miR-34a抑制SW480细胞增殖的机制研究

MIR-34A INHIBITORY EFFECTS ON SW480 CELL PROLIFERATION

目的探索miR-34a的生物学功能及其对肠癌细胞的抑制作用。方法通过检索miRTar Base和miRBase数据库,筛选出已经实验验证的miR-34a靶基因,使用Cytoscape软件对筛选出的靶基因进行功能富集分类,构建靶基因相互作用网络,用DAVID数据库对miR-34a的靶基因进行信号通路富集分析;免疫印迹技术检测miR-34a对SIRT1、NOTCH1蛋白表达的影响;MTT法检测转染miR-34a对SW480细胞增殖的影响。结果共有56个基因被确定为miR-34a的靶基因,对靶基因进行功能富集分析包含Wnt Signaling Pathway and Pluripotency、Pathways in cancer、MicroRNAs in cancer、Notch signaling pathway等;对靶基因进行信号通路富集分析(p<0.001;FDR<0.05),共富集到8条通路,包含Pathways in cancer、Small cell lung cancer、Colorectal cancer等。免疫印迹结果显示SW480细胞转染miR-34a类似物后,SIRT1、NOTCH1蛋白的表达水平较对照组明显下降(p<0.01);SW480细胞转染miR-34a类似物后生长速度明显减慢,培养5天后检测两组有显著统计学差异(p<0.05)。结论 miR-34a靶基因参与多个信号通路;miR-34a可能通过负性靶向调控SIRT1、NOTCH1基因的表达而抑制SW480细胞的增殖。

Objective To analyze the biological function and the effect of miR -34a on SW480 cell proliferation.Methods Target genes of miR-34a were filtrated by TarBase and miRBase, and the intersection of the target genes was performed by Cyto-scape; Pathway analysis was performed by DAVID.The expression level of SIRT1,NOTCH1 protein affected by miR-34a were de-tected by Western blotting.The proliferation of SW480 cells was measured by MTT assay.Results 56 verified target genes of miR-34a were filtrated, which were enriched in Wnt Signaling Pathway and Pluripotency, Pathways in cancer, MicroRNAs in cancer, Notch signaling pathway et al;Pathway analysis showed the genes set mainly mapped in 8 pathways （p〈0.001;FDR〈0.05）, inclu-ding pathways in cancer, small cell lung cancer, colorectal cancer, et al.Western blotting results demonstrated that the SIRT1 and NOTCH1 protein level in the SW480 cells transfected with miR-34a mimics decreased compared with the negative control group （p〈0.01）;The growth of SW480 cells transfected with miR-34a mimics was much slower than negative control group （p〈0.01）. Conclusion miR-34a may be involved in the different signaling pathways and inhibit SW480 cell proliferation by downloading the expression of SIRT1 and NOTCH1.