Metformin Induces Apoptosis By MiR-26a-mediated Down-regulation Of Myeloid Cell Leukaemia-1 In Human Oral Cancer Cells

Aims:To explore the effects of metformin on proliferation and apoptosis in human oral cancer cell line KB in vitro.Methods:1. Observe the change of cell morphological characteristics induced by metformin under a light microscopy, MTT assay was conducted to detect the changes in cell vialibity. Colony-forming assay was conducted to detect the growth inhibition induced by metformin.2. Apoptosis was assessed using double staining with annexin V-FITC/PI and The mitochondrial membrane potential was measured by JC-1 assay. Western blot test Mcl-1, Bim, Bax and caspase-3 protein expression.3. After transfection with cDNA-Mcl-1, the expression of Mcl-1,Bim,Bax and Caspase-3 were detected by western blot.4. Quantitative reverse transcription-polymerase chain reaction(qRT-PCR) was performed for mi RNA detection. Cells at 60–80% confluency were transfected with micro RNA(mi R)-26 a mimics and mi R-26 a inhibitor, Cells were harvested later for PI viability assays, Colony-forming assay and western blot.Results:Effect of different concentrations of metformin on proliferation and apoptosis in KB1. Compared with the control group, KB cells were displaying significant morpholo-gical changes, such as body swelling round, smaller size and increased floating cells after treatment with metformin in different concentrations.2. MTT assay showed that the proliferation of KB cells were significantly inhibited after treated with metformin in a time and concentration dependent manner.3. Colony formation assay showed that the clonogenic capacity of KB cells was significantly inhibited after treated with metformin.4. Relative proportions of red and green fluorescence decrease in JC-1 fluorescence detection suggested the decrease of mitochondrial membrane potential.5. Annexin V-FITC / PI staining showed that metformin could induce KB cells apoptosis.6. Western blot showed that metformin significantly reduced the expression of Mcl-1 in oral cancer cells and stimulated the expression of Bim, Bax and the activation of caspase-3.Overexpression of Mcl-1 inhibited the sensitivity of metformin in oral cancer KB cells1. KB cells were transfected with cDNA Mcl-1 plasmid, after 6 h, then change the medium with FBS, continue to developed the cells, finally, cell lysates were examined by western blotting. The expression of Mcl-1 was pronounced increased by c DNA Mcl-1 plasmid.2. MTT assay showed that cell viability of cells transfected with c DNA Mcl-1 was significantly increased compared with non-transfected cells, suggested the sensitivity of metformin was decreased after c DNA Mcl-1 transfection. JC-1detection showed that transfection of c DNA Mcl-1 could inhibit the decline of metformin-induced mitochondrial membrane potential.3. Over-expression of Mcl-1 significantly increased cell viability in response to metformin treatment and decreased the expression of Bim and Bax compared with their respective controls. In addition, the cleaved products of caspase-3 were scarcely activated in the Mcl-1-overexpressing KB cells.Regulation of metformin on mi R-26 a in KB cells and the effect of mi R-26 a on proliferation and apoptosis of oral cancer KB cells.1. mi R-26 a was signi?cantly up-regulated in metformin-treated cells compared with non-treated cells.2. mi R-26 a mimics obviously inhibited the proliferation of KB cells. PI analysis revealed that transfection of mi R-26 a could induce apoptosis. And the level of Mcl-1 decreased in cells transfected with mi R-26 a mimics compared with cells transfected with the corresponding negative control. In contrast, Mcl-1 was increased in KB cells transfected with anti-mi R-26 a inhibitor. These experiments confirm that the expression of Mcl-1 can be regulated by mi R-26 a.Conclusion:1. Metformin could inhibit the KB cells proliferation and induce apoptosis of KB cells mainly through mitochondria-mediated pathways.2. Up-regulation of Mcl-1 protects KB cells from apoptosis induced by metformin.3. Metformin increased the expression of mi R-26 a.4. Overexpression of mi R-26 a reduced cell viability and regulated Mcl-1 expression in KB cells.