miR-449a/b负反馈调节E2F1抑制胃癌细胞增殖

摘要：目的 探讨miR-449a/b在胃癌发生中的可能作用机制。方法 采用qRT-PCR方法检测了miR-449a/b和E2F1基因在胃癌细胞BGC-823和胃粘膜细胞GES-1表达情况；采用瞬时转染技术将miR-449a/b模拟物(mimic)以及mimic阴性对照分别转入胃癌细胞BGC-823中，qRT-PCR验证转染成功后，通过CCK-8法检测胃癌细胞的增殖能力，流式细胞术检测胃癌细胞凋亡的变化，细胞划痕实验检测胃癌细胞的迁移能力，Western blot方法检测miR-449a/b上调后其靶基因蛋白的表达水平；并通过转染E2F1过表达质粒和空质粒对照入胃癌细胞BGC-823，Western blot确定转染效率后，分别用qRT-PCR和数字PCR检测miR-449a/b的表达水平。结果 相比于正常胃粘膜细胞株GES-1，miR-449a/b在胃癌细胞株BGC-823中表达均下调（P<0.01）；过表达miR-449a/b可抑制胃癌细胞BGC-823增殖和迁移，并促进其凋亡（P<0.05）；过表达的E2F1可上调miR-449a/b的表达（P<0.001），而上调miR-449a/b的表达，其直接靶基因CDK4、CDK6表达下调，并可通过CDKs-pRb-E2F1信号通路，间接下调E2F1蛋白的表达（P<0.01）。结论 miR-449a/b的低表达和E2F1的高表达均参与了胃癌的发生、发展，并且miR-449a/b能负反馈调节E2F1抑制胃癌细胞增殖。

miR-449a/b negatively regulates E2F1 to suppress proliferation of gastric cancer cells

Abstract: Objective To investigate the possible role of miR-449a/b in the occurrence of gastric cancer. Methods The expressionof miR-449a/b and E2F1 mRNA in gastric cancer cells BGC-823 and gastric mucosal cells GES-1 were detected with qRT-PCR.miR-449a/b mimics or a negative control was transiently transfected into BGC-823 cells, and the changes in cell proliferation,apoptosis, and migration ability were assessed using CCK-8 assay, flow cytometry, and scratch wound healing assay,respectively. Western blotting was used to observe the effects of miR-449a/b upregulation on its target gene expression. Theeffects of transfection with an E2F1-over-expressing plasmid or an empty plasmid were analyzed on the expression level ofmiR-449a/b in BGC-823 cells using qRT-PCR and digital PCR. Results The gastric cancer cell line BGC-823 showedsignificantly a lowered expression of miR-449a/b compared with the normal gastric mucosal cell line GES-1 (P<0.01).Overexpression of miR-449a/b obviously inhibited the proliferation and migration and promoted apoptosis of BGC-823 cells(P<0.05). Overexpression E2F1 in the cells resulted in significantly up-regulated expression of miR-449a/b (P<0.001). Upregulation of miR-449a/b caused significant down-regulation of its direct target genes CDK4 and CDK6 and also of theexpression of E2F1 protein via the CDKs-pRb-E2F1 signaling pathway. Conclusion The low expression of miR-449a/b and thehigh expression of E2F1 are both involved in the occurrence and progression of gastric cancer, and miR-449a/b negativelyregulates E2F1 to inhibit the proliferation of gastric cancer cells.