摘要
目的:本课题利用RNA干扰(RNAi)技术,研究短发夹RNA(siRNA)抑制人端粒酶逆转录酶(hTERT)基因表达对鼻咽癌细胞生长的抑制作用。方法:根据hTERT cDNA序列构建表达hTERT mRNA的特异的、含荧光素基因的真核表达载体,包装成慢病毒。实时定量PCR及Western blot分析染细胞hTERT mR-NA及蛋白表达水平。MTT实验检测转染细胞的体外增殖速度。流式细胞仪检测细胞的生长周期。体外细胞侵袭实验检测比较各组的侵袭能力。结果:RT-PCR和Western blot检测显示,hTERT siRNA能显著下调hTERT mRNA和蛋白水平,尤其hTERT siRNA1,siRNA处理后的肿瘤细胞增殖受到抑制,细胞迁移、侵袭能力也显著降低。siRNA组和空病毒组(NC组)分别与空白细胞组(control组)之间比较,siRNA组与control组瘤细胞生长差异有统计学意义(P<0.01),NC组与control组之间瘤细胞生长差异无统计学意义(P>0.05)。结论:慢病毒载体沉默hTERT基因可以显著抑制鼻咽癌细胞的生长;hTERT siRNA可有效抑制鼻咽癌细胞的增殖、迁移和侵袭;提示hTERT可能成为抑制鼻咽癌细胞增殖和转移的新的分子靶点。
Objective：This study is to explore the inhibitory effect of silencing hTERT gene by short-hairpin RNA on growth of nasopharyngeal carcinoma xenograft in nude mice with RNAi technique.Method：Construction and expression of hTERT cDNA sequence according to the specific hTERT mRNA,including fluorescein eukaryotic expression vector,packaged into a lentivirus.qPCR and Western blot analyzed hTERT mRNA and protein levels in transfected cells.Proliferation rate of transfected cells was determined by MTT assay in vitro.Cell growth cycle was detected by flow cytometry.The invasiveness of each group was compared using in vitro cell invasion assay.Result：RT-PCR and Western blot analysis showed that,hTERT siRNA significantly reduced hTERT mRNA and protein levels,especially hTERT siRNA1.siRNA treatment inhibited tumor cell proliferation,and cell migration and invasiveness were significantly lower.Tumor cell growth rate was significantly different between control group and siRNA group（P0.01） while tumor cell growth rate in empty virus group（NC group） and control group was not significantly different（P0.05）.Conclusion：Lentivirus containing specific sequences of hTERT gene could significantly inhibit the growth of nasopharyngeal carcinoma cells line.hTERT siRNA expression vector can effectively inhibit NPC cell proliferation,migration and invasion,which may provide a novel molecular targets for gene therapy of nasopharyngeal carcinoma.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2011年第20期939-943,共5页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
国家自然科学基金资助项目(No:30973300)
关键词
鼻咽肿瘤
人端粒酶逆转录酶
肿瘤
鳞状细胞
慢病毒
基因沉默
nasopharyngeal neoplasms
human telomerase reverse transcriptase
neoplasms
squamous cells
lentivirus
siRNA
