Chen Shi-ming, Tao Ze-zhang, Xiao Bo-kui, Pan Song, Liu Dan, Chi Hua-ming
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Zhonghua Bing Li Xue Za Zhi. 2005 Dec;34(12):796-800.
To investigate the effect of RNA interference by targeting human telomerase reverse transcriptase (hTERT) mRNA in the larynx cancer cell line, Hep-2.
The primary structures of hTERT cDNA were found in GenBank. Then the structure analysis were done according to RNAi strategy which determined the specific base sequences to design shRNA plasmid. Two types of plasmid, pshRNA1 and pshRNA2, involved in fluorescein gene were synthesized based on the specific base sequences. Control pshRNA3, a random sequence, and control pshRNA4, without additional specific sequence were also constructed. Cells were treated daily with pshRNA1-4 or normal culture medium respectively. The pshRNA1-3 was identified by electrophoresis. After administration of pshRNA1-4, fluorescence expression was detected by confocal microscopy, the expression of hTERT of the transfected cells was determined by Western blotting, telomerase activity was measured by TRAP-PCR ELISA, cell viability was determined by MTT assay, morphological changes and apoptosis were examined by inverted microscope and TUNEL respectively.
There was a 400 bp balteum in pshRNA1-3 after cut by SalI, which was identical with the size of the objective gene. Many cells presented green fluorescence after being treated by pshRNA1-4, but there are much more dead green fluorescent cells in the pshRNA1 and pshRNA2 group. hTERT protein and telomerase activity was significantly decreased after treated by pshRNA1 or pshRNA2. It was observed that treatment with pshRNA1 or pshRNA2 in the presence of a valid transfection reagent could reduce cell viability of Hep-2 cells within 96 h (P < 0.01). Under the same culture conditions, cells grew more sparsely and the number of apoptotic cell increased significantly.
shRNA plasmid directed against human telomerase reverse transcriptase can effectively transfect Hep-2 cells. shRNA targeted hTERT gene can significantly inhibit the growth and proliferation of Hep-2 cells, which results in apoptotic cell death. RNA interference may be a promising strategy for the treatment of laryngeal cancer.
研究针对人端粒酶逆转录酶（hTERT）mRNA的RNA干扰对喉癌细胞系Hep-2的影响。
在GenBank中查找hTERT cDNA的一级结构。然后根据RNAi策略进行结构分析，确定特定碱基序列以设计shRNA质粒。基于特定碱基序列合成了两种携带荧光素基因的质粒pshRNA1和pshRNA2。还构建了对照pshRNA3（随机序列）和对照pshRNA4（无额外特定序列）。细胞分别每天用pshRNA1 - 4或正常培养基处理。通过电泳鉴定pshRNA1 - 3。给予pshRNA1 - 4后，用共聚焦显微镜检测荧光表达，用蛋白质免疫印迹法测定转染细胞中hTERT的表达，用端粒重复序列扩增 - 聚合酶链反应酶联免疫吸附测定法（TRAP - PCR ELISA）测量端粒酶活性，用MTT法测定细胞活力，用倒置显微镜和TUNEL分别检测形态变化和细胞凋亡。
用SalI切割后，pshRNA1 - 3中有一条400 bp的条带，与目的基因大小一致。用pshRNA1 - 4处理后，许多细胞呈现绿色荧光，但pshRNA1和pshRNA2组中死亡的绿色荧光细胞更多。用pshRNA
