For inhibition experiments amiRs or scrambled control were added at 30-nM concentration

s PTEN and p27 expression. In addition to direct regulation by miR-141, p27 can also be regulated by PTEN. These findings critical 21602423 insight into the mechanism of EBV-mediated NPC development and implicate that SPLUNC1 could be exploited in the development of targeted therapies and serve as a diagnostic and prognostic factor for NPC. Materials and Methods Ethics Statement Before study initiation, ethical approval was obtained from the Cancer Hospital of the Hunan province in Changsha, and the Central South University Ethics Review committees/Institutional Review Boards. NPC samples, nontumor nasopharyngeal epithelial tissues as well as peripheral blood lymphocytes from normal volunteers were collected at the Cancer Hospital of the Hunan province. Written informed consent was obtained from all patients and volunteers. All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Central South University. Animals were allowed access to standard chow diet and water ad libitum and were housed in a pathogen-free barrier facility with a 12L:12D cycle. The mice were sacrificed by CO2 asphyxiation. Plasmid and miRNA The coding region of the SPLUNC1 gene and its BPI domain -deleted mutant were inserted into the pEGFP vector as previously described. The plasmid p2089, which contained the complete EBV genome of the B95-8 strain was kindly provided by Professor Hammerschmidt SPLUNC1 Signal Pathway Can Be Hindered by LMP1 . Two eukaryotic expression vectors with the EBVrelated 26841170 BZLF1 and BALF4 genes /BZLF1 and pcDNA3.1 /BALF4) were constructed as previously described. miR-141 mimics, negative control mimics, miR-141 inhibitors, and negative control inhibitors were purchased from Shanghai Gene-Pharma Co. ward 59-CGTTTCT TGGAGTATAGC-39, CAJA-DRBI reverse 59-CACTAGGAACCTCTCTGA-39. Antibodies and Western Blot Analysis Antibodies specific for human PTEN, Akt, GSK3b, MDM2, Bcl-2, BAD, caspase-3, caspase-8, ERK, JNK, NFkB, Ika, p38, p27, CCND1, CCND2, CCND3, CCNE1, CCNE2, CDK2, bactin, GAPDH, and Phospho-PTEN, Phospho-Akt, PhosphoGSK3b, Phospho-MDM2, Phospho-BAD, Phospho-ERK, Phospho-p38, and Phospho-Ika were purchased from Cell Signal Inc and R&D system. Antibody for SPLUNC1 was previously produced by us. Anti-LMP1 mouse monoclonal antibody was purchased from Dako. HRPlabeled goat antirabbit, mouse and rabbit antigoat secondary antibodies were purchased from Sigma. Tissue Array and Immunohistochemistry Tissue array was constructed as described in our previous study. The tissue core numbers on each section were slightly different because of additional losses suffered from block trimming and staining procedures. 17 normal nasopharyngx, 65 chronic inflammation of nasopharyngeal mucosa, 59 atypical hyperplasia, 195 newly diagnosed NPC, 95 epithelium adjacent to glandular adjacent to NPC, and 37 NPC after radiotherapy were placed in one TMA block. Immunohistochemistry was MedChemExpress PP 242 performed using the peroxidase antiperoxidase technique after a microwave antigen retrieval procedure. The sections were incubated at 4uC overnight using mouse anti-human SPLUNC1 antibody. A semiquantitative scoring criterion for IHC was used, in which both staining intensity and positive areas were recorded. Tumor Formation Assay in Nude Mice Male BALB/c nude mice at 46 weeks of age were used and divided into two groups 10 mice. To assess the effect of SPLUNC1 on tumorigenicity in vivo, 56106 pEGFP-C2SPLUNC1 or pEGFP-C2 plasmids