The observations made in this study suggest that the
H2AFX gene undergoes CNA in patients with sporadic breast cancer, as well as in studied cancer cell lines; however, the expression status does not correspond with the CNA status. Two recent studies in rats and mice at a genome-wide scale have described the effect of CNVs on gene expression, exhibiting negative correlation in 2% to 15% of the genes with their expression [
3,
4]. We provide evidence for one of the possible mechanisms of such a nonconcordant relation between expression and the number of gene copies based on specific miR regulation of expression. One such miR, hsa-miR-24-2, that has been reported to be a strong regulator of
H2AX expression [
24] was confirmed in our study, both in cell lines and in sporadic breast tumor samples, irrespective of CNA. Interestingly, it was observed that overexpression of miR-24-2 downregulated the transcript expression of
H2AFX alongwith
BCL-2, MDM2and
P21, with a corresponding increase in apoptotic cell death, suggesting an adoption of a new paradigm in therapeutic designs to overcome apoptotic resistance in cancer cells. The role of miR-24-2 in regulation of apoptosis has been shown by a few studies, but the regulation of pro- or antiapoptotic genes by this miR is not known, except for FAF1 [
30]. Our study provides the mechanistic insight into the apoptotic induction mediated by miR-24-2 and identifies BCL-2 as the novel cellular target of miR-24-2 (Figure
4e). We propose that while downregulation of H2AX results in impaired DNA repair, channeling the cells into the apoptotic pathway, downregulated BCL-2, encoding an integral outer mitochondrial membrane protein and known to block the apoptotic death in a variety of cell systems [
40], could contribute further to apoptotic cell death [
41]. It has been shown that H2AX is required for the p53/p21 pathway [
42], and it is expected that the lower level of H2AX expression could prevent the cells from cell cycle arrest and promote induction of apoptosis. We have also observed that
MDM2 and
P21 possibly could emerge as other key genes that promote apoptotic induction and whose expression is modulated by miR-24-2, either directly or indirectly. This, however, would require experimental confirmation through reporter gene assays in future studies. Nevertheless, on the basis of our findings, we propose that miR-24-2 is a strong inducer of apoptotic pathway in MCF-7 cells by controlling the expression of important genes involved in apoptotic regulation. MDM2 and p21 are known as key players in regulating the p53 response to induce apoptosis or growth arrest [
43]. MDM2 acts as an oncoprotein that promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein [
44]. Also, low levels of MDM2 have been shown to induce the transcription of proapoptotic genes and the translocation of p53 from nucleus to mitochondria, resulting in apoptosis [
45]. p21 is a cyclin-dependent kinase inhibitor (CDKN1A) and functions as a regulator of cell cycle progression to G1 in response to p53 checkpoint pathway [
46]. Its role in apoptosis is not very clear, but the possibility is that low expression of p21 would prevent the cells from p53/p21-mediated cell cycle arrest pathway and result in induction of apoptosis [
47]. Since p21 transcripts do not have a miR-24-2 binding site, we surmise that the expression of p21 gets reduced as a result of secondary effect and could possibly be a secondary target of miR-24-2 [
48]. Interestingly, we have also tested the apoptotic potentiating activity of miR-24-2 in the presence of a mitotic inhibitor drug, docetaxel, and observed a significant increase in cell death in MCF7 cells that have received combination treatmentof docetaxel (2 nmol/l) and miR-24-2 over-expression (500 ng of pEP-miR-24-2) as compared to MCF7 cells that have received docetaxel treatment or mir-24-2 over-expression alone (data not shown). We propose that the lower expression of these genes as a result of miR-24-2 overexpression could independently, or in association with other proteins, target different apoptotic pathways and provide an alternative window for effective tumor cell killing, either alone or in combination with anticancer drugs such as cisplatin and docetaxel.