摘要
目的应用RNA干扰技术将FAK基因表达沉默后,观察人舌鳞癌细胞株CAL-27凋亡和侵袭能力的变化。方法使用RNAi技术构建3对FAK siRNA后,瞬时转染细胞。运用实时定量聚合酶链反应(qPCR)法检测FAK mRNA的表达情况,筛选出沉默效率最佳的siRNA用于后续实验。运用蛋白免疫印迹法(Western blot)法检测细胞FAK蛋白的表达情况,流式细胞仪观察细胞的凋亡情况,Transwell侵袭实验观察细胞体外侵袭能力的变化。结果 qPCR实验结果显示转染siRNA-1组、siRNA-2组、siRNA-3组FAK mRNA表达水平明显低于阴性对照组和空白对照组(P<0.01),其中以siRNA-3沉默效率最高;Western blot结果显示转染组细胞FAK蛋白表达水平明显低于阴性对照组和空白对照组(P<0.05);流式细胞仪检测结果显示转染组细胞凋亡率明显高于阴性对照组和空白对照组(P<0.01);Transwell法实验结果显示转染组细胞穿膜的数量明显低于阴性对照组和空白对照组(P<0.05)。结论沉默FAK基因表达可诱导舌鳞癌细胞CAL-27的凋亡,有效抑制其侵袭能力。
Objective To investigate the impacts of FAK gene silencing on the invasion and apoptosis of human tongue squamous cell carcinoma cell lines CAL-27.Methods Three pairs of FAK siRNA were constructed by the siRNA interference technology,then transiently transfected cells.The expression of FAK mRNA was detected by real-time quantitative polymerase chain reaction(qPCR),selected the most efficient silencing siRNA for follow-up experiments.The expression of FAK protein was detected by Western blot the cell apoptosis was measured by using flow cytometry,the change of cell invasion ability was detected by using Transwell insert.Results The relative levels of FAK mRNA in the siRNA-1 group,the siRNA-2 group and the siRNA-3 group were lower than that in the negative control group and the blank control group(P<0.01).Among them,siRNA-3 was the most efficient.The relative level of FAK protein in the transfected group was lower than that in the negative control group and the blank control group(P<0.05).The apoptosis rate in the transfected group was higher,compared with the negative control group and the blank control group(P<0.01).Transwell insert invasion assay shouwed that the number of transmembrane cells in the transfected group was less than thoes in the negative control group and the blank control group(P<0.05).Conclusion FAK gene silencing can effectively induce the apoptosis and inhibit the invasion of human tongue squamous cell carcinoma cell lines CAL-27.
作者
陈凯
廖晓颖
吴妹娟
康成容
潘宣
CHEN Kai;LIAO Xiaoying;WU Meijuan;KANG Chengrong;PAN Xuan(Department of stomatology,the First Affiliated Hospital of Guangdong Pharmaceutical University,Guangzhou,Guangdong 510080,China)
出处
《重庆医学》
CAS
2018年第20期2645-2648,共4页
Chongqing medicine
基金
广东省科技发展专项基金项目(2016A020215154)
广东省医学科研基金项目(C2015031)
广东省省级科技计划项目(2016ZC0177)
关键词
舌鳞癌细胞株
黏着斑激酶
RNA干扰
凋亡
侵袭
tongue squamous cell carcinoma
focal adhesion kinase
RNA interference
apoptosis
invasion
