Prediction of potential targets and lncRNAs for miR-378c
The genes highly expressed in STAD were downloaded from the GEPIA database [19] and intersected with the top 500 survival genes in this database and the target genes predicted by starBase [20] for miR-378c. The overlapped genes were potential target genes for miR-378c in STAD. Highly expressed lncRNAs in STAD were extracted from TCGA database and intersected with lncRNAs predicted to adsorb miR-378c by starBase. The overlapped genes were potential lncRNAs adsorbing miR-378c in STAD.
Cell culture and transfection
Stomach adenocarcinoma cell lines MGC-803 and MKN-28, and Human gastric mucosal cells GES-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and were incubated at 37 °C in DMEM (Gibco, Thermo Fisher Scientific, Inc., Jiangsu, China) with 10% FBS (cat. no. 12483020, Gibco, Thermo Fisher Scientific, Inc., Jiangsu, China).
The pcDNA-NORAD, pcDNA-NRP1, miR-378c-mimics, and miR-378c-inhibitors were obtained from Genomeditech (Shanghai, China), and NORAD or NRP1 shRNA was inserted into the lentiviral vector pLKO.1 (Genechem, Shanghai, China) to knock down NORAD or NRP1. Then, they were transfected into MGC-803 and MKN-28 cells based on the protocols of lipofectamine (11668-019, Invitrogen, USA).
Colony formation
To determine cell proliferation, cells were seeded into 6-well plates at a density of 1000 cells per well and cultured for 2 weeks. Then 4% paraformaldehyde was added for fixation and 0.1% crystal violet was added for dye. Finally, the cloned cells were observed and calculated.
BrdU staining
BrdU incorporation assay was performed for the estimation of MGC-803 and MKN-28 cell proliferation. In brief, MGC-803 and MKN-28 cells were incubated in coverslips, followed by mixing with BrdU (20 μM) for 4 h. Then, PBS containing 0.1% Triton X-100 was added to permeabilize these cells and 3% FBS solution was used for blocking. DNasel treatment was used to denature cellular DNA. The Alexa Fluor 647 anti-BrdU monoclonal antibody (BD Biosciences, USA) was added for incorporated BrdU staining, and a Carl Zeiss fluorescence microscope was used for visualization.
Transwell assay
Transwell chambers (BD Bioscience, US) were used for determination of MGC-803 and MKN-28 cell migration and invasion. In brief, 100 μL serum-free DMEM containing a total of 105 MGC-803 or MKN-28 cells was added into the upper chambers pre-coated with Matrigel (BD Bioscience). Meanwhile, complete medium was added into the lower chambers to induce cell invasion. After 24-h incubation, cells in the lower surface were fixed by paraformaldehyde, and stained by 0.1% crystal violet. The results were observed under a microscope (Olympus) at least 6 fields of view.
Wound healing assay
To monitor the migration of MGC-803 and MKN-28 cells, a 200-µL sterile pipette tip was used to generate the scratch, and the treated cells were incubated on DMEM containing 10% FBS. At 0 and 24 h, the wound area was quantified using ImageJ software version 1.8.0–112 (National Institutes of Health, MA).
Luciferase reporter assay
To quantify the luciferase activity, miR-378c mimics and NC-mimics were co-transfected with NRP1-MUT or NRP1-WT for 24 h, and the LncNORAD-MUT or LncNORAD-WT was transfected into HEK293T cells. The dual-luciferase reporter assay system (Promega Corp, Madison, US) was used to quantify the luciferase activity.
Xenograft and lung metastasis models and HE staining
To construct xenograft and lung metastasis models, we first constructed miR-378c-agomir + NRP1, miR-378c-agomir + LncNORAD, miR-378c-agomir and miR-378c-agomir + Vector MGC-803 cells, and these cells were respectively injected into the flank or tail vein of BALB/c nude mice (female, 6 weeks old). 4 weeks after injection, these nude mice were sacrificed after intravenous injection of barbiturate at a final concentration of 100 mg/kg and the xenografts tumor and lung metastatic nodules were collected. The volume of xenograft tumor was monitored using the formula below: volume (mm3) = (L × W2)/2 (L: length, W: width), and the tumor weight and number of lung metastatic foci were measured. The collected lung metastatic tissues were fixed, dehydrated and embedded using 4% paraformaldehyde, ethanol and wax, respectively. Based on the manufacturer manual, HE staining was performed, and the tissues were observed histologically.
Immunohistochemistry (IHC) assay
In brief, the paraffin embedded xenograft tumor tissues were boiled in citrate buffer for antigen retrieval, and the permeabilization was performed using PBS which contained 0.2% Triton-X. Subsequently, the treated tissues were incubated at 4 °C overnight with antibodies of Ki67 (ab92742, 1:500) and cleaved caspase-3 (ab32042, 1:500), and then cultured with secondary antibody. 3,3′-diaminobenzidine substrate (DAB) staining was performed, and Ki67-positive cells in tissues were observed under a light microscope.
RNA immunoprecipitation (RIP)
EZ-Magna RIP kit (Merck KGaA) was used to determine the relationship of miR-378c with NRP1 and LncNORAD. In brief, after washed with PBS, MGC-803 cells were lysed by RIP lysis buffer, and then the lysates were exposed to the magnetic beads-coated AGO2 antibody or IgG antibody. Then the total RNA was extracted after the RNA complex was eluted from magnetic beads, and the expressions of LncNORAD and NRP1 were detected using qRT-PCR.
Western blotting assay
MGC-803 and MKN-28 cells were lysed by RIPA lysis buffer, and the collected lysates were added with protease inhibitor PMSF. Then BCA method was used for the determination of protein concentrations, and SDS-PAGE electrophoresis was performed to separate protein samples. Next, the isolated samples were transferred onto skim milk powder blocked PVDF membranes for 1 h, and then mixed with these membranes in a hybridization box overnight at 4 °C, followed by incubation with secondary antibody for 1 h. The bands were visualized using ECL luminescent solution. The primary antibodies NRP1 (ab81321,1/2000), NRP2 (ab273584,1/1000), E-cadherin (ab76055,1:200), N-cadherin (ab76011,1:5000), Vimentin (ab92547,1:2000), bcl-2 (ab32124,1:1000), bax (ab32503,1:2000), cleaved caspase-3 (ab32042,1:500), caspase-3 (ab32351, 1:5000), cleaved caspase-9 (ab2324, 1 µg/ml), and caspase-9 (ab32539,1:1000) were used.
qRT-PCR
For RNA quantification, TRIzol reagent (Invitrogen, Carlsbad, California) was utilized for the extraction of total RNA, and the cDNA was obtained after RNA was reversely transcribed using PrimeScript RT Reagent kit (Invitrogen, Carlsbad, California), while mirPremier microRNA Isolation Kit (Sigma, St. Louis, Missouri) was used for miRNA extraction. SYBR Prime Script RT-PCR kit (Takara, Dalian, China) was used for the quantitative real-time PCR based on the ABI 7300 rapid real-time PCR system (Applied Biosystems, Foster City, California). For results calculation, the 2−ΔΔCt formula was adopted, and the primer sequences are displayed in Additional file 1: Table S1.
Statistical analysis
In our work, SPSS version 20. 0 (SPSS Inc., Chicago, Illinois) was utilized for statistical analysis and all data were presented as mean ± SEM. Kaplan–Meier method was employed for the overall survival assessment, and differences between two groups or multiple groups were respectively analyzed by Student’s t-test and analysis of variance (ANOVA). P < 0.05 indicated significant difference.