蜂胶黄酮PB3A作用下结肠癌细胞株中miRNA-198和miRNA-296-5p的表达及功能预测

摘要

AIM: To explore the effect of pinobanksin-3-acetate (PB3A) on microRNA (miRNA) expression profile of human colon cancer cells for providing new methods of treatment of colon cancer and development of targeted drug.METHODS: The method of miRNA expression profiling was used to observe the miRNA differential expression in human colon cancer SW480 cells after treated with PB3A.The expression of miRNA-198 and miRNA-296-5p in the SW480 cells was detected by RT-qPCR.The network databases of miRWalk, MicroT, miRanda and so on were used to predict the target genes regulated by these miRNAs, and pathway significant enrichment analysis was performed.RESULTS: miRNA microarray analysis showed that after treated with propolis flavonoid PB3A for 24 h, 267 miRNAs with differential expression twice or more in the SW480 cells were observed.Among them, there were 30 miRNAs with 10-fold or more differential expression, in which 28 were up-regulated and 2 were down-regulated.The results of RT-qPCR showed that the expression levels of miRNA-198 and miRNA-296-5p were consistent with the results of miRNA microarray analysis, and the difference was statistically significant (P<0.05).Bioinformatic analysis revealed that miRNA-198 has 859 target genes, and miRNA-296-5p has 906 target genes.The target genes of miRNA-198 were clustered in pathways in cancer, axon guidance, Wnt signaling pathway, regulation of actin cytoskeleton, insulin signaling pathway and MAPK signaling pathway, while the target genes of miRNA-296-5p were clustered in axon guidance, Wnt signaling pathway, MAPK signaling pathway, endocytosis, melanogenesis, insulin signaling pathway and calcium signaling pathway.CONCLUSION: Propolis flavonoid PB3A affects the expression of miRNA in colon cancer SW480 cells.The abnormal expression of miRNA-198 and miRNA-296-5p may be involved in the inhibitory effect of PB3A on colon cancer.%目的: 探讨蜂胶黄酮短叶松素-3-乙酸酯(pinobanksin-3-acetate,PB3A)对结肠癌SW480细胞微小RNA(microRNA, miRNA)表达谱的影响,为结肠癌的治疗及靶向药物研发提供理论依据.方法: 使用miRNA芯片技术分析检测蜂胶黄酮PB3A处理人类结肠癌SW480细胞后miRNA表达谱的变化.通过实时荧光定量PCR方法检测miRNA-198和miRNA-296-5p的表达水平,以此来验证miRNA芯片结果的准确性和可靠性.利用miRWalk、MicroT、miRanda等12个网上数据库预测这2条miRNAs的靶基因并进行靶基因功能富集分析.结果: miRNA芯片分析结果显示,蜂胶黄酮PB3A干预24 h后结肠癌SW480细胞中差异表达倍数在2倍及以上的miRNA有267条,其中差异表达倍数达10倍及以上的miRNA有30条,28条为上调表达,2条下调表达;RT-qPCR实验结果显示miRNA-198和miRNA-296-5p的表达量趋势跟miRNA芯片结果一致,表达差异有统计学意义(P<0.05).miRNA靶基因预测发现miRNA-198有859个靶基因,miRNA-296-5p有906个靶基因;对这些可能被调控的靶基因进行Gene Class分析,结果显示miRNA-198和miRNA-296-5p的靶基因功能主要为转录因子、拷贝数变异、细胞分化、癌基因、蛋白激酶、组蛋白、转移癌基因、肿瘤抑制基因等(P<0.05).信号通路富集分析结果显示,miRNA-198靶基因显著富集于肿瘤通路、Wnt信号通路、胞吞通路、ErbB信号通路、黏着斑通路、黑素生成通路等信号通路,而miRNA-296-5p靶基因在MAPK信号通路、胞吞通路、轴突导向通路、Wnt信号通路、胰岛素信号通路、钙离子信号通路等信号通路中出现聚集(P<0.05).结论: 蜂胶黄酮PB3A 影响结肠癌SW480细胞的miRNA表达谱.PB3A作用下miRNA-198和miRNA-296-5p的异常表达可能参与PB3A抗结肠癌的过程.