miRNA Cluster Induce Apoptosis Figure 3. p(23a,27a,24-2) modulates pro and anti-apoptotic proteins in HEK293T cells. A. Effect of overexpressed miRNA cluster on the protein expression of FADD, Caspase 8, Caspase 3, Bcl-2, PARP, Bax and Bid in HEK293T cells. GAPDH/b-actin was used as an internal control. Data are representative of a typical experiment repeated three times with similar results. B. Release of Cytochrome-c from mitochondria to cytoplasm after the over expression of miR-23a,27a,24-2. Mitochondrial and cytoplasmic fractions were separated as described in the method section. The purity of the fraction was determined by the expression of Cox 4 (mitochondrial specific protein). C. Immunocytochemistry of AIF (red) after over expression of miRNA cluster in HEK293T. Nuclei were counterstained with the DNA-binding dye DAPI (49,6-diamidino-2-phenylindole; blue). Merged images are shown in iv and viii, bright field images are shown in iii and vii. doi:10.1371/journal.pone.0005848.g003 (p,0.04) decrease in PARP protein (116 kD fragment), 0.60 fold (p = 0.023) decrease in Bcl-2 level, 0.67 fold (p = 0.011) decrease in Bax level, 3.98 fold (p = 0.014) increase in cleaved caspase-8 level, 0.59 fold decrease (p = 0.04) in uncleaved Bid level and 0.50 fold decrease in FADD protein level (p = 0.02). To further confirm that miR-23a,27a,24-2 cluster induces apoptosis by caspase-dependent pathway we did western blot analysis for cytochrome-c. As shown in Fig. 3B, the cytochrome-c levels in the cytosol increased by 3.42 fold (p = 0.03) after the over expression of this cluster. It is known that apoptosis-inducing factor (AIF), perhaps the best-studied example of a caspase- independent cell death (CICD) mediator, is slowly released from mitochondria following mito- chondrial outer membrane permeabilization in a caspase-inde- pendent manner although caspase activity may accelerate the release [38]. Following release, AIF translocates to the nucleus. To confirm that the over expression of this cluster induces apoptosis by caspase- independent pathway also, we next performed immuno- staining to look for nuclear translocation of AIF (Apoptosis Inducing Factor) and confocal imaging was used to determine the location of AIF in HEK293T cells before and after cluster over expression. We observed that AIF translocates from mitochondria to the nucleus after the cluster over expression (Fig. 3C). The results obtained here suggest that over expression of this cluster induces FADD independent caspase-8 activation as there was decrease of FADD in miR-23a,27a,24-2 transfected HEK293T cells as compared to untransfected cells. Hence, it was confirmed that over expression of this cluster induces apoptosis by both caspase-dependent and -independent pathways. FADD is a potential target of hsa-mir-27a MicroRNAs regulate gene expression through decreased translation, increased degradation of the target message, or both [39]. Prediction of animal miRNA targets is challenging because there is only a partial pairing of miRNAs with their targets, and the mechanism of miRNA-mediated regulation is poorly under- stood. To improve the accuracy of binding site prediction, we used three target prediction algorithms, miRanda [40], RNAhybrid [41] and Target Scan [42]. Only those miR-target pairs predicted by all the three softwares were used in the analysis. The transcript PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e5848