去甲斑蝥素降低人胃癌细胞程序性细胞死亡因子4的表达(英文)

Norcantharidin downregulates programmed cell death 4 expression in human gastric cancer cells

目的研究去甲斑蝥素(NCTD)降低程序性细胞死亡因子4(PDCD4)表达的机制。方法 MTT法测定NCTD 5～640μmo.lL-1与人胃癌BGC-823细胞作用24,48和72 h细胞存活率;Western蛋白质印迹法测定NCTD 0,6,30和60μmol.L-1作用BGC-823细胞24 h PDCD4蛋白表达水平;NCTD60μmo.lL-1作用20 h后加入MG132 10μmo.lL-1作用4 h对PDCD4蛋白表达的影响;逆转录PCR法测定NCTD 60μmo.lL-1作用BGC-823细胞24 h后PDCD4 mRNA表达的变化;实时荧光定量PCR(qRT-PCR)测定NCTD 60μmol.L-1作用BGC-823细胞6,12和24 h后microRNA-21(miR-21)的表达。Western蛋白质印迹法测定细胞转染miR-21抑制剂对PDCD4蛋白表达的影响。结果 NCTD作用后BGC-823细胞存活率明显下降,NCTD作用BGC-823细胞24,48和72 h IC50分别为74.5,35.0和10.3μmo.lL-1。NCTD 6,30和60μmol.L-1作用于BGC-823细胞24 h,PDCD4蛋白分别降低9%,47%和62%。NCTD对PDCD4 mRNA表达无影响。与NCTD处理组相比,MG132和NCTD共处理对PDCD4蛋白表达无明显影响。NCTD 60μmo.lL-1作用BGC-823细胞12和24 h后,细胞中miR-21的表达显著升高(P<0.01)。细胞转染miR-21抑制剂后,可抑制NCTD降低PDCD4蛋白表达的作用。结论 NCTD通过调控miR-21降低PDCD4蛋白的表达。

OBJECTIVE To investigate the mechanisms by which programmed cell death 4（PDCD4） is down-regulated by norcatharidinon（NCTD） in human gastric cancer cells.METHODS Cell viability was detected by MTT assay in human gastric cancer BGC-823 cells treated with NCTD 5,10,20,40,80,160,320 and 640 μmol·L-1 for 24,48 and 72 h.The effect of NCTD 0,6,30 and 60 μmol·L-1 on PDCD4 protein expression was detected by Western blotting.BGC-823 cells were treated with NCTD 60 μmol·L-1 for 20 h and proteasome inhibitor MG132 10 μmol·L-1 for 4 h,then the PDCD4 protein was detected using Western blotting.The PDCD4 mRNA level were detected by RT-PCR after BGC-823 cells were treated with NCTD 60 μmol·L-1 for 24 h.The level of microRNA-21（miR-21） in BGC-823 cells was analyzed with RT-qPCR after treatment by NCTD 60 μmol·L-1 for 6,12 and 24 h.The PDCD4 protein level was detected by Western blotting after BGC-823 cells were transfected with miR-21 inhibitor and the cells treated with NCTD 60 μmol·L-1 for 24 h.RESULTS Cell viability obviously decreased in NCTD groups.The IC50 of NCTD in BGC-823 cells was 74.5,35.0,and 10.3 μmol·L-1 at 24,48 and 72 h,respectively.NCTD 6,30 and 60 μmol·L-1 down-regulated PDCD4 protein by 9%,47%,and 62%,respectively.The level of PDCD4 mRNA did not change in NCTD-treated BGC-823 cells.Compared with the cells treated with NCTD alone,the level of PDCD4 protein did not change in cells treated with both MG132 and NCTD.The miR-21 expression in NCTD-treated cells increased dramatically compared to that in control cells.The expression of PDCD4 protein was up-regulated dramatically by miR-21 inhibitor in NCTD-treated cells.CONCLUSION NCTD downregulates PDCD4 expression in BGC-823 cells through activation of miR-21.