shRNA沉默eIF4E基因表达对人喉癌细胞Hep-2生物学行为的影响

Effects of shRNA-mediated Downregulation of eIF4E Expression on Biological Behaviors in Human Laryngeal Carcinoma Cell Line

目的构建靶向真核细胞翻译起始因子4E(eIF4E)的shRNA表达载体,观察eIF4E表达抑制对喉癌细胞生物学行为的影响。方法根据人eIF4E mRNA序列设计并构建2个靶向人eIF4E基因的shRNA真核表达载体(pSilencer4.1-s1和pSilencer4.1-s2);然后以脂质体转染的方法将shRNA表达质粒转染至人喉癌细胞系Hep-2,经G418筛选出稳定转染的阳性细胞。分别通过反转录-聚合酶链反应(RT-PCR)和Western blotting技术对构建成功的2个shRNA干扰质粒进行有效性筛选,分析其eIF4EmRNA及蛋白表达水平,并筛选出表达抑制最强的稳定转染喉癌细胞系(Hep-2-s2)。通过噻唑蓝(MTT)试验检测Hep-2-s2细胞的体外增殖活性,流式细胞仪技术(FCM)检测其细胞周期和凋亡变化情况。结果经双酶切证实转录shRNA的目的基因片段已成功克隆入pSilencer4.1-CMVneo真核表达载体中,经测序证明插入序列完全正确;eIF4E-shRNA2可以显著下调eIF4E mRNA及蛋白表达水平;eIF4E基因的表达下调明显抑制Hep-2细胞的体外增殖(P<0.05);Hep-2-s2细胞的G0/G1期细胞比例显著增加了(14.7±1.1)%,而S期细胞比例减少了(13.2±1.6)%(P<0.05);Hep-2-s2的细胞凋亡率显著增加到(18.3±1.7)%(P<0.05)。结论shRNA介导的eIF4E基因表达下调可显著降低喉癌细胞的增殖活性,同时诱导其细胞周期阻滞和凋亡率增加,为喉癌基因治疗筛选出了新的靶点。

Objective To construct the potent shRNA expressing vectors which target to human eukaryotic initiation factor 4E（eIF4E） and to investigate the effect of shRNA- mediated downregulation of eIF4E expression on the biological behaviors of laryngeal carcinoma cell line （Hep-2）. Methods Two shRNA expressing vectors （pSilencer4. 1-sl and pSilencer4.1- s2） targeting to human eIF4E mRNA were designed and successfully constructed. Then, the plasmids were transfccted into Hep-2, and the stable transfectant were selected by G418. The levels of eIF4E mRNA and protein expression were analyzed by RT-PCR and Western blotting assays. Next,MTT assays were performed to detect the effect of eIF4E downregulation on the in vitro proliferation and colony formation of Hep-2-s2. Followingly,FCM was performed to detect the changes of cell cycle and apoptosis. Results The two recombinant plasmid vectors expressing shRNA targeting eIF4E were identified by restriction enzyme assay and sequence analysis. RT-PCR and Western blotting assays showed that eIF4E- shRNA2 could significantly downregulate eIF4E expression in Hep-2 cells. The downregulation of eIF4E expression obviously led to in vitro tumor proliferation （P 〈 0. 05）. Moreover, the results of cell cycle detection showed that the percentage of Hep-2-s2 cells in the G1/G0 phase obviously increased by （14.7± 1.1）%, while the percentage of Hep-2- s2 cells in the S phase significantly reduced by （13.2± 1.6）% （P 〈0. 05）. Furthermore,analysis of apoptosis by FCM assay indicated that the apoptosis rate of Hep-2-s2 significantly increased to approximately （18.3 ± 1.7）% （ P 〈 0. 05）. Conclusion shRNA - mediated downregulation of eIF4E expression could significantly reduce the activity of cell proliferation both in vitro and in vivo,induce cell arrest and enhance apoptosis in Hep-2 cells. This lays a foundation for the potential of eIF4E as a therapeutic target in the treatment of human laryngeal carcinoma.