靶向Cdc6 RNAi抑制舌癌CAL-27细胞的增殖

Suppression of Cdc6-targeted RNA Interference on Proliferation of CAL-27 Tongue Cancer Cells

【目的】应用RNA干扰技术,靶向抑制舌癌细胞中Cdc6的表达,观察舌癌细胞增殖、凋亡等改变,初步探讨下调Cdc6表达抑制舌癌细胞增殖的分子机制。【方法】实验分4组:空白对照组CON,阴性对照组NC,实验组KD1和KD2。应用前期构建的Cdc6的慢病毒载体,转染舌癌CAL-27细胞,通过Real time RT-PCR和Western blot法检测Cdc6 mRNA及蛋白的表达;以MTT法测定活细胞数并绘制细胞生长曲线;利用流式细胞仪观察细胞凋亡。【结果】慢病毒载体转染CAL-27细胞后,Real time RT-PCR和Western blot实验结果显示:舌癌CAL-27细胞中Cdc6的表达明显被抑制,相对NC组,KD1、KD2组对Cdc6基因敲减效率分别为55.60%和64.38%。蛋白表达分别降低44.44%和66.67%;细胞生长曲线表明,细胞生长明显减缓,KD1和KD2组细胞生长抑制率分别为33.68%和35.79%;流式细胞仪检测细胞周期显示:KD1组、KD2组两组细胞凋亡率较NC组均有显著上升,分别为57.55%和83.81%。【结论】靶向Cdc6慢病毒载体RNA干扰能有效抑制舌癌CAL-27细胞的DNA复制,阻止舌癌细胞的增殖。

【Objective】 RNAi technique was applied to inhibit the expression of the targeted Cdc6 in the tongue cancer cells to observe the proliferation and apoptosis of the tongue cancer cells.【Methods】 The experiment divides to four groups： CON,NC,KD1,and KD2.Lentiviral vector transfects CAL-27 cells,real-time PCR and Western blot were used to examine the knock-down effects of the RNA interference on the levels of expression of Cdc6 mRNA and protein.The MTT method was used to examine the growth levels of CAL-27 cells.Flow cytometry was used to examine the cell cycle changes and apoptosis rates.【Results】 The real-time PCR and Western blot results showed that KD1 and KD2 targets had significant knock-down effects on the expression of Cdc6.Compared with the NC group,the knockdown efficiencies of KD1 and KD2 on the Cdc6 gene were 33.68% and 35.79%,the levels of expression of Cdc6 protein were reduced by 44.44% and 66.67%,respectively.The cell growth levels in the KD1 and KD2 groups were inhibited,the cell growth inhibition rate in the KD1 and KD2 groups were 34.78% and 36.52%.Compared with the NC group,the apoptosis rates of CAL-27 cells were raised by 57.55% and 83.81% in the KD1 and KD2 groups.【Conclusions】 The Cdc6-targeted lentiviral vector RNAi can effectively inhibit DNA replication of CAL-27 cells,preventing the proliferation of tongue cancer cells.