Clinical samples

Tissue specimens of GC and normal samples were collected from patients in Hospital of Chengdu University of TCM (Chengdu, China). The clinical information of these patients is presented in Table 1. Throughout the experiment, informed consent was obtained from all patients. The Institutional Review Board of the Hospital of Chengdu University of TCM (Chengdu, China) (approval no. 2018KL-023) approved all experimental procedures, which were in compliance with the World Medical Association's Declaration of Helsinki.

Table 1 Clinical characteristics of GC patientsCell culture and transfection

Four GC cell lines (SGC7901, AGS, MGC803, MKN-78) and human gastric epithelium GES-1 cell line were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). AGS cells was cultured in F12K medium (Gibco, NY, USA) along with 10% fetal bovine serum (FBS, Invitrogen, MA, USA). Other cells were grown in RPMI-1640 medium (Invitrogen, MA, USA) with 10% FBS. Lentiviral plasmids overexpressing LINC00853/ PDZK1IP1/FOXP3 were constructed using pcDNA3.1 vector (GenePharma, Shanghai, China) following the producer’s instructions. The primers sequences used for plasmid construction were listed in Table S1. A co-transfection of lentiviral plasmids with packaging plasmids was used to produce lentiviral particles in 293 T cells. The shRNA lentivirus targeting LINC00853/ PDZK1IP1/FOXP3 were all constructed using pLKo.1 vector by Shanghai GenePharma Biotechnology company. An overview of all shRNA sequences had been provided in Table S2. The cells were harvested 48 h after cell transfection and subjected to efficiency determination via RT-PCR experiment.

CCK-8

Using theCCK-8 assay (KeyGEN, Nanjing, China), cell proliferation was measured. Lentivirus infected MGC803 and MKN-78 cells were plated on 96-well plates (1 × 104 cells/well) in triplicate and cultured for 48 h. Following putting in 10 µL CCK-8 reagent, further incubation for 1.5 h was completed. A microplate reader (BioRad 680, BioRad Laboratories, Hercules, CA, USA) was utilized to clarify the optical density under 450 nm.

Transwell assay

Transwell assay was applied using 24-well Transwell chambers (Corning, NY, USA). Following transfection for 48 h, MGC803 and MKN-78 cells (1 × 105 cells) were seeded on the upper chamber and 200 µL RPMI-1640 medium with 10% FBS were supplemented to the lower chamber to act as a chemoattractant. A paraformaldehyde (4%) solution was applied to fix the cells on the bottom chambers and subjected to stain using 1% crystal violet (Sigma, Shanghai, China) for 5 min at room temperature. Finally, each group was observed with three fields using an optical microscope (BX60, Olympus, Tokyo, Japan).

Serum-free spheroid formation assay

Serum-free spheroid formation experiment was carried out in accordance with previous descriptions [17, 18]. Briefly, MGC803 and MKN-78 cells (1000 cells/well) were grown in 6 well ultra-low adherent culture plates (Corning-Co-Star) with serum-free culture medium containing B27 (2%, Sigma), EGF (20 ng/ml, Sigma) and FGF (10 ng/ml, Sigma). After 7 days, a light microscope (BX60, Olympus, Tokyo, Japan) was applied to photograph and count the cell spheres.

Flow cytometry detection of CD44

CD44 positive cell proportions were investigated by flow cytometry using a FACSCalibur (BD Biosciences, California, USA). Briefly, after harvesting the cells with 0.25% trypsin–EDTA, they were washed and resuspended in PBS along with 0.5% FBS. Cell suspension and anti-CD44 antibody (dilution 1:200, cat. no. 550989, BD Biosciences, USA) were mixed and cultured for 1 h at room temperature. Flow cytometry was used to measure cells after collecting and suspending them in PBS.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR)

Following the producer’s protocol, RNA was separated via Trizol reagent (Invitrogen, MA, USA). After reverse transcription through PrimeScript RT Reagent Kit (Takara, Dalian, China) of RNA, complementary DNA (cDNA) was obtained. Next, PCR amplification was performed on 5 μL of cDNA using GAPDH as an internal parameter for LNC00853 and mRNA. The levels of genes were measured via applying the 2 − ΔΔCT method [19]. All primers applied for RT-PCR were designed and constructed via Sangon Biotech (Shanghai, China) and a list of the sequences was provided in Table S3.

