08±3.08 −2.49±3.56 0.241 hsa-miR-508-5p

−5.49±1.64 −7.48±1.96 0.069 hsa-miR-30c 4.37±3.70 2.27±5.47 0.058 hsa-miR-134 0.92±4.48 −1.50±4.19 0.022* hsa-miR-337-3p −3.67±3.32 −6.04±2.73 0.005* Gastric cancer (GC) vs. lymph nodes (LN); n=3; *P<0.05. RNA isolation and miRNA microarray profiling Total cellular RNA was isolated from tissue specimens using TRIzol® reagent (Invitrogen, Carlsbad, CA). Briefly, the frozen tissues were homogenized by using a biopulverizer with Mini-Bead-Beater-16 and added to TRIzol® reagent for RNA isolation according to the manufacturer’s instructions. The RNA purity was assessed by measuring the absorption rate at 260 nm and at 280 nm in a NanoDropND-1000 spectrophotometer (A260/A280 ratio of 1.8–2.1 was selleckchem considered acceptable), and the RNA integrity number (RIN) was detected by an Agilent 2000 analyzer (RIN≥8.0). Next, these RNA samples of human primary gastric cancer Nepicastat chemical structure and the corresponding metastatic tissues were reversely transcribed into cDNA, labeled with Hy3 and Hy5, and used as probes for miRNA profiling using the miRCURYTM LNA system (MicroRNA

array V10.0 whole list, LC Sciences, Houston, TX). After bioinformatics analysis of the primary gastric cancer and metastatic tissue samples, the differentially expressed miRNAs were identified. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) To confirm some of these differentially expressed miRNAs, tumor tissues were harvested and stored in RNAlater

solution (Ambion, Austin, TX). Total cellular RNA was isolated from RNAlater-fixed tumor tissues or fresh cultured cells by using the Dimethyl sulfoxide mirVana™ miRNA isolation kit (Ambion, Austin, TX) and reversely transcribed into cDNA with the TaqMan® MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Taqman gene expression assays (Applied Biosystems) were used to assess expression levels of hsa-miR-508-5p, hsa-miR-337-3p, hsa-miR-30c, hsa-miR-483-5p, hsa-miR-134, and U6 in tissues or cultured cells by the 7900HT fast real-time PCR system (Applied Biosystems, Darmstadt, Germany). Relative expression levels of each miRNA were calculated using the ΔΔCT method after normalization with U6 levels (an internal control). Cell lines and culture A nonmalignant GES cell line and nine human gastric cancer cell lines (SNU1, SNU5, AGS, HGC-27, BGC-823, MGC-803, SGC-7901, MKN-28, and MKN-45) were originally purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China), stored, recovered, and used at an early passage from cryopreservation in liquid nitrogen. These cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL). All cell lines were cultured in 6-well plates in humidified air supplemented with 5% CO2 at 37°C. After cell culture for 48 h, total RNAs were isolated and used for qRT-PCR, respectively.