Yao Xiaobao, Wang Xiaoria, Zhang Shaoqiang, Yan Liying, Zhu Hongliang
Department of Otolaryngology, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, 710061, China.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2011 Oct;25(20):939-43.
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.
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 hTER.T 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.
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 (P < 0.01) while tumor cell growth rate in empty virus group(NC group) and control group was not significantly different (P > 0.05).
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.
本研究旨在利用RNA干扰技术，探讨短发夹RNA沉默hTERT基因对裸鼠鼻咽癌移植瘤生长的抑制作用。
根据hTERT特异性mRNA构建并表达hTERT cDNA序列，包括荧光素真核表达载体，包装成慢病毒。采用qPCR和蛋白质免疫印迹法分析转染细胞中hTERT mRNA和蛋白质水平。通过MTT法体外测定转染细胞的增殖率。采用流式细胞术检测细胞生长周期。利用体外细胞侵袭试验比较各组的侵袭能力。
RT-PCR和蛋白质免疫印迹分析表明，hTERT siRNA显著降低hTERT mRNA和蛋白质水平，尤其是hTERT siRNA1。siRNA处理抑制肿瘤细胞增殖，细胞迁移和侵袭能力显著降低。对照组与siRNA组肿瘤细胞生长速率差异有统计学意义（P < 0.01），而空病毒组（NC组）与对照组肿瘤细胞生长速率差异无统计学意义（P > 0.05）。
携带hTERT基因特异性序列的慢病毒可显著抑制鼻咽癌细胞系的生长。hTERT siRNA表达载体可有效抑制鼻咽癌细胞的增殖、迁移和侵袭，可能为鼻咽癌的基因治疗提供新的分子靶点。
