蜂胶黄酮Pinobanksin-3-acetate对大肠癌细胞信号转导通路作用机制的研究

Propolis flavonoids Pinobanksin-3-acetate (PB3A) has a strong growth inhibitory effect, cell cycle arrest and induces cell apoptosis of human colorectal cancer (CRC) cell line HCT116 and SW480. However, the PB3A's anticancer signaling pathway remains to be further elucidated. The profiles of gene expression of CRC cell line SW480 control groups and PB3A intervention groups were analyzed by Human Genome Gene Chip Array U133, of the 54613 probe sets, compared with the control group cells, in result 551 genes were up-regulated by more than two folds and 848 genes were down-regulated by more than two folds in the PB3A intervention group cells. The differential expressed genes were correlated with cell cycle, apoptosis, and colorectal carcinogenesis etc 129 pathways of signal transduction. In our research, we have intend to choose Wnt/β-catenin and apoptosis related signaling pathways differential expressed genes, by using real time polymerase chain reaction(RT-PCR) and Western blotting assays to confirm the mRNA and protein expression level both in PB3A treated and untreated CRC cells. By using RNA interference (RNAi) to suppress drug untreated CRC cell's PB3A specific down-regulated genes. And using methylation-specific polymerase chain reaction (MSP) to determine PB3A specific differential expressed genes. Aims to identify PB3A specific gene targets and CRC signal transduction pathways. Furthermore to clarify the function of PB3A anti-cancer activity and to develop it's anti-cancer effect, provide a theoritical and experimental basis for the implementation of new therapeutic strategies.

蜂胶黄酮Pinobanksin-3-acetate （PB3A）对大肠癌HCT116和SW480细胞具有明显的增殖抑制、细胞周期阻滞及诱导凋亡作用，但其抗肿瘤作用信号转导途径仍不甚清楚。通过人类全基因组表达谱芯片检测的54613个探针组中，SW480细胞 PB3A药物干预组与对照组相比表达上调的有551个基因，表达下调的有848个基因,其涉及细胞周期、凋亡、大肠癌发生等129个信号通路。我们拟选择Wnt/β-catenin及凋亡信号通路差异表达基因，采用实时荧光PCR和蛋白质免疫印迹法检测PB3A对基因转录和蛋白质表达的影响， RNA干扰技术沉默PB3A特异性下调表达基因，甲基化特异性PCR检测PB3A作用下差异表达基因的甲基化水平，旨在找出PB3A对大肠癌细胞作用的信号转导途径和基因靶点，阐明PB3A的抗肿瘤作用途径和开发其抗肿瘤效应，为新型肿瘤治疗策略的实施提供理论和实验依据。

蜂胶黄酮 Pinobanksin-3-acetate（PB3A）是有很高研发价值的单体黄酮类活性物质。以往工作中，我们建立了PB3A提取制备工艺，开展了生物学活性研究，明确该药抑制肿瘤细胞增殖、细胞周期阻滞和诱导细胞凋亡等作用机理。本课题首先通过应用高通量人类基因组基因芯片，探讨PB3A对大肠癌细胞SW480基因表达谱的影响，推测PB3A对大肠癌细胞作用的信号转导途径和特异性基因靶点。本课题还通过结直肠癌SW480细胞的PB3A干预及人类miRNA表达谱分析，筛选出该药作用特异的候选差异表达miRNA，并预测出该药对细胞周期、凋亡和信号转导网络的影响等一系列研究。（1）应用高通量人类全基因组表达谱芯片观察了PB3A 对结直肠癌细胞SW480的mRNA 表达谱的影响，研究发现PB3A对SW480作用后引起1400 条基因的差异表达，其中有34 条基因的差异表达倍数研究超过10倍，将基因组表达谱芯片结果进行聚类和网络分析表明，PB3A诱导差异表达3倍或3倍以上的1400条基因联系到129条信号通路，其功能涉及复杂的增生抑制调控网络。差异表达基因相互作用通路网络中PB3A对MAPK信号通路、Wnt通路、细胞因子感受器相互作用、细胞周期调控、细胞凋亡、Hedgehog通路、大肠癌发生、p53、TGF-β和钙离子等10条通路的作用最明显。为验证芯片可靠性，通过qRT-PCR 和Western blot 方法，对差异表达倍数高的上调基因进行验证，结果表明差异倍数与其基因芯片结果相比较，候选基因mRNA表达量的改变趋势符合芯片结果。（2）通过miRNA 芯片检测蜂胶黄酮PB3A 干预人结直肠癌SW480 细胞后miRNAs 表达谱的影响情况，发现实验组与对照组比较发现结直肠癌细胞SW480中267条miRNAs 表达有显著性差异，其中218 条miRNAs 上调表达和49条下调表达，差异表达倍数3 倍及以上的miRNAs 有129 条，其中121条上调表达和8条下调表达，差异表达倍数10 倍及以上的miRNAs 有30条，其中28条上调表达和2条下调表达。经生物信息学分析，差异表达microRNA调控的靶基因参与的相关信号转导通路和特定靶基因的工作正在进行。