Twenty fresh GC tissue samples from GC patients and their matched adjacent normal gastric mucosal tissues were immediately snap frozen in liquid nitrogen and stored at −80 °C until total RNA was extracted. The samples were collected from consenting individuals according to the protocols approved by the Ethics Committee at First Affiliated Hospital of South China University. The association of miR-199a/b-3p relative expression with the clinicopathological characteristics in 20 GC patients are showed in Additional file 1: Table S1.
Human normal gastric mucosa cell line GES-1 (CBP60512) and human gastric cancer cell lines MGC-803 (CBP60485) and SGC-7901 (TCHu 46) were all purchased from Nanjing Cobioer Biotechnology and Shanghai Cell Bank of Chinese Academy of Sciences, and was cultured in RPMI 1640 medium (HyClone) supplemented with 10% fetal calf serum (HyClone), 2 mM L-glutamine, 100 U/ml Penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The cells were used between passages 10 and 20. The complete medium was refreshed every 24 h.
In vivo tumor xenograft studies
Four-week-old male athymic BABL/c nude mice were used under conditions approved by the Institutional Animal Care and Use Committee of First Affiliated Hospital of South China University. To assess the anti-proliferation capacity of miR-199a/b-3p in vivo, 2 × 106 MGC-803 cells transfected with negative control (NC) or 50 nM miR-199a/b-3p mimics respectively, were suspended in 0.2 ml sterile saline and subsequently implanted subcutaneously into the axillary fossae of each mouse. After 2 week implantation, mice were killed, and xenograft tumors were removed intactly. The volume of xenograft tumors was calculated as follows: length × width2 × 1/2. And fresh tissues from xenograft tumors were immediately snap frozen in liquid nitrogen and stored at −80 °C until total RNA and protein were extracted.
Oligonucleotides and transfection
MiR-199a/b-3p mimics and negative control (NC) were chemically synthesized by RIBOBIO and transfected with Lipofectamine 2000 (Invitrogen) in MGC-803 and SGC-7901 cells at a final concentration of 10 or 50 nM. PAK4-spedific siRNA and transfection reagent were purchased from Santa Cruz Biotechnology. Both MGC-803 and SGC-7901 cells were transfected with transfection reagent only (Mock) or the mixture of transfection reagent and 50 nM PAK4 siRNA according to the manufacturer’s protocol.
Real-time quantitative RT-PCR
Total RNA was extracted from the prepared cells and tissues using the RNAiso Plus Kit (Takara), then cDNA synthesis was performed using the PrimeScript RT reagent Kit (Takara), and finally Real-time PCR was performed in triplicate using the LightCycler 480 System (Roche) according to the manufacturer’s protocol. The PCR primers were shown in Additional file 1: Table S2. U6 and β-actin were used as internal controls. The 2−ΔΔCT method was used to analyze real-time PCR data.
The prepared cells and tissues were lysed for 30 min on ice in RIPA lysis buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholate, supplemented with the protease inhibitor PMSF. After being centrifuged at 14,000×g for 30 min at 4 °C the supernatants were collected. SDS-polyacrylamide gelelectrophoresis and western blot were performed according to standard protocols. Human and mouse monoclonal antibodies of anti-PAK4, anti-p-MEK, anti-p-ERK and anti-β-actin (Cell Signaling Technology) were all diluted at 1:1000. Secondary antibodies were all diluted at 1:4000.
Cell proliferation assay
Cell proliferation was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-.
diphenyltetrazolium bromide, Sigma) assay. In brief, the prepared cells were seeded into 96-well plates, of which 20 μl MTT (5 mg/ml) was added into each well, and subsequently the plates were incubated in 5% CO2 at 37 °C incubator. After 4 h, the liquid was thrown away and 150 μl dimethyl sulfoxide (Sigma) was added into each well. After vibrating 10 min, the optical density was measured in a microplate reader at the wave length of 570 nm.
The results are shown as the mean ± standard deviation. Statistical significance was determined by one-way analysis of variance with Tukey’s multiple comparisons under equal variances or with Dunnett T3’s multiple comparisons under unequal variances; Statistical significance of gene expression between normal and cancerous tissues was determined by Pearson chi-square test; a value of P < 0.05 was considered statistically significant.