miR-15a/miR-16 induces mitochondrial dependent apoptosis in breast cancer cells by suppressing oncogene BMI1.

AIMS: miRNAs are small non-coding RNA molecules that regulate post-transcriptional gene expression. Here we have made an endeavor to search whether any miRNAs are involved in the regulation of BMI1 in breast cancer that leads to mitochondrial dependent apoptotic cell death.

MAIN METHODS: Renilla luciferase reporter assay was performed to detect the ectopically expressed miRNAs that regulate the expression of 3' UTR of BMI1. MTT assay was performed to check the cytotoxicity level. Western blotting and qRT-PCR were performed to check the expression of BMI1, pro-apoptotic, anti-apoptotic proteins and mRNA expression levels respectively. JC-1 staining, Caspase-3, Caspase-6/9 assay and mitochondrial cytosolic fractionation were performed to monitor mitochondrial dependent apoptosis. Wound healing assay was performed to investigate migration. All experiments were performed upon miR-15a and miR-16 overexpression in MCF-7, MDAMB-231 breast cancer cells.

KEY FINDINGS: In MCF-7, MDAMB-231 breast cancer cells luciferase reporter assay confirmed the significant reduction of reporter activity upon co-transfection of 3' UTR of BMI1 along with miR-15a and miR-16. miR-15a and miR-16 significantly down-regulated BMI1 protein and mRNA expression levels as well as anti-apoptotic protein BCL2 and up-regulated pro-apoptotic proteins. Ectopic expression of miR-15a, miR-16, increased mitochondrial ROS resulting in impaired mitochondrial membrane potential, followed by cytochrome-C release into the cytosol that activated Caspase-3 and Caspase-6/9 leading to intrinsic apoptosis. Additionally, it also inhibits migration.

SIGNIFICANCE: Our results suggest that overexpression of miR-15a and miR-16 mediates down-regulation of BMI1, and leads to mitochondrial mediated apoptosis.