摘要
目的:探讨靶向沉默肝细胞生长因子受体(c-Met)基因对膀胱癌T24细胞放射敏感性的影响及作用机制。方法:通过脂质体将特异性c-Met siRNA1和c-Met siRNA2转染至膀胱癌T24细胞,设为si-c-Met1组和si-c-Met2组,转染非特异性c-Met siRNA的T24细胞设为NC组,同时设置空白对照组(Blank组),采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)检测各组T24细胞c-Met的表达,噻唑蓝(MTT)法检测各组细胞增殖情况,克隆形成实验检测各组细胞对放射敏感性的影响,流式细胞术检测放射后细胞凋亡变化,Western blot检测放射对细胞中剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、Bcl-2相关X蛋白(Bax)、磷脂酰肌醇-3激酶(PI3K)和磷酸化的蛋白激酶B(p-AKT)蛋白表达的影响。结果:si-c-Met1组和si-c-Met2组细胞中c-Met mRNA和蛋白表达较Blank组明显降低(P<0.05),转染c-Met siRNA2干扰效果更好。靶向沉默c-Met基因对细胞增殖能力无显著影响。si-c-Met2组细胞克隆形成数、D0值和SF2值均低于Blank组(P<0.05)。放射后si-c-Met2组细胞凋亡率显著高于Blank组(P<0.05)。放射后si-c-Met2组细胞中Cleaved Caspase-3和Bax的表达显著高于Blank组(P<0.05),PI3K和p-AKT的表达显著低于Blank组(P<0.05)。NC组和Blank组间以上各指标比较均无显著差异(P>0.05)。结论:靶向沉默c-Met基因可增加膀胱癌T24细胞对放射的敏感性,其作用机制可能与抑制PI3K/AKT信号通路的激活有关。
Objective:To investigate the effect and mechanism of targeted silencing of c-Met gene on radiosensitivity of bladder cancer T24 cells.Methods:Specific c-Met siRNA1 and c-Met siRNA2 were transfected into bladder cancer T24 cells by liposome,and were set as si-c-Met1 group and si-c-Met2 group,transfected with non-specific c-Met siRNA T24 cells were set as NC group,and blank control group(Blank group)was set up.Real-time quantitative PCR and Western blot were used to detect the expression of c-Met in each group of T24 cells.The cell proliferation of each group were detected by the MTT method.The effect of each group on the radiosensitivity was detected by colony formation assay.The apoptosis of the cells was detected by flow cytometry.The effect of radiation on the expression of Cleaved Caspase-3,Bax,PI3K and p-AKT protein in cells was detected by Western blot.Results:The expression of c-Met mRNA and protein in si-c-Met1 group and si-c-Met2 group were significantly lower than that in Blank group(P<0.05).The effect of transfection with c-Met siRNA2 was better.Targeted silencing of the c-Met gene had no significant effect on cell proliferation.The number of cell clone formation,D0 value and SF2 value in si-c-Met2 group were lower than those in Blank group(P<0.05).The apoptosis rate of si-c-Met2 group was significantly higher than that of Blank group(P<0.05).The expression of Cleaved Caspase-3 and Bax in the si-c-Met2 group was significantly higher than that in the Blank group(P<0.05),and the expression of PI3K and p-AKT was significantly lower than that in the Blank group(P<0.05).There was no significant difference between the NC group and the Blank group(P>0.05).Conclusion:Targeted silencing of c-Met gene can increase the sensitivity of bladder cancer T24 cells to radiation,and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway activation.
作者
李建伟
田文静
刘民(指导)
LI Jian-Wei;TIAN Wen-Jing;LIU Min(The Second Department of Urology,Cangzhou Central Hospital,Cangzhou 061001,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第7期842-847,共6页
Chinese Journal of Immunology
