cells as the active caspase-8 cleaves a BH3-domain-only subfamily protein, Bid. This truncated Bid (tBid) translocates to mitochon- dria and mediates cytochrome-c release [22]. Caspase- indepen- dent pathway occurs via the translocation of AIF (Apoptosis Inducing Factor) from the mitochondria towards the nucleus; ultimately resulting in apoptosis [23]. Resistance towards apoptosis is a key factor for the survival of a malignant cell. Cancer results if there is too little apoptosis and cells grow faster and live longer than normal cells. In addition, defects in apoptosis signaling contribute to drug resistance of tumor cells. Thus, one of the main goals for oncologic treatment is to overcome resistance of tumor cells towards apoptosis. The exciting challenge in oncology is to translate the growing knowledge of apoptotic pathways into clinical applications. The objective of the present study was to examine the role of the miRNA cluster (miR-23a,27a,24-2) in apoptosis. Understand- ing the role of miRNA in apoptosis is likely to provide deeper insights into various disease processes and thus totally revolution- ize the scientific approach towards developing therapies against these diseases. Since their discovery, a number of bioinformatic softwares have come up predicting the targets of miRNAs, yet very few miRNA-target interactions have been validated experimen- tally. 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 -independent pathway. We also show that hsa-miR-27a negatively regulated the expression of FADD. FADD/Mort1 is a cytosolic adaptor molecule which is critical for signaling from CD95 (FAS/Apo1) and certain other members of the tumor necrosis factor receptor (TNF-R) family (called death receptors). Two protein interaction domains have been identified in FADD. The C-terminal ‘‘death domain’’ (DD) is needed for recruitment of FADD/Mort1 to ligated death receptor and N- terminal ‘‘death effector domain’’ (DED) mediates oligomerization and activation of caspase-8 zymogens. Caspase-8 activates other caspases by cleavage and this starts a proteolytic cascade which constitutes the ‘‘point of no return’’ in apoptotic signaling. Besides its role in apoptosis, this protein has also been implicated in survival/proliferation and cell cycle progression. FADD can also function as tumor suppressor gene [25]. Many reports also reveal the underexpression of FADD in several cancers like thyroid adenoma and acute myeloid leukemia [26,27]. Lack of FADD protein confers survival/growth advantages in tumor cells. The diverse roles of FADD ranging from cell survival to cell death and little knowledge of regulation of FADD expression makes it an interesting subject to study and we believe that finding a miRNA targeting this gene would add a new dimension to the ongoing research in the cancer therapeutics. Materials and Methods Sequences retrieval, Primer Designing and Cloning The sequences of miRNAs were retrieved from miRbase (http://microrna.sanger.ac.uk/sequences/), the 39 UTR sequence was retrieved from NCBI (http://www.ncbi.nlm.nih.gov/sites/ entrez/) and for primer designing primer 3 software was used (http://frodo.wi.mit.edu/). We cloned 39 UTR of FADD gene into pMIR-REPORT miRNA Expression Reporter Vector (Ambion, Austin, TX, USA) between SpeI and HindIII restriction sites using forward primer CCG CCG ACT AGT CAG CCT GGA CTT TGG TTC TC and reverse primer CCG CCG AAG CTT CTG CCT TGG CAA TTC TGT TA. miR-23a,27a,24- 2 sequence was amplified by PCR from genomic DNA using the following primers: CCG CCG AAG CTT CAT CTC TGC TCC miRNA Cluster Induce Apoptosis AAG CAT CA and CCG CCG GGA TCC TCT CTT TCT CCC CTC CAG GT and cloned in pSilencer4.1 vector (Ambion, Austin, TX, USA). The resulting plasmids were sequenced to ensure accuracy. Cell Culture and Transfection A549 (NSCLC, lung adenocarcinoma), HepG2 (hepatocellular adenocarcinoma), HeLa (cervical carcinoma), H520 (lung carci- noma), HEK293T (human embryonic kidney), MCF7 (breast carcinoma), Hep2 (laryngeal carcinoma) and Neuro2A (rat neuronal) cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10% (v/v) fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin in a humid- ified 5% CO2 atmosphere. For luciferase assay, the cells were cotransfected in 6-well plates by using lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol with 1 mg of the luciferase reporter vector, 1 mg of pSilencer4.1 and 0.3 mg of the b-gal vector (for normalization of transfection, Ambion Austin, TX, USA). For each well 40 nM/80 nM anti- miR-27a (Ambion Austin, TX, USA) was used as indicated. Luciferase and b-gal assays were done simultaneously on the cells 24 h after transfection using Promega kits. For over expression studies, the cells were cotransfected in 6-well plates by using lipofectamine 2000 with either 2 mg or 4 mg of the pSilencer 4.1 expressing the miRNA cluster. Semiquantitative RT-PCR analysis Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA). Reverse transcription was carried out with M-MuLV reverse transcriptase (MBI Fermentas, USA) according to the manufacturer’s protocol using 2 mg of total RNA. Primers for miR-23a (CCG CCG GGA TCC ACG GCC GGC TGG GGT TCC and CCG CCG AAG CTT CAG AGC TCA GGG TCG GTT GG), mir-27a (CCG CCG GGA TCC AGC TCT GCC ACC GAG GAT and CCG CCG AAG CTT AGG ATG GCA GGC AGA CAG), miR-24-2 (CCG CCG GGA TCC CTG TCT GCC TGC CAT CCT and CCG CCG AAG CTT CAT CTC TGC TCC AAG CAT CA) and b-actin (TGC GTG ACA TTA AGG AGA AG and CAT TGC CGA CAG GAT GCA G) were designed. The PCR conditions used were as follows: initial denaturation at 94uC for 5 min followed by 30 cycles of 94uC for 30 sec, 56uC for 45 sec, 72uC for 45 sec, and an additional cycle with extension at 72uC for 5 min. The normalization was done using b-actin RT-PCR. RT-PCR and real-time TaqMan PCR Total RNA was extracted using Trizol reagent and reverse transcription was carried out with M-MuLV reverse transcriptase according to the manufacturer’s protocol. Primers for pre-miR-24- 2 (CTC CCG TGC CTA CTG AGC T and CCC TGT TCC TGC TGA ACT GAG), pre-miR-27a (GCA GGG CTT AGC TGC TTG and GGC GGA ACT TAG CCA CTG T) and 18 s (GTA ACC CGT TGA ACC CCA TT and CCA TCC AAT CGG TAG TAG CG) were designed. Analyses were carried out using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Results were normalized with 18 s rRNA. TaqMan microRNA assays (Applied Biosystems, Foster City, CA) that include specific RT primers and TaqMan probes were used to quantify the expression of mature miR-27a (PN4373287) and miR-24-2 (PN4373072). 18 s rRNA (PN 4333760F) was used for normalization. The real time PCR data was normalized using Pfaffl’s method [28]. PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5848