Cell lines and cultivation
GC cell lines (AGS, NUGC4, and MKN74) and human normal epithelial cell line (GES-1) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in the RPMI1640 medium (CD-02168-ML, GIBCO, USA) added with 10% Fetal Bovine Serum (FBS; 10270-106, GIBCO) and 100 U/mL Penicillin/Streptomycin solution in humidified incubators. The air and temperature for the cell cultivation was required of 5% CO2 and 37 °C respectively.
Vector construction and cell transfection
For the overexpression of TFAP2A-AS1, NISCH, SOX12, FOXq1, ZNF740, KLF15, ZNF281 and IRF3, the full length of these of these RNAs were inserted into pcDNA3.1 vectors to construct pcDNA3.1-TFAP2A-AS1, pcDNA3.1-NISCH, pcDNA3.1-SOX12, pcDNA3.1-FOXq1, pcDNA3.1-ZNF740, pcDNA3.1-KLF15, pcDNA3.1-ZNF281, and pcDNA3.1-IRF3 plasmids. For the knockdown of TFAP2A-AS1, sh-TFAP2A-AS1-1/2/3 plasmids were chemically synthesized by RiboBio (China). For the overexpression or silence of miR-3657, miR-3657 mimics or inhibitor were synthesized with the sequence or complementary bases of miR-3657. For the luciferase report assay, the wild-type or mutant TFAP2A-AS1 or NISCH-3′UTR was sub-clone into pmirGLO to form pmirGLO + TFAP2A-AS1/pmirGLO + TFAP2A-AS1-MUT or pmirGLO + NISCH-3′UTR (3′ untranslated region)/pmirGLO + NISCH-3′UTR-MUT, TFAP2A-AS1 promoter or TFAP2A-AS1 promoter-MUT being sub-cloned into pGL3 vectors to construct pGL3-TFAP2A-AS1 promoter or pGL3-TFAP2A-AS1 promoter-MUT. All the transfections were conducted with the application of Lipofectamine 2000 (XFSJ16444, GIBCO).
After being extracted from GC cells via Trizol (abs60154, Absin, Shanghai, China), total RNAs were reversely transcribed into complementary DNAs (cDNAs) using 1st Strand cDNA Synthesis Kit (11141ES10, Takara, Japan). Then, qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, USA) was used for PCR and evaluation of the relative expression levels of different RNAs: TFAP2A-AS1, RRAGD, CDK16, ZNRF1, NISCH, SOX12, FOXq1, ZNF740, KLF15, ZNF281, IRF3, hsa-miR-876-3p, hsa-miR-4516, hsa-miR-9-5p, hsa-miR-5703, hsa-miR-3131, hsa-miR-4687-3p, hsa-miR-6762-5p, hsa-miR-4434, hsa-miR-3142, hsa-miR-3657 and hsa-miR-1245b-5p. The results were calculated based on 2−ΔΔCt method. β-actin and GAPDH served as the internal reference for this analysis.
For the extraction of total proteins from GC cell, ProteoPrep® Total Extraction Sample Kit (PROTTOT-1KT, Sigma-Aldrich, USA) was implemented in this assay. Then, proteins were subjected to electrophoresis in SDS-PAGE Kit (P1200, Solarbio, China) before being transferred to PVDF membranes. Next, membranes were blocked with 5% skim milk at room temperature for 1 h, followed by the incubation with Anti-GAPDH (ab8245, Abcam, UK), Anti-NISCH (ab240588, Abcam, UK) or Anti-β-actin (ab8226, Abcam) at 4 °C for the whole night. After the subsequent cultivation of membranes with secondary antibodies, the protein levels were analyzed with the application of ECL substrates. β-actin and GAPDH served as the internal reference for this analysis. The results were quantified using ImageJ.
Dual-luciferase reporter assay
For the luciferase reporter assay assessing the interaction among RNAs, miR-3657 NC and miR-3657 mimics were co-transfected into 1 × 104 GC cells with different luciferase vectors (pmirGLO/pmirGLO + TFAP2A-AS1/pmirGLO + TFAP2A-AS1-MUT or pmirGLO + NISCH-3′UTR/pmirGLO + NISCH-3′UTR-MUT). For the verification of transcription of TFAP2A-AS1, pcDNA3.1 or pcDNA3.1-KLF15/pcDNA3.1-SOX12/pcDNA3.1-FOXq1/pcDNA3.1-ZNF740/pcDNA3.1-ZNF281/pcDNA3.1-IRF3 was co-transfected with pGL3, pGL3-TFAP2A-AS1 promoter or pGL3-TFAP2A-AS1 promoter-MUT into GC cells. The luciferase activity was observed through the XSP-63B fluorescence microscopy (Shanghai Optical Instrument Factory, Shanghai, China).
