Upregulation of miR-23a,27a,24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells Ravindresh Chhabra., Yogita K. Adlakha . , Manoj Hariharan, Vinod Scaria, Neeru Saini* Institute of Genomics and Integrative Biology, Delhi, India Abstract miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a,27a,24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 39UTR based reporter construct. Moreover, we also showed that over expression of miR-23a,27a,24-2 sensitized HEK293T cells to TNF-a cytotoxicity. Taken together, our study demonstrates that enhanced TNF-a induced apoptosis in HEK293T cells by over expression of miR- 23a,27a,24-2 cluster provides new insights in the development of novel therapeutics for cancer. Citation: Chhabra R, Adlakha YK, Hariharan M, Scaria V, Saini N (2009) Upregulation of miR-23a,27a,24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells. PLoS ONE 4(6): e5848. doi:10.1371/journal.pone.0005848 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received December 16, 2008; Accepted May 15, 2009; Published June 9, 2009 Copyright: 2009 Chhabra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Council of Scientific and Industrial Research, India [NWP0036]. RC was supported with fellowship from CSIR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nkhanna@igib.res.in . These authors contributed equally to this work. Introduction miRNAs are a class of highly conserved, non-coding single- stranded RNAs (18–24 nt) derived from the endogenously produced pre-miRNA (precursor) having a hairpin (stem-loop) structure [1–3]. miRNAs refine gene expression post-transcrip- tionally either via the cleavage of target mRNAs or the inhibition of translation of target mRNAs depending upon complementarity between miRNA and the 39 untranslated regions (UTRs) of targeted mRNA in animals and plants [4–8]. The emerging role of miRNAs in the regulation of fundamental set of cellular mechanisms such as proliferation, apoptosis, development, differ- entiation and metabolism [9–16] clearly suggests that any aberration in miRNA biogenesis pathway or its activity contributes to the human disease pathogenesis including cancer [17]. Recent findings have also demonstrated that miRNAs could act either as potential oncogenes or tumor suppressor genes [18,19]. Apoptosis, or programmed cell death, is a highly regulated, morphologically and biochemically distinct physiological cell death event which is accompanied by cytoskeletal disruption, cell shrinkage, membrane blebbing, chromatin condensation, and internucleosomal DNA fragmentation [20]. Caspases, a family of aspartate-specific cysteine proteases, play a critical role in the execution of apoptosis. Broadly caspases can be divided into two types - Initiator and Effector. Initiator caspases, with long prodomains, such as caspases-8, -9 and -10, either directly or indirectly activate effector caspases, such as caspases-3, -6 and -7. These effector caspases then cleave intracellular substrates, including structural and regulatory proteins such as cytokeratin, poly (ADP-ribose) polymerase (PARP) and are directly responsible for many of the morphological features of apoptosis [21]. Literature also shows that cells can undergo apoptosis in caspase-dependent and/or -independent manner. Caspase-depen- dent pathway can be further divided based on the initiator caspases into the extrinsic pathway or intrinsic pathway. The extrinsic pathway (the death receptor-mediated pathway) is triggered by the members of the tumor necrosis factor (TNF) receptor super family. Binding of the ligand to its cognate receptor results in receptor trimerisation and subsequent recruitment of the dual adaptor molecule FADD leading to rapid activation of caspase-8 through recruitment of procaspase-8. Intrinsic pathway (the mitochondrial-mediated pathway) is activated in response to a variety of stress conditions, such as cytotoxic drugs, UV irradiation or growth factor withdrawal, which induce mitochondrial perturbation and the cytoplasmic release of pro-apoptotic mitochondrial proteins such as cytochrome-c leading to the activation of caspase-9. Both pathways finally lead to activation of caspase-3, the executioner caspase responsible for the final morphological changes observed during apoptosis. Accumulating evidences suggest that a cross-talk between two pathways exists in PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5848