沉默Livin基因增加5-氟尿嘧啶诱导Cal27凋亡敏感性的研究

Study on silencing Livin gene to improve the apoptosis sensitivity of Cal27 to 5-fluorouracil

目的:通过shRNA降低Livin基因的表达,研究口腔癌细胞Cal27对化疗药物5-氟尿嘧啶(5-fluorouracil,5-FU)敏感性的改变,并探索其可能的机制,为口腔癌化疗耐受的靶向治疗提供新的治疗思路。方法:设计针对Livin基因的shRNA序列,构建慢病毒shRNA表达载体,包装慢病毒颗粒并感染Cal27细胞,获得稳定沉默Livin基因的细胞株,使用20、40、60μmmol/L的5-FU处理Livin沉默细胞和对照细胞24 h,通过流式细胞术测定细胞凋亡率,MTT法检测细胞活力。结果:成功构建了针对Livin的shRNA质粒,并将其包装为慢病毒;降低Livin的表达水平后,Cal27细胞对5-FU的敏感性上升,凋亡率提高,细胞活力下降,Caspase 3表达量增高(均P<0.05)。结论:通过慢病毒介导的shRNA降低了Cal27细胞Livin基因的表达,增加了Cal27对5-FU的敏感性,其机制与Livin对Caspase 3的抑制效果有关,这为口腔癌化疗耐受问题提供了新的治疗靶点。

Objective:To explore changing the sensitivity of Cal27 to 5-fluorouracil(5-FU)by downregulating the expression of Livin gene through shRNA and its potential mechanism,thus to provide new ideas to solve the chemoresistance of oral cancer.Method:ShRNA sequence targeting Livin was designed,and constructed into lentivirus expressing vector.Then,lentivirus was packed for affecting into Cal27 to get the stable Livin-silencing cell strain.After Cal27 cells in both control group and Livin-silencing group were treated with concentrations of 20,40 and 60μmmol/L 5-FU for 24 hours,the cell apoptosis rate and cell viability were measured by flow cytometry and MTT respectively.Results:The designed shRNA plasmid targeting Livin were successfully constructed and packed into lentivirus.After the expression level of Livin was knocked down,the sensitivity and cell apoptosis rate of Cal27 to 5-FU were elevated dramatically,and the cell viability decreased significantly and the expression of Caspase 3 was significantly decreased(all P<0.05).Conclusion:The expression level of Livin gene in Cal27 was downregulated by transfection with shRNA lentivirus,which increased the sensitivity of Cal27 to 5-FU.The underlying mechanism may be associated with the inhibitory effect of Livin on Caspase 3,which will provide a new target for solving chemoresistance of oral cancers.