小RNA干扰技术靶向沉默CDK6对喉癌细胞生物学特性的影响  

Effect of Small RNA Interference Technique Targeting Silencing CDK6 on Biological Characteristics of Laryngeal Cancer Cells

作　　者：何克剑 侯晓强[1,2] 尹龙[1,2] HE Kejian;HOU Xiaoqiang;YIN Long(The First College of Clinical Medical Science,China Three Gorges University,Yichang,Hubei,443000,China;Department of Rheumatology,Yichang Central People's Hospital,Yichang,Hubei,443000,China)

机构地区：[1]三峡大学第一临床医学院,湖北省宜昌市443000 [2]宜昌市中心人民医院风湿免疫科,湖北省宜昌市443003

出　　处：《医学分子生物学杂志》2019年第6期582-586,共5页Journal of Medical Molecular Biology

摘　　要：目的研究小RNA干扰技术靶向沉默周期蛋白依赖性激酶6(CDK6)对喉癌细胞生物学特性的影响.方法在喉癌细胞中依次转染CDK6 siRNA1和CDK6 siRNA2,用荧光定量PCR和Western印迹测定转染效率,筛选干扰效果较好的CDK6 siRNA2作为下调CDK6的小RNA.以CCK-8法测定细胞的增殖情况,以流式细胞术测定细胞周期分布情况及细胞凋亡情况,以Western印迹法测定细胞中细胞周期蛋白依赖性激酶4(CDK4)、细胞周期蛋白D1(CCND1)、剪切型Caspase-3(Cleaved Caspase-3)、细胞周期蛋白依赖性激酶抑制剂1A(p21)蛋白水平.结果CDK6 siRNA1和CDK6 siRNA2都可以下调喉癌细胞中CDK6的转录和表达,并且CDK6 siRNA2转染后下调水平更多.下调CDK6后的喉癌细胞的增殖能力下降,细胞G1期比例增多,同时细胞凋亡率也增加,细胞中周期促进因子CDK4、CCND1蛋白水平降低,周期抑制因子p21蛋白水平升高,细胞中Cleaved Caspase-3水平升高,与没有转染的细胞比较有显著性差异(P<0.05).结论沉默CDK6能够在体外抑制喉癌细胞生长,阻滞喉癌细胞周期,诱导喉癌细胞凋亡.Objective To study the effect of small RNA interference targeting silencing CDK6 on the biological characteristics of laryngeal cancer cells.Methods CDK6 siRNA1 and CDK6 siR-NA2 were transfected in the larynx cells,the transfection efficiency was measured by fluorescence quantitative PCR and Western blotting,the CDK6 siRNA2 with better interference effect was used as a small RNA for down-regulation of CDK6.The proliferation of cells was measured by CCK-8 method.Cell cycle distribution and apoptosis were measured by flow cytometry.The levels of CDK4,CCND1,Cleaved Caspase-3 and p21 in cells were measured by Western blotting.Results Both CDK6 siRNA1 and CDK6 siRNA2 downregulated the transcription and expression of CDK6 in the larynx cells,and the level of CDK6 siRNA2 was more downregulated after transfection.The prolifer-ation ability of laryngeal cancer cells decreased after CDK6 reduction,the proportion of cells at G1 phase increased,at the same time,the rate of cell apoptosis was also increased,the level of cycli-cal promoter CDK4 and CCND1 protein in cells decreased,the level of cyclical inhibitory factor p21 protein increased,the level of Cleaved Caspase-3 in the cells was elevated,compared with the cells without transfection,the differences were statistically significant(P<0.05).Conclusion Silen-cing CDK6 can inhibit the growth of laryngeal cancer cellsin vitro,block the cell cycle of larynx cancer and induce the apoptosis of laryngeal cancer cells.

关 键 词：喉癌 细胞周期 凋亡 CDK6 

分 类 号：Q522[生物学—生物化学]