miRNA Cluster Induce Apoptosis Figure 6. Effect of p(23a,27a,24-2) on JNK and NF-kB. A. Western blot analysis for p-JNK before and after p(23a,27a,24-2) over expression in HEK293T cells. 50 mg protein was loaded on the gel and blot was developed using BCIP/NBT. Tubulin was used as a loading control. B. Transcriptional activity of NF-kB in transiently transfected HEK293T cells after p(23a,27a,24-2) over expression. HEK293T cells were treated with TNF-a after 8 h of transfection and luciferase activity was measured and normalized per mg of protein. C. Western Blot analysis for NF-kB after overexpression of cluster in presence of TNF in HEK293T cells. The fold change in expression of NF-kB is also presented after densitometric analysis. doi:10.1371/journal.pone.0005848.g006 factor-beta-induced tumor-suppressive activities in human hepa- tocellular carcinoma cells. Contrary to their findings is our study where for the first time it is shown that upregulation of miR- 23a,27a,24-2 cluster induces caspase-dependent and caspase- independent apoptosis in human embryonic kidney cells. Consis- tent to our findings there are reports in the literature where it has been shown that the same microRNA can act as an oncogene under one set of conditions and as a tumor suppressor in another [18,36]. In this investigation, several observations suggest that miR-27a regulates FADD expression. For example, over expression of miR- 23a,27a,24-2 cluster in HEK293T cells induces dose-dependent increase of hsa-miR-27a and dose dependent decrease of FADD as seen by northern and western analysis, respectively. This effect was direct, as p(23a,27a,24-2) cluster negatively regulated the expression of FADD 39 untranslated region (UTR)-based reporter construct. Our data shows that over expression of this cluster induces down regulation of FADD and up regulation of caspase-8 thereby showing that there is FADD-independent caspase-8 activation in HEK293T cells. In regard to caspase-8 activation there are mixed reports. Caspase-8 activation has been extensively studied in apoptosis mediated by members of the death domain receptor family such as Fas/CD95 and TNFR [44]. Interactions between Fas and FADD via their C-terminal death domains expose the N- terminal death effector domain (DED) of FADD, which can interact with DED domains in the caspase-8 proform, resulting in the oligomerization of this protease and its subsequent auto- cleavage and activation [45–48]. Recent studies have also suggested that alternative pathways may also be responsible for caspase-8 activation. Indeed, it has been observed that TGF- mediated caspase-8 activation in Burkitt cells does not involve death domain receptors [49], and FADD-independent caspase-8 activation by anticancer drugs has also been reported by Wesselborg et al [50]. The data here suggests that there are at least two different pathways through which miR-23a,27a,24-2 cluster induces apoptosis in HEK293T cells: conventional caspase-dependent apoptotic cell death and caspase-independent cell death. Both occur via the mitochondrial membrane disruption pathway. Here, we propose a model (Fig. 7) for miR-23a,27a,24-2 cluster induced apoptosis in HEK293T cells. There is FADD-indepen- dent caspase-8 activation which in turn is followed by cleavage of Bid, the loss of mitochondrial membrane potential, induced reactive oxygen species (ROS) generation, release of apoptotic proteins from mitochondria, and subsequent caspase-dependent apoptosis. Our findings were confirmed by the upregulation of active form of caspase 3, down regulation of 116 kD PARP PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e5848