CAJ | 학술논문

目的研究EB病毒核抗原1(Epstein-Barr virus nuclear antigen 1,EBNA1)对人鼻咽癌细胞增殖及细胞周期的影响.方法构建特异性的短发夹RNA(short hairpin RNA,shRNA)EBNA1慢病毒干扰载体,包装慢病毒后感染过表达EBNA1的鼻咽癌细胞C666-EBNA1(简写为CE).应用四甲基偶氮唑蓝法(MTT)和细胞计数法检测细胞增殖状态,流式细胞术、荧光定量PCR及蛋白免疫印迹法检测细胞周期及相关调控因子变化.结果成功构建含shRNA EBNA1的重组慢病毒,其感染可显著下调CE细胞中EBNA1表达.细胞计数和MTT均证明CE-shRNA细胞的增殖能力较CE-对照组显著下降(P值均＜0.05).细胞周期提示干扰EBNA1后CE细胞G0-G1期比例由(62.43±6.62)%升高到(89.66±0.64)%,两者比较差异有统计学意义(t=-7.091,P=0.002),S期比例由(34.93±7.36)%下降为(7.82±2.44)%,两者比较差异有统计学意义(t=6.095,P=0.004),G2-M期无明显变化(t=0.090,P=0.933).抑制EBNA1后,c-myc、CDK4、CDK6、pRb的mRNA水平分别下调65.60%、34.06%、41.05%、55.29%.蛋白免疫印迹法证实抑制EBNA1后4种蛋白的表达亦下调.结论沉默EBNA1基因可抑制鼻咽癌细胞增殖,其机制之一可能是通过影响c-myc、CDK4、CDK6、pRb等基因的表达使鼻咽癌细胞阻滞于G0-G1期.
목적 연구EB병독핵항원1(Epstein-Barr virus nuclear antigen 1,EBNA1)대인비인암세포증식급세포주기적영향.방법 구건특이성적단발협RNA(short hairpin RNA,shRNA)EBNA1만병독간우재체,포장만병독후감염과표체EBNA1적비인암세포C666-EBNA1(간사위CE).응용사갑기우담서람법(MTT)화세포계수법검측세포증식상태,류식세포술、형광정량PCR급단백면역인적법검측세포주기급상관조공인자변화.결과 성공구건함shRNA EBNA1적중조만병독,기감염가현저하조CE세포중EBNA1표체.세포계수화MTT균증명CE-shRNA세포적증식능력교CE-대조조현저하강(P치균＜0.05).세포주기제시간우EBNA1후CE세포G0-G1기비례유(62.43±6.62)%승고도(89.66±0.64)%,량자비교차이유통계학의의(t=-7.091,P=0.002),S기비례유(34.93±7.36)%하강위(7.82±2.44)%,량자비교차이유통계학의의(t=6.095,P=0.004),G2-M기무명현변화(t=0.090,P=0.933).억제EBNA1후,c-myc、CDK4、CDK6、pRb적mRNA수평분별하조65.60%、34.06%、41.05%、55.29%.단백면역인적법증실억제EBNA1후4충단백적표체역하조.결론 침묵EBNA1기인가억제비인암세포증식,기궤제지일가능시통과영향c-myc、CDK4、CDK6、pRb등기인적표체사비인암세포조체우G0-G1기.
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.