The Role Of MiR-23a On Autophagy Of Human Breast Cancer Cells

Objective: To investigate mi RNAs involve in modulating autophagy of human breast cancer cells.Methods:1. By first detecting the expression of LC3 protein using western-blot assasy, we select four mi RNAs for further study on autophagy regulation.2. The effects of mi R-23 a on autophagy were determined by western blot, electronand fluorescence-microscopy after T47 D and MCF-7 cells transfected with mi R-23 a mimic and mi R-23 a ASO, respectively, both compare with negative control.3. Flow cytometry assay was used to detect the role of mi R-23 a on apoptosis on human breast cancer cells.4. Bioinformatics tools were used to predict the potential target genes of mi R-23 a.Results:1. The result of western-blot showed that MCF-7 cells were transfected with mi R-24 mimic, mi R-7 mimic, mi R-23 a mimic and mi R-513a-5p mimic. Forty-eight hours later, compared with negative control group, mi R-23 a mimic significantly increased LC3 II/LC3 I conversion ratio.2. The results of autophagy experimental in vitro were summarized as follows:(1) Western-blot assay showed that forced expression of mi R-23 a significantly decreased the expression of SQSTM1/p62 protein and increased LC3 II/LC3 Iconversion ratio in T47 D cells. In contrast, mi R-23 a ASO significantly increased the expression of SQSTM1/p62 protein and decreased LC3 II/LC3 I conversion ratio in MCF-7 cells, both compared with negative control group.(2) Fluorescence microscope showed that there was a significant increase 22.6% of GFP-LC3 puncta in mi R-23 a mimic transfected cells and a decrease 3% of GFP-LC3 puncta in mi R-23 a ASO transfected cells, both compare with negative control group(P<0.05), after mi R-23 a mimic and mi R-23 a ASO, respectively, were transfected into MCF-7 cells together with the GFP-LC3 plasmid.(3) Electron microscopy showed that accumulation of autophagosomes was increased in mi R-23 a mimic transfected cells, compare with negative control group.3. Flow cytometry assay showed that the apoptosis rate significantly decreased 42% in mi R-23 a mimic transfected cells, compare with negative control group(P<0.05).4. The possible target genes of mi R-23 a were predicted by bioinformatics tools and some of the potential target genes play roles in inhibiting autophagy and promoting apoptosis.Conclusion: mi R-23 a promotes autophagy and inhibits apoptosis of human breast cancer cells.