(ENST00000301838) arising from the FADD gene was identified as a target to hsa-miR-27a. We observed that there is perfect complementarity between miR-27a and the 39UTR of FADD over the seed region (2–9 bases of the mature miRNA). To ascertain if miR-27a regulates FADD, we transfected HEK293T cells with p(23a,27a,24-2) and did northern blot and western blot analysis. Northern blot analysis confirmed increased expres- sion of miR-27a following transfection (Fig. 4A). Similarly, HEK293T cells transfected with p(23a,27a,24-2) showed reduced FADD protein by western blot (Fig. 4B). Since we observed dose-dependent increase of miR-27a by northern blot and dose-dependent decrease of FADD by western blot, FADD appeared to be a potential target of hsa-miR-27a. To demonstrate that the negative effect of miR-27a on FADD expression was direct, we cloned the entire wild type 39UTR of the FADD gene into luciferase reporter vector and did luciferase reporter assay. The luciferase activity assay at 24 h post transfection demonstrated that miR-23a,27a,24-2 cluster sup- pressed luciferase reporter activity by 32% (Fig. 4C). To demonstrate that this suppression in luciferase activity was the effect of miR-27a and not the effect of other two miRNAs (miR- 23a and miR-24-2), we used anti-miRs at two different concentrations (40 nM and 80 nM). Results in the Fig. 4C show that suppression was relieved by anti-miR-27a but not by miRNA Cluster Induce Apoptosis anti-miR-23a and anti-miR-24,2. Thus, our data confirms that miR-27a directly inhibits expression of FADD by binding to its target sequence. miRNA cluster augments the effect of TNF-a As TNF-a is known to induce apoptosis via the extrinsic pathway and the miRNA cluster (hsa-miR-23a,27a,24-2) also induced apoptosis by down regulating FADD, we next investigated whether over expression of miR-23a,27a,24-2 cluster has any effect on the sensitivity of HEK293T cells towards TNF-a. Our annexin assay (Fig. 5A) showed that p(23a,27a,24-2) and TNF-a by themselves induced 30%60.3% (p = 0.04) and 8.4%60.5% (p = 0.02) apoptosis, as compared to 37.3%60.2% (p = 0.018) apoptosis when HEK293T cells were over expressed with the cluster and treated with TNF-a (20 ng/ml) for 18 h. We observed only 2.3% apoptosis in untransfected cells. We also studied their combined effect on the mitochondrial membrane potential (ym). Our DioC6 assay showed (Fig. 5B) that 72% cells moved towards left when TNF-a treatment was given to cells after over expression of the miRNA cluster as compared to 29% cells which shifted towards left after TNF-a treatment in HEK293T cells and 57% cells that moved towards left in HEK293T cells after over expression with the miRNA cluster. Because receptor interacting protein (RIP), TNF receptor (TNFR)-associated factor Figure 4. Dose dependent expression of miR-27a and FADD and luciferase assay confirms that miR-27a targets the FADD 39 UTR. A. Detection of mature miRNA after transfection of clone expressing p(23a,27a,24-2), the same blot was probed for U6 expression for normalization. B. Western blotting for FADD after transfection of clone expressing miR-27a, the same blot was probed for b-actin for normalization. The fold change in expression of FADD is also presented after densitometric analysis. C. Luciferase assay proves that 39UTR of FADD is a target of miR-27a. *p(23a,27a,24-2) (2 mg) – Transfection of 2 mg of p(23a,27a,24-2). p(23a,27a,24-2) (4 mg) – Transfection of 4 mg of p(23a,27a,24-2). doi:10.1371/journal.pone.0005848.g004 PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e5848