miRNA Cluster Induce Apoptosis Figure 7. Hypothetical model for p(23a,27a,24-2) induced apoptosis. The green arrows indicate down regulation and red arrows indicate up regulation. doi:10.1371/journal.pone.0005848.g007 protein, Bcl-2, Bax and release of cytochrome-c from mitochon- dria to cytosol after over expression of miR-23a,27a,24-2 cluster in HEK293T cells. Rapid ROS generation might be causing the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis by miR-23a,27a,24-2 cluster in HEK293T cells. There are several reports in the literature which support this fact [51,52]. Further dissection of miR-23a,27a,24- 2 induced apoptosis shows that there was increased TRAF2 expression after miR-23a,27a,24-2 cluster over expression. Literature suggests that trimerised TNFR1 can recruit proteins that engage various signal transduction pathways, some of which either abrogate or potentiate the apoptotic response [53] for example, the serine threonine kinase RIP and TRAF-2. Studies using RIP and TRAF-2 deficient cells indicate that RIP is most likely responsible for NF-kB activation, whereas, TRAF-2 preferentially activates c-Jun N- terminal kinase (JNK) [43]. Since in our study we observed increased TRAF-2 and decreased RIP levels we can hypothesize that in HEK293T cells TRAF-2 plays a major role in this cluster induced apoptosis. To prove this we did western blot analysis and observed that there was increased JNK activation as we observed increased p-JNK after miR- 23a,27a,24-2 cluster over expression in HEK293T cells. We also did not observe any NF-kB activation and the NF-kB expression was found to be decreased after over expression of this References 1. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294: 862–864. 2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297. 3. Carrington JC, Ambros V (2003) Role of MicroRNAs in Plant and Animal Development. Science 301: 336–338. 4. de Moor CH, Meijer H, Lissenden S (2005) Mechanisms of translational control by the 39 UTR in development and differentiation. Semin Cell Dev Biol 16: 49–58. cluster. In conclusion, we can say that over expression of miR- 23a,27a,24-2 cluster induces caspase-dependent and indepen- dent apoptosis via JNK. Our study extends the knowledge on FADD protein, a key death receptor adaptor molecule. In addition to death receptor ligand, FADD is also regulated at the transcriptional level by microRNA. Absence of FADD protein expression is a marker for tumor development and a prognostic factor for poor response to chemotherapy in humans. FADD deficient tumor cells resist death receptor-mediated apoptosis by chemotherapeutics drugs. Our data demonstrating that over expression of miR-23a,27a,24-2 sensitized HEK293T cells to TNF-a cytotoxicity could potentially be of value in cancer therapy. Acknowledgments The authors thank Prof. Samir K. Brahmachari for project conceptual- ization and for his valuable scientific inputs. Author Contributions Conceived and designed the experiments: RC YA NS. Performed the experiments: RC YA. Analyzed the data: RC YA NS. Contributed reagents/materials/analysis tools: RC YA MH VS NS. Wrote the paper: RC YA NS. 5. Lai EC (2002) Micro RNAs are complementary to 39 UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 30: 363–364. 6. Robins H, Press WH (2005) Human microRNAs target a functionally distinct population of genes with AT-rich 39 UTRs. Proc Natl Acad Sci U S A 102: 15557–15562. 7. Stark A, Brennecke J, Bushati N, Russell RB, Cohen SM (2005) Animal MicroRNAs confer robustness to gene expression and have a significant impact on 39UTR evolution. Cell 123: 1133–1146. PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e5848