title: miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.
abstract: BACKGROUND
  New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.
  
  
  METHODS
  Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2.
  
  
  RESULTS
  It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2.
  
  
  CONCLUSIONS
  mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.
authors.affiliation: National Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, Saraswatipuram, New Delhi 110 067, India.
authors.forename: Niloo
authors.initials: N
authors.lastname: Srivastava
authors.forename: Siddharth
authors.initials: S
authors.lastname: Manvati
authors.forename: Archita
authors.initials: A
authors.lastname: Srivastava
authors.forename: Ranjana
authors.initials: R
authors.lastname: Pal
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Kalaiarasan
authors.forename: Shilpi
authors.initials: S
authors.lastname: Chattopadhyay
authors.forename: Sailesh
authors.initials: S
authors.lastname: Gochhait
authors.forename: Raina
authors.initials: R
authors.lastname: Dua
authors.forename: Rameshwar N K
authors.initials: RN
authors.lastname: Bamezai
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002945:Cisplatin / Q000494:pharmacology; D056915:DNA Copy Number Variations; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D019254:Genes, bcl-2; D006367:HeLa Cells; D006657:Histones / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C468783:H2AX protein, human; D006657:Histones; C543845:MIRN24 microRNA, human; D035683:MicroRNAs; D019253:Proto-Oncogene Proteins c-bcl-2; D002945:Cisplatin