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目的构建沉默环氧化酶-2(COX-2)基因重组慢病毒,观察其体外侵袭的抑制作用,从而探讨干扰COX-2抑制喉癌细胞增殖的作用机理,为喉癌的治疗提供新的思路。方法逆转录聚合酶链反应(RT-PCR)检测COX-2基因在人表皮样喉癌细胞(Hep-2)中的表达情况。利用上海吉凯公司RNA干扰(RNAi)慢病毒表达载体系统,构建针对COX-2基因慢病毒RNAi表达载体。转染Hep-2细胞,干扰COX-2基因的表达,实时定量PCR检测干扰前后基因表达变化。利用生长曲线测定干扰载体转染前后细胞生长速度变化。流式细胞仪检测细胞的生长周期。Boyden侵袭小室法测定体外侵袭力。结果成功构建了COX-2慢病毒RNAi表达载体,并建立了干扰COX-2基因的Hep-2细胞系。实时定量PCR检测COX-2基因在Hep-2细胞系中过表达被显著抑制。生长曲线测定,COX-2基因干扰后细胞增殖明显变慢。流式细胞仪检测细胞的生长周期可见干扰组诱导Hep-2细胞凋亡,转染G0～G1期细胞数量明显上升,S期细胞减少,表明siRNA干扰Hep-2细胞后,细胞由G0～G1期进入到S期受到阻滞,细胞增殖速度下降。体外侵袭实验中,Hep-2-AS侵袭细胞数(31.0±1.8)显著低于Hep-2细胞(104.0±2.6)及Hep-2-P细胞(99.0±2.7),差异有统计学意义(P<0.05)。结论喉癌中过表达的COX-2基因被干扰后表达明显降低并显著抑制细胞的生长速度和侵袭能力。同时验证了COX-2基因RNA干扰在进行抗肿瘤的治疗中潜在的应用前景。
OBJECTIVE: To construct a recombinant lentivirus with COX-2 gene silencing and observe the inhibitory effect of COX-2 gene on the invasion of laryngeal squamous cell carcinoma Ideas. Methods The expression of COX-2 gene in human epidermoid laryngeal carcinoma cells (Hep-2) was detected by reverse transcription-polymerase chain reaction (RT-PCR). RNA interference (RNAi) lentiviral expression vector system was used to construct RNAi expression vector targeting COX-2 lentivirus. Transfection of Hep-2 cells interferes with COX-2 gene expression, real-time quantitative PCR detection of gene expression changes before and after interference. Growth curve was used to determine the change of cell growth rate before and after interference with vector. Flow cytometry to detect cell growth cycle. Boyden invasion chamber assay for in vitro invasiveness. Results The RNAi expression vector of COX-2 lentivirus was successfully constructed and the Hep-2 cell line which interfered with COX-2 gene was established. Quantitative real-time PCR detection of COX-2 gene over-expression in Hep-2 cell lines was significantly inhibited. Growth curve determination, COX-2 gene interference significantly slowed cell proliferation. The number of cells transfected with G0-G1 phase increased significantly and the number of S phase cells decreased when siRNA was used to detect Hep-2 cells. Flow cytometry showed that the number of cells transfected by G0-G1 S phase into the S phase was blocked, cell proliferation decreased. The invasiveness of Hep-2-AS cells in vitro (31.0 ± 1.8) was significantly lower than that in Hep-2 cells (104.0 ± 2.6) and Hep-2-P cells (99.0 ± 2.7) <0.05). Conclusion The COX-2 gene overexpression in laryngeal carcinoma was significantly down-regulated and significantly inhibited the growth and invasion ability of COX-2 cells. At the same time, the potential application of COX-2 RNA interference in the treatment of anti-tumor was validated.