A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. In this study, the effect of antisense oligonucleotides (ODNs) by targeting human telomerase reverse transcriptase (hTERT) mRNA in a laryngeal cancer cell line (Hep-2) was investigated.A 20mer antisense oligodeoxynucleotide targeting the most open part of hTERT mRNA (anti-hTERT) and a mismatched control sequence were synthesized. Cells were treated daily with oligonucleotides for up to 72 hours. hTERT mRNA expression was measured by the reverse transcription polymerase chain reaction (RT-PCR) assay; telomerase activity by the telomerase PCR ELISA assay kit (TRAP; Boehringer Mannheim, GmbH, Mannheim, Germany). Cell viability after administration of ODNs was determined using the MTT assay. Morphological changes were examined by haematoxylin and eosin staining.The cell cycle was analyzed using flow cytometry. It was found that antisense treatment induced a decrease in hTERT mRNA expression, telomerase activity, cell growth rate, cell viability, and an increase in apoptosis. The results suggest that inhibition of telomerase activity in Hep-2 cells by short-term antisense treatment against the mRNA of hTERT results in apoptotic cell death. The treatment with anti-hTERT may be useful as a treatment modality for laryngeal squamous carcinoma.

Objective: Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression and telomerase activity in laryngeal cancer cells. Design: Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against 2 different hTERT sites, control vectors that included mismatched shRNA and those without shRNA. The expression of hTERT was determined by rsetranscriptase polymerase chain reaction and Western blotting. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay. The cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay. Results: We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects. Conclusion: Our results suggest that shRNA directed against hTERT inhibits telomerase activity through suppression of the hTERT expression in laryngeal cancer cells and that RNA interfering technology may be a promising strategy for the treatment of laryngeal cancers.

目的：探讨RNA干扰hTERT（人端粒酶逆转录酶）基因对人喉鳞状细胞癌的治疗作用。方法：根据hTERT cDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1、pshRNA2。将shRNA质粒分别转染人喉癌Hep-2细胞株及荷瘤裸鼠瘤体内。以激光共聚焦显微镜观察质粒在Hep-2细胞及瘤体内的表达；以MTT法观察质粒对Hep-2细胞增殖的抑制作用；以蛋白印迹法（Western blot法）检测Hep-2细胞中hTERT蛋白的表达；以免疫组化SP 法检测裸鼠瘤体内hTERT蛋白的表达。结果：pshRNA1、pshRNA2转染Hep-2细胞及pshRNA1转染瘤体后，共聚焦显微镜下见大量的癌细胞表达绿色荧光，hTERT蛋白表达明显下降。细胞受pshRNA1、pshRNA2转染后, 其生长活性受到明显抑制。体内抑瘤实验表明，与空质粒载体组（pshRNA4）和生理盐水组相比，pshRNA1 组移植瘤生长明显受到抑制。结论：表达shRNA的、hTERT基因特异的真核表达质粒能有效转染体内、外喉鳞癌细胞，并可有效抑制人喉鳞癌细胞的生长，为今后应用RNA干扰基因治疗喉癌提供重要的实验参考。

目的：探讨应用RNA干扰技术抑制Hep-2细胞人端粒酶逆转录酶（hTERT）基因对C-myc蛋白表达的影响。方法：根据hTERT eDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1，随机选取一段与人类基因无同源性的碱基序列构建表达shRNA 的含荧光素基因的对照质粒pshRNA2，采用METAFECTENE作为转染试剂。分别以质粒pshRNA1、pshRNA2及空白培养液处理喉癌Hew2细胞。应用RT-PCR法检测hTERT基因的表达，Western Blot法检测hTERT和C-myc蛋白表达水平。结果：质粒pshRNA1转染组hTERT mRNA及蛋白表达水平显著低于其他处理组及空白对照组（P<O.05）；质粒pshRNA1转染组C-myc蛋白表达水平显著高于其他处理组及空白对照组（P<O.01）。结论：RNA 干扰抑制人端粒酶逆转录酶活性使喉癌Hep-2细胞C-myc蛋白表达升高，其确切的机制有待进一步研究。

目的：探讨核因子κB/p65（NF-κB/p65）和环氧合酶2（COX-2）在喉鳞状细胞癌（LSCC）中的表达和临床意义及两者间可能存在的相互关系。方法：采用免疫组织化学SP法检测41例LSCC和10例声带息肉组织中NF-κB/p65和COX-2蛋白的表达水平。结果：NF-κB/p65和COX-2蛋白在喉癌组与对照的声带息肉组比较有显著性差异。进一步定性分析发现COX-2与喉癌的组织病理学分化程度有关。Spearman相关分析显示NF-κB/p65与COX-2高度相关。结论：①喉癌中NF-κB/p65与COX-2的表达均升高。②NF-κB/p65可能促使COX-2的表达。③NF-κB/p65可能成为喉癌治疗的新靶点。