛋白18( Keratin 18)的mRNA水平分彆上調1.77倍、1.58倍,間質標誌物p-連環蛋白(β-catenin)、神經性鈣黏附蛋白(N-cadherin)分彆下調18.04％、41.18％；蛋白免疫印跡證實此四者的蛋白水平錶達亦齣現相同趨勢.結論 EZH2可顯著促進鼻嚥癌細胞的體外增殖和侵襲能力,其機製可能是通過誘導上皮間質轉化促進鼻嚥癌轉移.
목적 탐토과승zeste기인증강자인류동원물(enhancer of zeste homolog 2,EZH2)대비인암세포증식급침습능력적영향급기분자궤제.방법 구건특이성적단발협상(short hairpin RNA,shRNA)EZH2만병독간우재체,전염293FT세포포장성만병독후,감염비인암세포주5-8F.채용사갑기우담서람법、평판극륭형성실험화류식세포술검측세포증식상태급세포주기적개변,이용세포화흔실험급Invasion Chamber침습소실측정법검측천이화침습능력변화,형광정량PCR급단백면역인적법검측전염EZH2기인후적mRNA화단백표체수평개변급상피간질전화상관표지물적변화.결과 성공구건EZH2 shRNA만병독간우재체,포장적만병독감염비인암세포주5-8F후,EZH2적mRNA화단백표체수평현저하조.사갑기우담서람법현시,세포접충96 h후,5-8F/shEZH2조적생장속도교공재조현저하강(P＜0.001),평균(x±s,하동)세포극륭형성솔위(31.56±7.49)％명현소우공재조적(84.44±7.12)％,차이유통계학의의(P=0.001)；세포주기결과표명,침묵EZH2표체적세포증식수억,주요통과유도G0/G1기조체,감소S기세포적비례；기인전염침묵EZH2후,비인암세포적운동천이능력현저하강,5-8 F/shEZH2조세포화흔48 h적상대천이솔위(0.58±0.05)명현저우공재조적(0.81±0.02),차이유통계학의의(P ＜0.000)；체외침습능력명현강저,5-8F/shEZH2조천막세포수(72.23 ±4.08)개,여기상대응공재조(150.95 ±16.27)개비교,명현감소(P＜0.000)；5-8F/shEZH2세포적상피표지물상피성개점부단백(E-cadherin)、세포각단백18( Keratin 18)적mRNA수평분별상조1.77배、1.58배,간질표지물p-련배단백(β-catenin)、신경성개점부단백(N-cadherin)분별하조18.04％、41.18％；단백면역인적증실차사자적단백수평표체역출현상동추세.결론 EZH2가현저촉진비인암세포적체외증식화침습능력,기궤제가능시통과유도상피간질전화촉진비인암전이.
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-SF/ShEZH2 cells and control cells was 0.58 ± 0.05,and 0.81 ± 0.02,respecptively(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. Conclusion EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro,which might be mediated by inducing EMT.
