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

Abstract

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. Key words: Colon cancer; miR-302b; Apoptosis; E2F1