miRNA Cluster Induce Apoptosis microRNA can act as an oncogene in one biological condition and as a tumor suppressor gene in another [18,36]. Recent report suggests that upregulation of miR-23a,27a,24-2 cluster decreas- es transforming growth factor-beta induced tumor suppressive activities in human hepatocellular carcinoma cells [37]. In the present study we wanted to investigate the biological effects of over expression of miR-23a,27a,24-2 cluster in HEK293T cells. As shown in Fig. 2A, the annexin positive cells increased to 33.2%60.8% (p = 0.024) after over expression of miRNA cluster in HEK293T as compared to 2.5%60.3% (p = 0.016) in untransfected HEK293T cells. Moreover, in the presence of caspase inhibitor (BOC-D-FMK) the annexin positive cells came down to 12.3%60.4% (p = 0.015). The results of annexin assay were also validated with a positive control (data not shown). Our findings show that the over expression of this miR-23a,27a,24-2 cluster induces apoptosis in HEK293T cells by caspase-dependent as well as caspase-independent pathway. Since mitochondria plays a major role in both the types of apoptosis, we examined disruption in the mitochondrial mem- brane potential (Dym) by DiOC6 and ROS production by DCFH-DA in untransfected and p(23a,27a,24-2) transfected HEK293T cells. As indicated in Fig. 2B in comparison to controls where only 18.24% (p,0.002) of cells shifted towards left, approximately 74.64% cells (p,0.04) shifted towards left in miR-23a,27a,24-2 transfected HEK293T cells. Our results indicate that over expression of miR-23a,27a,24-2 cluster induces significant disruption of Dym in HEK293T cells. mCICCP was used as positive control for DiOC6 experiments. The DCFH-DA assay (Fig. 2C) also shows that over expression of miR-23a,27a,24-2 cluster increases ROS production as we observed an increase in fluorescence intensity from 26.80% (p,0.03) in untransfected HEK293T cells to 57.78% (p,0.02) in miR-23a,27a,24-2 cluster over expressed HEK293T cells. p(23a,27a,24-2) modulates expression of proteins of apoptotic pathways In order to better understand the molecular basis of this cluster induced apoptosis, we checked the expression of various proteins involved in apoptosis before and after the over expression of this cluster in HEK293T cells. Our western blot analysis (Fig. 3A) showed 0.70 fold (p = 0.02) decrease in procaspase-3 level, 2.20 fold (p = 0.01) increase in cleaved caspase-3 level, 0.72 fold Figure 2. Biological effect of over expression of p(23a,27a,24-2) in HEK293T cells. A. Annexin V-PE binding in HEK293T cells i) untransfected cells, ii) cells transfected with 4 mg p(23a,27a,24-2) for 24 h, and iii) cells transfected with p(23a,27a,24-2) for 24 h and treated with caspase inhibitor. B. Mitochondrial membrane potential in HEK293T cells, i) untransfected cells, ii) 24 h after transfection with 4 mg p(23a,27a,24-2), and iii) positive control, detected by DiOC6 staining and flow-cytometry analysis. C. ROS production in HEK293T cells i) untransfected cells, ii) 24 h after transfection with 4 mg p(23a,27a,24-2), and iii) positive control. doi:10.1371/journal.pone.0005848.g002 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5848