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


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.


RESULTS
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.


CONCLUSIONS
Our results suggest that overexpression of miR-15a and miR-16 mediates down-regulation of BMI1, and leads to mitochondrial mediated apoptosis.
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State 500007, India.
Nibedita
N
Patel
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State 500007, India.
Koteswara Rao
KR
Garikapati
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State 500007, India; School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamilnadu 613401, India.
M Janaki
MJ
Ramaiah
Department of Genetics, Osmania University, Hyderabad, Telangana State 500007, India.
Kavi Kishor
KK
Polavarapu
Gene Silencing Group, Centre for Cellular and Molecular Biology, Hyderabad, Telangana State 500007, India.
Utpal
U
Bhadra
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State 500007, India. Electronic address: manikapb@gmail.com.
Manika Pal
MP
Bhadra
D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000503:physiopathology; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D063150:Polycomb Repressive Complex 1 / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction
C069077:BMI1 protein, human; C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D063150:Polycomb Repressive Complex 1
Apoptosis; BMI1; miR-15a and miR-16