Western blot

Protein concentrations were quantified via a BCA protein concentration kit (Beyotime, Shanghai, China) after extracting the total protein with RIPA lysis buffer (Sigma, Shanghai, China). Using a 10% SDS-PAGE gels, we separated 25 μg of proteins and then transferred them to PVDF membranes (Merk, Darmstadt, Germany). Incubation with primary antibodies, including PDZK1IP1 (1:1500, ab156014, Abcam, Shanghai, China), FOXP3 (1:2000, ab4728, Abcam, Shanghai, China) and GAPDH (1:1000, AF7021, Affinity, Shanghai, China), was performed overnight at 4 °C after membranes had been blocked for 1 h using 5% fat-free milk. Following incubation with the horseradish peroxidase-labeled secondary antibody (1:1000, A0208, Beyotime, Shanghai, China) for 4 h at room temperature, the signal was detected via ECL reagents (Pierce, MA, USA) and subjected to image by the FluorChem imaging system (BioRad Laboratories, Hercules, CA, USA).

Fluorescence in situ hybridization (FISH)

FISH test was applied to examine the localization of LINC00853 and FOXP3 in GC cell lines of MGC803 and MKN-78. Briefly, Specific probes to LINC00853 and FOXP3 were prepared and labeled with cy3 (GenePharma, China) and FITC (GenePharma, China), respectively. After mixing the probes with the pre-made hybridization buffer, samples were incubated overnight in this buffer. Finally, DAPI was applied to counterstain cell nuclei. The images were captured via a confocal laser-scanning microscopy (Zeiss, Jena, Germany).

Immunohistochemistry (IHC)

As soon as the xenograft tumor tissues were collected, they were immersed in a paraformaldehyde solution for fixation. Then a paraffin embedding process followed after the tissues were dehydrated, cleared, and cleared. At 4 °C overnight, 4-μm paraffin sections were coated with specific antibody against Ki67 (1:200, AF0189, Affinity, Shanghai, China), followed by 1 h incubation with goat anti-mouse horseradish peroxidase (1:50, A0216, Beyotime, Shanghai, China). Subsequently, the slides were stained with DAB and counterstained with hematoxylin for 30 s, followed by conventional treatments. Finally, under the observation of a fluorescence microscope (BX60, Olympus, Tokyo, Japan), photographs were taken. By using ImageJ software, we calculated the positive staining area.

RNA pull-down assay

LINC00853 and its antisense RNA were synthesized by Sangon (Shanghai, China) and biotin-labeled via the Biotin Labeling Kit (E-LK-B002, Elabscience, Texas, USA). Then a Pierce Magnetic RNA–Protein Pull-Down Kit (20,164, Thermo Scientific, USA) was applied to conduct RNA pull-down experiment. Briefly, biotin-labeled LINC00853, along with its anti-sense RNA, were incubated with cell lysates and streptavidin magnetic beads. Next, the precipitate was centrifuged, eluted via high-salt buffer, and then supernatant was gathered. Finally, the input and pulldown complex were subjected to western blot analysis.

RNA immunoprecipitation (RIP) assay

RIP was applied via a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore, Massachusetts, USA). Cells were lysed with of RIP lysate (P0013B, Beyotime, Shanghai, China) on ice for 30 min, then centrifuged at 14,000 rpm to collect supernatants. Then, the magnetic beads were thoroughly mixed with RIP Wash Buffer and subjected to incubate with rabbit anti-Ago2 antibody (ab186733, 1:50, Abcam, Shanghai, China) for 6 h at 4 °C. Re-suspended magnetic beads-antibody complex were incubated with 100 μL of cell supernatant overnight at 4 °C after washing in 900 μL of RIP Wash Buffer. Immunoprecipitated RNA was detected through RT-PCR analysis. Rabbit anti-IgG antibody (ab172730, 1:100, Abcam, Shanghai, China) was uses as a negative control (NC).

Xenograft tumorigenesis

BALB/c nude mice (6–8 weeks old) were purchased from the Shanghai Laboratory Animal Center of China (Shanghai, China). The animals were randomly allocated into groups and kept on a normal diet with free access to water. A number of three mice were used per group. MGC803 and MKN-78 cells (4 × 105) stably expressing sh-LINC00853 or LINC00853 OE were transplanted subcutaneously into the side of the back of each mouse. We monitored tumor growth weekly, and tumor size was calculated via applying the formula (length × width × width /2) [20]. Following the euthanasia of all the mice after four weeks, the tumors were collected and weighed. Three representative tumors were shown. All the animal experiments were approved by the Institutional Review Board of the Hospital of Chengdu University of TCM (Chengdu, China) (approval no. 2018KL-023).

Statistical analysis

A statistical analysis of the data was carried out using GraphPad prism 7.0 version (GraphPad Software, CA, USA). All data are displayed as mean ± standard deviation (SD). An analysis of statistical significance was carried out through Student's t-test or one-way ANOVA. Correlations were quantified using Pearson correlation coefficients, and survival was evaluated using Kaplan–Meier analysis. Normal distribution was assumed for all data. It was set to 0.05 for statistical significance.