All study protocols were performed with the approval of the Ethics Committee of The People’s Hospital of Kaizhou District and conducted in strict adherence with the Declaration of Helsinki. All patients and/or legal guardians signed informed consent documentation prior to enrollment into the study. All animal experiments were approved by the Animal Ethics Committee of The People’s Hospital of Kaizhou District. Extensive efforts were made to minimize the suffering and number of animals during the study.
The human immune genes were obtained from ImmPort (https://www.immport.org/home). Following differential analysis of stomach adenocarcinoma (STAD) dataset in The Cancer Genome Atlas (TCGA) using the R package limma (http://www.bioconductor.org/packages/release/bioc/html/limma.html), GC-related differentially expressed immune genes (∣logFoldChange∣ > 1, p < 0.05) were screened followed by single-factor Cox analysis with GC clinical data in the TGCA (http://www.bioconductor.org/packages/release/bioc/html/limma.html) using “survival” package (https://cran.r-project.org/web/packages/survival/index.html) to identify the genes associated with GC prognosis . The hazard ratio (HR) and p value of each differential immune gene with the survival of GC patients were calculated, and the immune genes significantly related to the prognosis of GC patients were screened out based on the criteria of p < 0.05. Survival-related genes in the multivariate Cox regression analysis were inferred using the least absolutes shrinkage and selection operator (LASSO) by the R package glmnet. Risk scores were obtained according to genes expression multiplied by a linear combination of regression coefficient acquired from the multivariate Cox regression. Subsequently, according to the risk scores, the patients were classified into 158 high-risk cases and 159 low-risk cases based on the optimal cut-off point of risk score using the R package survminer. A survival curve was then constructed via survival analysis to determine the difference in terms of survival between high-risk cases and low-risk cases, while its credibility was confirmed by plotting the relative operating characteristic (ROC) curve. Through comparing differential expression of the genes involved in the GC prognostic risk model, the most significantly differentially expressed genes were subsequently selected as the key genes. The downstream pathways of the key genes were then identified based on previous literature. LncBase (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index) was applied in an attempt to confirm he binding of lncRNA to microRNA (miRNA) and the downstream genes of miRNA were predicted by the databases including TargetScan (Total context + + score < − 0.05) (http://www.targetscan.org/vert_71/), RAID (Score > 0.5) (http://www.rna-society.org/raid2/index.html), mirDIP (Number of Sources > 3, Score Class: Medium) (http://ophid.utoronto.ca/mirDIP/) and miRWalk (energy < -20, accessibility < 0.01, au > 0.45) (http://mirwalk.umm.uni-heidelberg.de). After the intersection of the predicted downstream genes with the top 500 differentially expressed genes in TCGA, key downstream genes were obtained through the construction of a protein–protein interaction (PPI) network based on String (https://string-db.org) and calculating core degree of them via Cytoscape (https://cytoscape.org) while downstream pathways were analyzed by the co-expression analysis based on MEM (https://biit.cs.ut.ee/mem/index.cgi).
Tissue sample collection
GC tissues and adjacent normal tissues (over 5 cm away from the tumors) were excised from 64 male patients and 51 female patients, aged between 16 and 76 (mean age: 52.62 ± 13.32, median age: 55). All patients underwent surgery at The People’s Hospital of Kaizhou District from 2015-03-02 to 2017-03-02. After the tissues had been collected, they were immediately stored in − 80 °C liquid nitrogen. All patients had yet to receive any treatment with chemotherapy or radiotherapy prior to surgery, and had their diagnosis confirmed via a pathology test. According to the pathological staging criteria (7th edition) of the Union for International Cancer Control, 18 patients were at stage I, 32 in stage II, 39 at stage III, and 26 at stage IV; 44 were poorly differentiated, 33 were moderately differentiated, and 38 were well-differentiated; 61 cases had lymph node metastasis.
Cell culture, grouping, and transfection
Human GC cell line SGC-7901 (American Type Culture Collection, ATCC, Manassas, VA, USA) was maintained in the Roswell Park Memorial Institute (RPMI)-1640 (South Logan, UT, USA) with 10% fetal bovine serum (Tianhang Biotechnology Co., Ltd., Zhejiang, China) and 100 units/ml penicillin/streptomycin in an incubator with 5% CO2 at 37 °C. The medium was renewed every 24—48 h. The cells exhibiting logarithmic growth were subsequently detached using 0.25% trypsin and transfected with CCL21 (150 μg·L−1 CCL21, ZSGB-Bio, Beijing, China), oe-MALAT1-1, si-MALAT1-1, si-MALAT1-2, or si-SRSF1 (the above plasmids designed, synthesized and constructed by Shanghai GenePharma Co. Ltd., Shanghai, China) in accordance with the instructions of the LipofectamineTM2000 (Invitrogen, Carlsbad, CA, USA).
