MicroRNA?452?5p on proliferation and apoptosis of breast cancer cells and the possible mechanism

TANG Xiaoqiao; SANG Jianfeng; ZHANG Yin; SHI Xianbiao; SU Lei.

Abstract：investigate the effect of microRNA?452?5p ( miR?452?5p) on proliferation and apoptosis in breast cancer cell lines and the possible mechanism. Methods The expression level of miR?452?5p in breast cancer cells MDA?MB?231, MCF?7, MDA?MB?361 and HCC70, and normal breast cell line MCF?10A was detected by the realtime?PCR( QPCR), and the cell line with the lowest expression was chosen for the following experiment. The miR?452?5p mimic and negative control were transfected into human breast cancer cell line with the lowest expression of miR?452?5p by lipofection, and blank control was set. Cell proliferation was examined by EdU proliferation assay and colony formation assay. Flow cytometry was employed to detect cell apoptosis. The expres?sion levels of apoptosis?related proteins procaspase?3 and procaspase?9 were detected by Western blotting. Bioinformatics the software TargetScan predicted the binding site of miR?452?5p and vascular endothelial growth factor A(VEGF?A), and Western blotting and du? al luciferase reporter gene experiment were used to verify the targeting relationship. Results The expression of miR?452?5p in breast cancer cells MDA?MB?231 (0. 26±0. 02), MCF7 (0. 35±0. 04), MDA?MB?361 (0. 49±0. 05) and HCC70 (0. 67±0. 07) cells were significant lower than that of normal breast cell MCF?10A (1. 00±0. 11) with significance ( P＜0. 05). The expression level was the lowest in breast cancer cell line MDA?MB?231 (P＜0. 05).QPCR detection showed that the expression of miR?452?5p in MDA?MB?231 cells was up?regulated in the miR?452?5p group compared with negative control group (0. 95±0. 09 vs.4. 28±0. 44, P＜0. 05), and the difference had no significance between negative control group and blank control group (P＞0. 05). EdU test showed that the cell prolif?erative rate in miR?452?5p group was decreased compared with negative control group ( P＜0. 05), and the difference had no signifi?cance between negative control group and blank control group ( P＞0. 05). The clone formation assay showed that the number of cell clones in miR?452?5p group decreased (77. 65±7. 84 vs.53. 22±6. 16, P＜0. 05) compared with negative control group, and the differ?ence had no significance between negative control group and blank control group (P＞0. 05). Flow cytometry showed that the apoptotic rate of miR?452?5p group was higher than that of negative control group [(2. 21±0. 46)% vs.(20. 61±3. 09)%, P＜0. 05)], and the difference had no significance between negative control group and blank control group (P＞0. 05).Western blotting analysis showed that the expression of apoptosis?related proteins procaspase?3 (1. 20±0. 13 vs.0. 49±0. 05) and procaspase?9 (1. 49±0. 20 vs.0. 38±0. 04) in miR?452?5p group were down?regulated compared with negative control group (P＜0. 05), and the difference had no significance be?tween negative control group and blank control group (P＞0. 05). Western blotting analysis showed that the expression of VEGF?A pro?tein in miR?452?5p group was decreased compared with negative group ( P＜0. 05). The dual luciferase reporter gene showed that the relative luciferase activity of miR?452?5p was decreased in the VEGF?A?Wt group compared with negative control group ( P＜0. 05), while luciferase activity had no significant change in VEGF?A?Mut group ( P＞0. 05). Conclusion MiR?452?5p is down?regulated in breast cancer cell lines. MiR?452?5p affects the proliferation and apoptosis of breast cancer cells by regulating the expression of VEGF?A.