2.1. Cell culture and transfection
Human normal gastric epithelial cell line GES-1 and gastric cancer cell lines BGC-823, SGC-790, AGS, MKN45 and MKN28 were all purchased from Cell Bank of The Chinese Academy of Sciences (Shanghai, China). DMEM with 10% FBS was used for cell culture. MiR-496 mimics, miRNA mimics negative control and LYN kinase (LYN) overexpression plasmid were purchased from GenePharme (Shanghai, China) and transfected into AGS cells using lipofectamine2000 (Invitrogen, CA, USA) according to manufacturer’s protocol.
2.2. Fluorescence quantitative PCR (qPCR)
After the transfection for 24 h, total RNA was extracted from AGS cells by TRIzol reagent (Invitrogen, USA) and reverse transcript to cDNA using M-MLV Reverse transcriptase and TaqMan™ MicroRNA Reverse Transcription Kit (Invitrogen, USA). QPCR was performed to detect the level of miR-496 and LYN using the qPCR kit according to manufacturer’s protocol (Invitrogen, USA). The relative expression of miR-496 and LYN was analyzed using 2-ΔΔCt method.
2.3. Western blot
After the transfection for 48 h, whole-cell protein was extracted using a RIPA lysis buffer. The protein concentrations were detected by bicinchoninic acid (BCA) method. Then, protein extracts were separated using SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with skimmed milk, the protein was incubated with the primary antibodies for 1 h, followed by secondary antibodies for 1 h. primary antibodies were all purchased from Cell Signaling Technology (Danvers, MA, USA), including anti-Bcl-2, anti-Bax, anti-total Caspase-3, anti-Cleaved Caspase-3, anti-GAPDH, anti-vimentin, anti-Cyclin D1, anti-Snail2, anti-p-AKT (Ser473), anti-p-mTOR(Ser2448), and anti-p70S6K (1:1000).
2.4. CCK8 assay
The proliferation of AGS cells was detected by Cell Counting Kit-8 (CCK8). After transfection, cells were transfferd into a 96-well plate (1000 cells/well). CCK-8 reagent was added into cells at a series of time points after the transfection (0 h, 24 h, 48 h and 72 h). Cell viability was represented by OD value at 450 nm.
2.5. Clonogenic assay
After the transfection, about 500 cells were transferred into a 6 cm dish and cultured at 37 ℃ for 2 weeks. Then, the colonies were fixed with methanol for 20 min and stained by 0.1% crystal violet for 30 min. Visible colonies were counted separately by two researchers.
2.6. Transwell
For cell migration, AGS cells were transferred into the up chamber of transwell inserts after transfection (2×105 cells). Serum-free medium was added into the lower chamber of transwell inserts. After incubation for 48 h, AGS cells were fixed with methanol for 10 min and stained with 0.1% crystal violet for 10 min. The migrated cells were counted in 5 random fields. For cell invasion, transwell inserts were pre-coated with Matrigel.
2.7. Flow cytometry analysis
After transfection for 48 h, the apoptosis of AGS cells was detected using Annexin-V and PI (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s protocol. The percentage of apoptotic cells was analyzed on a FACScalibur flow cytometer using CellQuestPro software (Becton Dickinson, USA).
2.8. Statistical analysis
A one-way analysis followed by post hoc Bonferroni test was performed to analyze the differences between groups. All data in the present study were analyzed and plot with GraphPad Prism 8 (GraphPad Software Inc., San Diego, California, USA). All experiments were performed in triplicate. P<0.05 was considered statistically significant.