A phase-contrast microscope (Olympus imt-413, Olympus Optical Co, Ltd., Tokyo, Japan) was used to observe and photograph the morphology of the SGC-7901 cells.
Following 48 h of transfection, the GC cells were confirmed to have reached 90% confluence. Straight lines were drawn on the bottom of the culture plate with a Marker pen and a sterilized 200 µL pipette tip was used to create a wound along the marked line. The cells were added into a serum-free medium after which the wound was measured and recorded under an optical microscope at 0 and 48 h, respectively, to assess the wound healing status.
Next, to identify cell migration, the resuspended cells (3 × 104/mL) with the Opti-MEMI (Invitrogen) containing 10 g/L bovine serum albumin were added to the apical side of a 24-well Transwell chamber (8 μm aperture, Corning, USA), while 600 μL 10% RPMI-1640 medium was added to the basolateral side. After 48 h, the chamber was fixed with 4% paraformaldehyde for 30 min and processed by 0.2% Triton X-100 (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 15 min, followed by the application of gentian violet staining. The migrated cells were counted under an inverted microscope with five randomly selected fields. The cell invasion assay was performed by coating the chamber with 50 μL Matrigel (Sigma) prior to the experiment with the subsequent steps performed in an identical manner as the aforementioned procedures.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from GC tissues using a RNA extraction kit (Invitrogen) and then reversely transcribed into cDNA based on the instructions of the PrimeScript RT kit. MALAT1, SRSF1, CCL21, miR-202-3p, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and U6 primers were designed and synthesized by the Takara (Additional file 1: Table S1). Fluorescence qPCR was performed as per the instructions of the SYBR® Premix Ex Taq™ II kit. GAPDH served as the internal reference for the relative expression of MALAT1, SRSF1, and CCL21 while U6 was regarded as the internal reference for miR-202-3p. Next, in order to identify the expression of mature miRNAs, the TaqMan® microRNA Assay (Applied Biosystems, Foster City, CA) was used based on the manufacturer’s instructions. The quantitative analysis of the change in expression levels was calculated by ABI 7300 real-time PCR machine (Applied Biosystems). The relative transcriptional level of MALAT1 and mRNA were calculated by the 2−△Ct and 2−ΔΔCt methods respectively. RT-qPCR was performed according to the aforementioned protocol for the cell experiment.
Western blot analysis
Total protein was extracted from SGC-7901 cells with lysis buffer (Shanghai Beyotime Biotechnology Co. Ltd., Shanghai, China) containing a protease inhibitor, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was subsequently blocked using 5% skimmed milk powder at 4 °C overnight, and probed with diluted primary antibodies to SRSF1 (1 µg/mL, ab38017, Abcam, Cambridge, UK), β-catenin (1: 5000, ab32572, Abcam), cyclinD1 (1: 500, ab226977, Abcam), E-Cadherin (1: 50, ab1416, Abcam), Vimentin (1: 10000, 60330-1-Ig, Proteintech, Rosemont, IL, USA), Snail (0.1 µg/mL, ab53519, Abcam), Slug (1: 500, ab27568, Abcam), Twist (0.5 µg/mL, ab50581, Abcam), and GAPDH (1: 500, ab8245, Abcam) at room temperature overnight. Additionally, the membrane was re-probed with secondary antibodies of goat anti-rabbit antibody (1: 5000, ab97080, Abcam) and goat anti-mouse antibody (1: 10000, ab97258, Abcam) for 1 h followed by visualization using enhanced chemiluminescence working fluid (Pierce, Rockford, IL, USA). Finally, ImageJ2x was used during the analysis with GAPDH employed as the internal reference.
Dual-luciferase reporter gene assay
The HEK-293 T (AT-1592, ATCC) cells were seeded in 24-well plates and cultured for 24 h. The dual-luciferase reporter gene vector for 3′-untranslated region (UTR) of SRSF1, pmiR-RB-Report-SRSF1-3′UTR, was constructed and co-transfected with oe-MALAT1, si-MALAT1, or negative controls (NCs) into HEK-293 T cells. The cells were then lysed at 48 h after transfection, and the dual-Luciferase® Reporter Assay System (E1910, Promega, Madison, WI, USA) was applied to detect luciferase activity. A total of 50 μL Firefly luciferase working fluid and 50 μL Renilla luciferase working fluid were added to each 10 μL cell sample and the ratio of Firefly luciferase activity to Renilla luciferase activity was regarded as the relative luciferase activity.
