蜂胶黄酮Pinobanksin-3-acetate对结直肠癌细胞miRNA指标的调控机制的研究

Propolis flavonoids Pinobanksin-3-acetate (PB3A) is a monomer flavonoid with bioactivity of high research value. In previous works, we established an extraction method for PB3A, and studied the biological activity, and found out several significant mechanisms of this drug, such as the inhibition of tumor cell proliferation, cell cycle arrest and induction of apoptosis. Recently，we studied miRNA expression profile of SW480 colon cancer cells in response to treatment with PB3A，and identified candidate differentially expressed miRNAs，and predicted the impact of the drug on cell cycle, apoptosis and signal transduction network, but further study is needed to validate the specificity of candidate miRNAs and functions of their potential target genes. In this study, we will treat SW480 colon cancer cells with PB3A, and analyze the specificity and sensitivity of previously identified candidate miRNAs and the expression and funtion of predicted target genes using methods like qRT-PCR and immuno blotting, for establishing the miRNA profile and revealing the signaling pathways of target genes changed in response to treatment with PB3A. The goal of this study is to further understand mechanisms and potential targets of this drug, and provide evidence for the development of a PB3A-based novel drug and its clinical application.

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。近期，我们通过结直肠癌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，尚需候选miRNA指标的验证及其潜在的靶基因功能等一系列研究。故本研究拟以PB3A干预结直肠癌SW480细胞，采用定量RT-PCR和免疫印迹等方法，验证候选miRNA指标的特异性和灵敏度，预测和分析潜在的靶基因表达水平和功能调控，明确PB3A作用与候选miRNA及靶基因表达调控网络和信号转导通路变化的关系，探讨该药作用机理和潜在靶点，为基于PB3A新药研发和临床应用提供依据。

本研究通过结肠癌细胞中显著差异表达的 miRNAs进行pathway enrichment分析，筛选关键miRNA，进行靶基因预测及靶基因功能富集分析，构建差异表达miRNAs与靶基因及基因功能调控网络。①PB3A干预结肠癌SW480细胞miRNA芯片结果中候选出12条差异表达miRNA，RT-qPCR法进行验证，发现10条miRNAs的表达差异有统计学差异。②利用miRWalk及Targetscan等数据库预测PB3A干预下差异表达miRNA的靶基因，GO功能富集分析对候选miRNA靶基因进行分析，显示14条miRNA通过调控其靶基因参与细胞组分的形成；参与Wnt受体信号转导等生物过程。③差异表达miRNAs靶基因基因功能主要富集于蛋白功能与Ras通路、Wnt信号通路、p53通路、凋亡信号通路有关。④利用lipo-3000载体把miR-4326模拟物转染到结肠癌SW480细胞中发现miR-4326在结肠癌SW480细胞内高表达。⑤miR-18a-5p和miR-802两条miRNA的模拟物，载体转染到结肠癌SW480细胞系，建立过表达miR-18a-5p和miR-802的SW480细胞模型。⑥利用lipo-3000载体把miR-18a-5p-eGFP模拟物转染到结肠癌SW480细胞中发现过表达miR-18a-5p与结肠癌增殖与凋亡相关。⑦RT-qPCR法检测miR-18a-5p调控的靶基因DUSP5、FZD3及CCND2，发现DUSP5和FZD3基因的mRNA转录水平上调表达，而且CCND2基因的mRNA转录水平下调表达。⑧miR-802调控的靶基因NKD1、HSPA6基因的mRNA转录水平上调表达，而且TCF7L2基因的mRNA转录水平下调表达，DUSP5、FZD3和CCND2等靶基因参与调控MAPK、Wnt和PI3K/Akt等信号通路，参与结肠癌的发生、发展过程。⑨差异表达基因相互作用通路网络中PB3A作用在MAPK信号通路、Wnt通路、细胞因子、细胞周期调控、细胞凋亡、Hedgehog通路、大肠癌发生、p53通路。PB3A 作用与候选 miRNA 及靶基因表达调控网络和信号转导通路变化的关系。