小RNA干扰细胞周期蛋白Y基因表达对喉癌Hep-2细胞增殖的影响

Influence on cell proliferation by small interfering RNA of Cyclin Y expression in laryngeal cancer cells

目的利用RNA干扰技术下调细胞周期蛋白Y（CyclinY，CCNY）基因的表达，观察其对喉癌Hep-2细胞增殖的影响。方法构建针对CCNY基因的短发夹RNA（shorthairpinRNA，shRNA）真核表达载体，包装慢病毒感染喉癌Hep-2细胞。将细胞分为2组，对照组和实验组（感染CCNYshRNA慢病毒组）。运用Real—timePCR检测CCNY在RNA水平的变化；四甲基偶氮唑蓝法和平板克隆形成实验检测CCNY表达下调对细胞增殖的影响。结果成功包装表达CCNYshRNA的慢病毒并感染Hep-2细胞，shRNA下调CCNY的表达后，与对照组相比CCNY在mRNA水平的表达显著下降，抑制率为75．3％（P〈0．05）。表达CCNYshRNA的慢病毒感染5d后，继续培养6d，细胞的生长明显受到抑制（P〈0．001）。细胞在体外培养10d后形成克隆的潜力降低了60％。结论靶向CCNY基因RNA干扰能够阻抑喉癌Hep-2细胞的CCNY表达，抑制细胞增殖，CCNY基因有望成为喉癌基因治疗靶点。

Objective The effects of lentivirus-mediated suppression of Cyclin Y （CCNY） expression on the proliferation of laryngeal cancer cells were investigated in vitro. Methods The lentivirus vectors containing a small hairpin RNA （shRNA） to target CCNY were constructed. Hep-2 cells were divided into the following two experimental groups：the negative control group （control lcntivirus infected cells） and CCNY knockdown group （CCNY shRNA-expressing lentivirus infected cells）. After Hep-2 ceils were infected, Real-time PCR was used to measure CCNY expression. The influence of CCNY on the proliferation of laryngeal cancer cells were assessed using MTT and colony formation experiments. Each experiment was performed in triplicate and repeated three times. Results Lentiviruses expressing shRNA against CCNY were constructed and Hep-2 cells were infected with above mentioned lentivirus at MOI （Muhiplicity of infection） of 120. Real-time PCR analysis showed that the mRNA expression of CCNY in Hep-2 cells in the knockdown group was significantly decreased （P 〈 0. 05 ） ; the mRNA level of CCNY was 75.3% lower in the si-CCNY group than in the si-CTRL group. After 5 days of lentiviral infection, the cell viability was significantly lower in ceils infected with the CCNY-shRNA lentivirus compared to cells infected with thecontrol lentivilaas following a 6-day incubation. The colony number was decreased by 60% in Hep-2 cells infected with the CCNY-shRNA-lentivirus infected cells following a 10-day incubation. Conclusions The results suggested that lentivirus-mediated downregulation of CCNY expression decreased the proliferation and growth potency of laryngeal cancer cells. Lentiviruses delivering shRNA against CCNY may be a promising tool for laryngeal cancer therapy.