Nude mouse xenograft experiment
Specific pathogens-free grade female BALB/C nude mice (age: 4–5 weeks, weight: 19.23 ± 0.92 g) for experimental use were purchased from the Experimental Animal Center of Yangzhou University Medical College and maintained under a controlled constant temperature (20–26 °C) with constant humidity (50–56%). SGC-7901 cells (logarithmic growth) infected with si-MALAT1 or NC lentivirus were selected and detached with mixed liquor containing 0.02% ethylene diamine tetraacetic acid-2Na and 0.25% trypsin followed by 5-min centrifugation for supernatant removal. The cells were resuspended, counted, and adjusted to a density of 5 × 105 /mL. A total of 200 μL cell suspension was subcutaneously injected into the nude mice with the xenografts formed one week later. The major and minor axis of the tumors were measured and recorded every two days. After 6 weeks, the mice were euthanized with the tumors excised and photographed. Half of the tumor tissues were frozen and stored in liquid nitrogen for RNA and protein extraction, while the remaining half was fixed with paraformaldehyde and embedded in paraffin for tissue sectioning. Finally, the tumor volume was calculated followed by plotting of a tumor growth curve.
RNA pull-down assay
The Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific) was employed to perform RNA pull-down assay. Biotinylated MALAT1 or miR-202-3p was incubated with streptavidin beads overnight at 4 °C. Cell lysate was added and incubated at 4 °C for 4 h. After washing and eluting, MALAT1 and miR-202-3p were analyzed by RT-qPCR.
RNA binding protein immunoprecipitation (RIP)
The RIP Kit (Merck Millipore, Billerica, MA, USA) was used to identify the binding of miR-202-3p to MALAT1 and SRSF1. The cells were washed with pre-chilled phosphate buffer saline with the supernatant discarded and lysed with Radio Immunoprecipitation Assay lysis (P0013B, Beyotime) followed by centrifugation at 14,000 rpm for 10 min at 4 °C to collect the supernatant. Half of the supernatant was taken as the input whilst the remaining half was incubated with antibodies for immunoprecipitation. Initially, 50 μL magnetic beads were washed and resuspended in 100 μL RIP Wash Buffer, in which 5 μg antibodies were added. The magnetic bead-antibody complex was subsequently resuspended in 900 μL RIP Wash Buffer followed by the addition of 100 μL cell extract for overnight incubation at 4 °C with the bead-protein complex collected. The RNA of the sample and input was detached using protease K and extracted, after which PCR was performed to detect MALAT1 and miR-202-3p expression. The antibody to Argonaute 2 (AGO2) (ab32381, 1: 1000, Abcam) and antibody to immunoglobulin G (IgG) (1: 100, abl09489) were used for the assay with the latter regarded as the NC.
Tissue blocks fixed in 4% paraformaldehyde were extracted, embedded in paraffin, and sectioned into 4-μm-thick sections. Then the paraffin-embedded sections were subjected to IHC staining using standard procedures. Sections were incubated with primary antibody followed by incubation with HRP-labeled secondary antibody. The diluted concentrations of antibodies used in IHC were as follows: Snail and Slug (1: 5000, ab224731), Vimentin (1: 200, ab92547) antibodies were from Abcam; SRSF1 (1: 500, PA5-30,220) antibody was from Thermo Fisher. The binding extent of the antibodies was visualized using DAB staining. Tissue sections were re-stained with hematoxylin. The cross-sectional images were taken by LeicaMicrosystems (model: DM2000, CMSGmbH, Wetzlar, Germany). Six fields per section were randomly selected for immunohistochemical scoring by ImagePro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining
Tissue sections were dewaxed and rehydrated, and then processed by antigen unmasking using 0.1 M sodium citrate (pH 6.2). Then, each section was incubated with 100 ul DNAse-free proteinase K for 30 min followed by addition of 500 ul diluent and 50 ul of TACS nuclease reaction mix (streptavidin-HRP solution) by capillary action for 30-min incubation. Afterwards, 50 ul of fluorescein-labeled solution and 450 ul of enzyme solution were prepared followed by adding 50 ul of each reagent to the 15 sections for 60-min incubation at 37 °C. Evans blue was used for counterstaining of the sections, which were then a covered with fluorescent mounting medium. Finally, an Olympus BX61 fluorescence microscope was used for examining reactions, and positivity was dichotomously categorized, with the results analyzed by X2 test.
RNAScope Fluorescent Multiplex (Advanced Cell Diagnostics) was employed according to manufacturer’s instructions accompanied by the following changes. Target Retrieval boiling time was altered to 12 min and incubation by Protease IV at a temperature of 40 °C was altered to 8 min. Sections were mounted with Slowfade Mountant + DAPI (Life Technologies, S36964) and sealed.
SPSS version 21.0 (IBM SPSS Statistics, Chicago, IL, USA) was performed for statistical analysis. Measurement data were expressed as mean ± standard deviation, and two groups of data obeying normal distribution and equal variance in paired design were compared by paired t-test, while data in unpaired design were compared by unpaired t-test. Data between multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Data between the groups at different time points were compared using repeated measures ANOVA and Bonferroni’s post hoc test. A value of p < 0.05 was considered to indicate a significant difference.