RNA pulldown assay
Structure buffer was added to biotinylated RNAs to form secondary structure. Afterwards, biotinylated RNAs were subjected to the heating and ice-bathing for denaturation. Denatured biotinylated (Bio-) TFAP2A-AS1, TFAP2A-AS1-MUT, NISCH-3′UTR or Bio-NISCH-3′UTR-MUT (1 µg) was incubated with 15µL Streptavidin beads for 2 h at 4 °C. Then, 2 × 107 GC cells were lysed and the cell lysate was incubated with Bio-NC, Bio-TFAP2A-AS1, Bio-TFAP2A-AS1-MUT, Bio-NISCH-3′UTR, or Bio-NISCH-3′UTR-MUT conjugated with the beads at 4 °C overnight. After the precipitation was fulfilled, RNAs were extracted from precipitates for qPCR analysis.
RNA Binding Protein Immunoprecipitation (RIP) assay
Imprint® RNA Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich, USA) was applied in this assay under the instruction of the manufacturer. First, Anti-IgG or Anti-Ago2 was cultured with 50 µg Protein A/G Agarose beads overnight at 4 °C. Meanwhile, 6 × 107 GC cells were lysed in RIPA lysis buffer (RIPA20110527, TBD, China) into 300 µL lysate. The cell lysate was separated into three groups: Anti-IgG (100 µL lysate), Anti-Ago2 (100 µL) and Input (10 µL). After the incubation of antibodies overnight at 4 °C, the precipitates containing Protein A/G Agarose beads, Anti-IgG/Ago2 and RNAs were precipitated. QPCR analysis was implemented for the analysis of RNA enrichment. Anti-Ago2 (ab186733) was bought from Abcam.
Chromatin immunoprecipitation (ChIP) assay
For this assay, 2 × 107 GC cells were fixed with formaldehyde and subjected to the fractionation of nuclei and cytoplasm. Then, sonication was applied for the fragmentation of chromatins. Subsequently, Anti-IgG and Anti-KLF15 were incubated with chromatins for the immunoprecipitation. When the precipitation was finished, DNAs were extracted from the antibody-DNA complexes for qPCR.
The FISH Kit (Bes1001, Biosense, China) was applied for FISH assays according the manufacturer’s protocol. 5 × 104 GC cells were fixed with PFA and permeabilized with Triton X-100. Then, TFAP2A-AS1 probes labeled with Digoxigenin (DIG) were incubated with GC cells. DAPI was used for the counterstaining of nuclei. Finally, LSM800 confocal laser scanning microscopy (Carl Zeiss, Germany) was applied for the observation and recording of images.
Cell Counting Kit-8 (CCK-8) assay
GC cells were plated into 96-well plate, with 1 × 103 cells to each well, for cultivation with 5% CO2 at 37 ℃. After 24, 48 or 72 h, CCK-8 solution was added to the cells. The cell samples were incubated with CCK-8 solution for another 4 h. OD value at 450 nm was detected via a microplate reader to manifest the proliferation of GC cells.
5-ethynyl-2′-deoxyuridine (EdU) assay
After the transfection, 1 × 105 GC cells were plated into 96-well plate for the cultivation overnight. Then, paraformaldehyde was used to fix cells and Triton X-100 to permeabilize cells. After that, EdU reagent was added to cells for staining. Nuclei were counterstained with DAPI. The cell viability was determined by a fluorescence microscope.
For the transwell-migration assay, 5 × 104 transfected GC cells were re-suspended in the upper chambers without serum. Meanwhile, the lower chambers were filled with DMEM added with FBS. After the incubation for 24 h, cells maintained in the upper chambers were slightly swiped with a cotton swab and cells migrating to the lower chambers were fixed with methanol and stained with 0.1% crystal violet. Then, cells in the lower chambers were counted under the microscopy.
Wound healing assay
5 × 105 transfected GC cells were seeded into the 6-well plated for cultivation and a straight wound was scratched with a sterile pipette tip. After 24 h, the closure of wounds were recorded and measured under the microscopy at the magnification of 10 × 10. The wound width at the 24th h was normalized to that at 0 h. The results were quantified using ImageJ.
All assays in this present study are required of triplicate and data were shown as mean ± standard deviation (SD) in each group. Statistical analysis was achieved with the application of SPSS 22.0 (IBM, Armonk, USA). For the statistical test, Student’s t test was used for the comparison between two groups and one-way or two-way analysis of variance (ANOVA) for that among more than two groups. The post hoc was achieved via Dunnett or Tukey. Data were significant only when the P value was less than 0.05.