miR-302b靶向E2F1对结肠癌细胞增殖和凋亡影响的机制研究

Effects of miR-302b on proliferation and apoptosis of colon cancer cells by targeted regulation of E2F1: a mechanistic study

目的研究miR-302b靶向调控转录因子(E2F1)对结肠癌细胞增殖和凋亡影响。方法以结肠癌细胞SW620作为体外实验对象,转染miR-302bmimics、mimicscontrol,Realtime PCR方法测定过表达效果,MTT方法测定细胞增殖变化,克隆形成实验测定细胞克隆能力,流式细胞术测定细胞凋亡变化,免疫印迹测定细胞中Cleaved Caspase-3蛋白水平。靶基因预测软件预测E2F1可能是miR-302b的靶基因,构建荧光素酶报告载体鉴定。将miR-302bmimics和pcDNA3.1-E2F1共转染至SW620细胞中,用上述方法测定细胞增殖、克隆、凋亡和Cleaved Caspase-3、E2F1蛋白表达水平。结果SW620细胞转染miR-302bmimics后,miR-302b表达增多,细胞增殖、克隆形成能力降低,细胞凋亡增多,细胞中CleavedCaspase-3蛋白表达水平升高。野生型E2F1荧光素酶报告载体与miR-302bmimics共转染后细胞荧光素酶活性降低。与单纯转染miR-302bmimics的细胞比较,miR-302bmimics和pcDNA3.1-E2F1共转染可以明显提高SW620细胞增殖、克隆能力和E2F1蛋白表达水平,并减少细胞凋亡和Cleaved Caspase-3蛋白表达水平。结论miR-302b靶向调控E2F1抑制结肠癌细胞增殖并诱导细胞凋亡。

Objective To investigate the effects of miR-302b on the proliferation and apoptosis of colon cancer cells by targeted regulation of the transcription factor E2F1. Methods Colon cancer cell-line SW620 was used for this in-vitro study, transfected with miR-302b mimics and mimics control, respectively. Real-time PCR was used to determine the over-expression of miR-302b;MTT assay was used to examine cell proliferation;colony formation assay was used to determine the cell clone formation. Cell apoptosis was measured by flow cytometry. Intracellular cleaved caspase-3 protein level was determined by Western blotting. Based on Targetscan, E2F1 was predicted to be the target gene of miR-302b and therefore, a luciferase reporter vector was constructed for the validation. Thereafter, miR-302b mimics and pcDNA3.1-E2F1 were co-transfected into the SW620 cells. The cell proliferation, clone formation, apoptosis, and cleaved caspase-3 and E2F1 protein levels were determined as described above. Results After transfected with miR-302b mimics, the SW620 cells showed increased expression of miR-302b, lower levels of cell proliferation and colony formation, increased apoptosis, and increased level of cleaved caspase-3 proteins.The luciferase activity was reduced after co-transfection of wild-type E2F1 luciferase reporter vector and miR-302b mimics. Compared with those transfected with miR-302b mimics alone, SW620 cells co-transfected with miR-302b mimics and pc DNA3.1-E2F1 showed significant increases in cell proliferation, clone formation, E2F1 protein level, and reductions in cell apoptosis and cleaved caspase-3 protein level. Conclusion miR-302b inhibits proliferation and induces apoptosis of colon cancer cells by targeted regulation of E2F1.