RNA干扰Chk1/2调控二烯丙基二硫诱导的G2/M期阻滞作用及相关周期蛋白表达

Interference of Chkl/2 by RNA Regulates G2/M Arrest and Expressions of Cell Cycle Related Proteins Induced by Diallyl Disulfide

在细胞周期检测点信号传导通路中,Chkl和Chk2起着重要作用,主要参与G2/M期细胞周期检测点信号传导.首先采用RNAi技术在BGC823细胞中将Chk1、Chk2基因沉默,Chk1、Chk2 siRNA转染BGC823细胞后24h加入15mg/L二烯丙基二硫(diallyl disulfide,DADS),接着通过Real-timePCR、Western blot分析Chk1、Chk2基因在转染前后的表达差异,然后运用流式细胞术和Western blot分析Chk1、Chk2基因沉默后对DADS诱导的细胞周期G2/M期阻滞作用及相关周期蛋白CDC25C和cyclinB1表达的影响.实验结果表明,与未转染对照组相比,转染Chk1、Chk2siRNA后BGC823细胞中Chk1、Chk2表达明显被抑制,二者的mRNA表达分别下降84.7%和69.0%,蛋白质水平分别下降73.4%和78.5%.流式细胞术分析结果发现,ChklsiRNA转染的BGC823细胞在15mg/LDADS处理24h后,G2/M期比例由单纯DADS处理组的58.1%降至10.4%(P<0.05).但Chk2 siRNA转染后加入15mg/LDADS,与单独15mg/LDADS作用相比,细胞周期G2/M期比例差异不明显(P>0.05).Chk1和Chk2下游分子的改变显示,DADS可抑制BGC823细胞中CDC25C和cyclinB1表达(P<0.05),但Chk1基因沉默后加入DADS,CDC25C和cyclinB1表达却不被抑制(P>0.05),Chk2基因沉默后对DADS抑制CDC25C和cyclinB1表达的作用无影响.由此得出结论,Chkl基因沉默可消除DADS诱导BGC823细胞G2/M期阻滞作用,DADS阻滞G2/M期可能是通过Chk1/CDC25C/cyclinB1信号通路起作用.

Chk1 and Chk2 play major role in cell cycle checkpoint signaling pathway,which are mainly involved in G2/M cell cycle checkpoint signal transduction.Firstly,the siRNA targeting at Chk1 or Chk2 gene was transfected into human gastric cancer BGC823 cells for 24 h before 15 mg/L DADS was added.Then,the mRNA and protein expression of Chk1 or Chk2 was detected by Real-time PCR and Western-blot respectively.Cell cycle rates and expressions of CDC25C and cyclinB1 were determined by FCM and Western blot respectively.The results showed that the Chk1 or Chk2 expression was inhibited in Chk1 or Chk2 siRNA-transfected group,in which the Chk1 or Chk2 expression at mRNA level was reduced 84.7% and 69.0% respectively and the protein expression of Chk1 or Chk2 was reduced 73.4% and 78.5% respectively as compared with that in empty control group（P0.05）.Then it was investigated whether Chk1 and Chk2 proteins could influence cell cycle regulation by knocking down their expressions in BGC823 cells.Further investigation revealed that inhibition of the Chk1 expression in Chk1 siRNA transfected group cut down the proportion of the cells in G2/M phase from 58.1% to 10.4% in BGC823 cells after 24 h induced by DADS（P0.05）.While inhibition of the Chk2 expression in Chk2 siRNA transfected group had little effect on G2/M arrest after treatment with DADS（P0.05）.Western blot showed that although DADS decreased expression of CDC25C and cyclinB1 in untransfected cells,inhibition of expression of CDC25C and cyclinB1 treated by DADS was blocked by Chk1 gene silence（P0.05）.On the contrary,Chk2 gene silence can not do so.These results suggest Chk1 gene silence could abrogate G2/M arrest induced by DADS in BGC823 cell line,and Chk1/CDC25C/cyclinB1 pathway was involved at the G2/M arrest induced by DADS.