Written informed consent was obtained from all participants. The study was approved by the Human Research Ethics Committee of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University (HREC 08–028), and the Laboratory Animal Ethics Committee of Ruijin Hospital. Research in human GC tissues was conducted in accordance with the Declaration of Helsinki. Animal procedures were carried out according to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
Cell lines and cell culture
Human GC cell lines SGC-7901, NCI-N87, BGC-823, and AGS were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). MKN-45 and MKN-28 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan), and KATO III and SNU-1 were originally purchased from the American Type Culture Collection (Manassas, VA, USA). GES-1, an immortalized gastric epithelial cell line, was a gift from Professor Feng Bi (Huaxi Hospital, Sichuan University, Chengdu, China). Cells were stored, recovered from cryopreservation in liquid nitrogen and used at early passages. All cells were maintained in RPMI-1640 medium plus 10% fetal bovine serum (FBS) and cultured in a 5% CO2 humidified atmosphere.
GC patient tissues and the adjacent non-tumor tissues were obtained from 140 GC patients undergoing radical gastrectomy at the Department of Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University. All patients provided consent and samples were confirmed by independent pathological examination. None of the patients received preoperative treatment. The pathologic tumor staging was determined according to the International Union Against Cancer (2009).
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After the quantitation of mRNA, 2 μg of total RNA were reverse transcribed with random primers following the manufacturer’s instructions (MBI Fermentas, Vilnius, Lithuania). The PCR amplifications were performed in triplicate using the SYBR Green Real Time PCR (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Quantification was performed using the ΔΔCt relative quantification method with human GAPDH as an internal control. The following primers were used: Gli1 [GenBank:NM_005269.2, GI: 224809486] (sense: 5′-GGA AGT CAT ACT CAC GCC TCG A-3′; antisense: 5′-CAT TGC TGA AGG CTT TAC TGC A-3′) , Zeb2 [GenBank: NM_001171653.1, GI: 224809486] (sense: 5′-AGC CAC GAT CCA GAC CGC AA-3′; antisense: 5′- GCT GTG TCA CTG CGC TGA AGG T-3′), OPN [Genbank: NM_000582, GI:38146097] (sense: 5′-GGA TCC CTC ACT ACC ATG AG-3′; antisense: 5′-AAG CTT GAC CTC AGA AGA TGC ACT-3′)  and GAPDH [GenBank:NM_002046.4, GI: 284413745] (sense: 5′-GGA CCT GAC CTG CCG TCT AG-3′; antisense: 5′-GTA GCC CAG GAT GCC CTT GA-3′).
The expression levels of miRNAs were assessed by the stem-loop RT-PCR method using the Hairpin-it™ miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) with specific primers for miR-133b and U6 small nuclear RNA (RNU6B). Relative miRNA expression of miR-133b was normalized against the endogenous control, U6, using the ΔΔCt method.
Transient transfection of miRNA mimics
MiR-133b mimic (dsRNA oligonucleotides) and negative control mimic (NC) (sense: 5′-UUC UCC GAA CGU GUC ACG UTT-3′, antisense: 5′-ACG UGA CAC GUU CGG AGA ATT-3′) were purchased from GenePharma (Shanghai, China). Transfection was carried out using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s procedures. MiRNA mimics were used at a final concentration of 100 nM.
At 16 h post-transfection with miRNA mimics, cells (1 × 106 cells/well) were seeded to 90% confluence in a 6-well plate for overnight culture. A scratch was made through the center of each well using a pipette tip, creating an open “wound” that was clear of cells. The dislodged cells were removed by three washes with culture media. Plates were then cultured with serum-reduced medium containing 1% FBS. Migration into the open area was documented at 72 h post-scratching. Each condition was tested in triplicate and each experiment was repeated at least three times.
Cell migration and invasion assays
At 16 h post-transfection with miRNA mimics, 5 × 104 cells in serum-free medium were introduced into the upper compartment of the BD BioCoat control inserts (Cat. # 354578, BD Discovery Labware, Bedford, MA, USA) fitted with membranes of 8 micron porosity separating the upper and lower compartments. The lower compartment was filled with normal culture medium supplemented with 10% FBS as the chemoattractant. Cells were incubated for 48 h for the migration assay and 72 h for the invasion assay. For the invasion assay, the inserts were previously coated with extracellular matrix gel (BD Biosciences, Bedford, MA, USA). At the end of the experiments, the cells on the upper surface of the membrane were removed, and the cells on the lower surface were fixed and stained with 0.2% crystal violet. Five visual fields of each insert were randomly chosen and counted under a light microscope. Each condition was assayed in triplicate and each experiment was repeated at least three times.
Construction of the reporter gene system and luciferase activity assay
The 203 bp full length wild-type (WT) Gli1-3′UTR containing the putative miR-133b binding site or mutant Gli1-3′UTR (mut) was synthesized (Sangon, Shanghai, China). After digestion by SpeI and HindIII, the fragments of wild-type and mutant Gli1-3′UTR were cloned into the SpeI and HindIII sites of the pMIR-Report luciferase vector (Applied Biosystems) and named pMIR/Gli1 and pMIR/Gli1/mut, respectively. Sequencing was used to verify the constructs.
For the relative luciferase reporter assay, cells were seeded in a 24-well Plate 24 h prior to assay performance. In each well, 100 ng pMIR/Gli1 or pMIR/Gli1/mut, 1 ng pRL-TK (Promega, Madison, WI, USA) containing Renilla luciferase and 100 nM miRNA mimics were cotransfected using Lipofectamine™ 2000 reagent. Relative luciferase activity was calculated 48 h after cotransfection using the Dual-Glo Luciferase assay (Promega) according to the manufacturer’s procedure. Firefly luciferase activity was normalized to Renilla luciferase activity.
Western blot analysis
Protein levels were quantified by standard western blot procedures with the following antibodies: Gli1 (1:1000, Cell Signaling Technology, Beverly, Massachusetts, USA), OPN (1:500, IBL, Japan), Zeb2 (1:1000, Prosci, Poway, CA, USA) and GAPDH (1:20000, Abcam, Cambridge, UK). Protein levels were normalized to total GAPDH levels.
Retroviral transfection for stable cell lines
As previously described , retroviruses containing miR-133b or no insert (NC, negative control) were produced. After infections of MKN-28 cells, positive cells were selected and named RV-miR-133b and RV-miR-NC. MiR-133b expression was confirmed by qRT-PCR.
In vivo metastasis peritoneal spreading assay
MKN-28, RV-miR-NC and RV-miR-133b cells were resuspended and injected intraperitoneally (2 × 106 cells/mouse) into 4-week-old male BALB/C nude mice (Shanghai Laboratory Animal Center of China). Ten mice were included in each group. On the 60th day after intraperitoneal injection, mice were euthanized by cervical dislocation, and peritoneal spreading of tumor lesions was assessed by necropsy. All experiments were performed in accordance with the official recommendations of the Chinese Animal Committee.
All tests of significance were two tailed. Continuous variables were compared using the Student’s t test for normally distributed variables and Wilcoxon rank-sum test for non-normally distributed variables. The relationship between the miR-133b expression levels and clinicopathologic parameters was analyzed using tertiles and the Pearson Chi-square test. All values are presented as mean ± SD. All statistical analyses were performed using PASW Statistics 18.0 software (IBM, Chicago, IL, USA). p <0.05 was considered to indicate a statistically significant result.