MiR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

Nibedita Patel,Koteswara Rao Garikapati,Raj K Pandita,Utpal Bhadra,Manika Pal Bhadra,Dharmendra Kumar Singh,Tej K Pandita

doi:10.1038/s41598-017-02800-2

Abstract

The B-lymphoma Moloney murine leukemia virus insertion region-1 protein (BMI1) acts as an oncogene in various cancers, including breast cancer. Recent evidence suggests that BMI1 is rapidly recruited to sites of DNA double strand breaks where it facilitates histone H2A ubiquitination and DNA double strand break repair by homologous recombination. Here we show that miR-15a and miR-16 expressionis decreased during the initial period after DNA damage where it would otherwise down-regulate BMI1, impairing DNA repair. Elevated miR-15a and miR-16 levels down-regulated BMI1 and other polycomb group proteins like RING1A, RING1B, EZH2 and also altered the expression of proteins associated with the BMI1 dependent ubiquitination pathway. Antagonizing the expression of miR-15a and miR-16, enhanced BMI1 protein levels and increased DNA repair. Further, overexpression of miR-15a and miR-16 sensitized breast cancer cells to DNA damage induced by the chemotherapeutic drug doxorubicin. Our results suggest that miR-15a and miR-16 mediate the down-regulation of BMI1, which impedes DNA repair while elevated levels can sensitize breast cancer cells to doxorubicin leading to apoptotic cell death. This data identifies a new target for manipulating DNA damage response that could impact the development of improved therapeutics for breast cancer.

Highlights

MicroRNAs are small non-coding regulatory RNA molecules (22 nucleotides in length) involved in diverse biological processes27–29. microRNAs negatively regulate gene expression at the post-transcriptional level by binding to complementary sequences in the coding 3′ untranslated region of their target messenger RNA(mRNA)[30,31,32]
In order to identify miRNAs involved in the double-stranded nucleic acid (DNA) damage response (DDR) and in modulating DNA repair gene expression, we created a DNA damage response model using etoposide, a topoisomerase-II inhibitor extensivelyused in DNA damage studies[42,43]
Our dose-dependent study showed 50% cell death at 5 μM concentration (Fig. S1) where it efficiently induced significant DNA damage and by changing the media of the etoposide-treated cells, the cells were able to repair the damage to a certain extent (Fig. S2), we restricted our further studies to this concentration

Summary

Introduction

MicroRNAs (miRNA) are small non-coding regulatory RNA molecules (22 nucleotides in length) involved in diverse biological processes27–29. microRNAs negatively regulate gene expression at the post-transcriptional level by binding to complementary sequences in the coding 3′ untranslated region of their target messenger RNA(mRNA)[30,31,32]. Results from the reporter assay of BMI1 3′UTR as well as levels of BMI1 protein expression upon ectopic expression of miR-15a, miR-16 or both showed a significant decrease, whereas inhibition of endogenous levels of miR-15a, mir-16 along with overexpression of BMI1 reversed the effect and resulted in the regain of DNA repair response that facilitated cell survival. We observed that in etoposide-induced DNA damage response, ectopic expression of miR-15a, miR-16 induced up-regulation of the phosphorylation of DNA damage related proteins like γ-H2AX, p-CHK2, p-ATM, p53BP and down-regulation of BMI1, RING1A, RING1B, ub-H2A, RNF8, RNF168, MEL18 and BRCA1. In the present study for the first time, we showed a significant role of miR-15a and miR-16 in DNA damage repair by targeting BMI1. We have shown the role of specific miRNAs involved in regulating the expression of BMI1 in response to DNA damage and BMI1 dependent ubiquitination pathway in breast cancer cells

Methods

Results

Conclusion