YB-1抑制三氧化二砷诱导的胃癌细胞自噬机制

目的研究YB-1抑制三氧化二砷(As2O3)诱导人胃癌BGC-823细胞自噬的机制。方法采用脂质体转染siRNA干扰YB-1和腺病毒转染pAd1Easy/YB-1过表达YB-1;实时荧光定量PCR检测Bcl-2和Beclin1基因转录;Western blot检测YB-1、Bcl-2、Beclin1、P62和LC3Ⅱ蛋白的表达;MDC染色、荧光显微镜观察自噬泡。结果与空病毒(AdGFP)组相比,pAd1Easy/YB-1(AdYB-1)组Bcl-2基因表达增高,而干扰质粒(siYB-1)组Bcl-2基因表达水平较空质粒(HK)组降低(P均<0.05),各组Beclin1基因表达比较无统计学差异。与AdGFP和HK组相比,AdYB-1组Beclin1和LC3Ⅱ蛋白减少,Bcl-2和P62蛋白增加(P均<0.01),siYB-1组Beclin1和LC3Ⅱ蛋白增加,Bcl-2和P62蛋白减少(P均<0.01)。与AdGFP组相比,AdYB-1组自噬泡聚集减少,加入Bcl-2抑制剂HA14-1后自噬泡重新聚集,Beclin1蛋白恢复表达,P62降解增加,LC3Ⅱ升高。结论 YB-1可能通过上调Bcl-2、抑制Bec-lin1,实现对As2O3诱导的BGC-823细胞自噬的抑制。

YB-1 inhibits autophagy in gastric cancer cells induced by arsenic trioxide

Objective To investigate the mechanisms of YB-1 inhibiting autophagy in gastric cancer BGC-823 cells induced by arsenic trioxide.Methods YB-1 and its siRNA recombined adenovirus vectors were transfected into BGC-823 cell lines,and the expression of YB-1 was tested after the transfection by Western-blot.Real time fluorescent quantitative PCR(qRT-PCR) was used to determine the mRNA levels of Bcl-2 and Beclin1.The protein expressions of YB-1,Bcl-2,Beclin1,P62 and LC3 were detected by Western-blot.MDC staining was used to observe the autophagic vacuoles.Results The expressions of Bcl-2 were enhanced after BGC-823 cells were transfected with YB-1 recombined adenovirus vectors.On the contrary,the expressions of Bcl-2 were reduced by transfecting YB-1 siRNA.The mRNA expression of Beclin1 had no obvious differences among groups.Compared with the control groups,the protein expressions of Bcl-2 and P62 were increased and the expressions of Beclin1 and LC3Ⅱ protein decreased after BGC-823 cells were transfected with YB-1.On the contrary,the results of proteins detection were opposite by transfecting YB-1 siRNA.MDC staining showed that the autophagic vacuoles were decreased in BGC-823 cells over expressed YB-1.And autophagic vacuoles were increased after adding HA14-1 in BGC-823 cells with over expressed YB-1.Compared with AdYB1 group,the protein expressions of Beclin1 and LC3Ⅱ increased,but P62 decreased after adding with HA14-1,which were detected by Western-blot.Conclusion YB-1 inhibits the autophagy of BGC-823 cell induced by As2O3 through up-regulating Bcl-2,down-regulating Beclin1.