Patients and tissue specimens
A total of 30 pairs of gastric cancer tissues and matched adjacent normal tissues that were collected by surgical excision from January 2015 to December 2019 and reserved in biological samples library of the Shijiazhuang People’s Hospital were used in this study. Each patient signed the informed consent and all procedures were approved by the Human Ethics Committee of the Shijiazhuang People’s Hospital. All patients were followed up for 5 years to evaluate the overall survival.
Human gastric cancer cell lines (MKN-28, SGC-7901, HGC-27, NCI-N87, AGS, BGC-823 and FU97) and gastric epithelial cell line (GES-1) were stored at the Digestive Disease Laboratory of the Shijiazhuang People’s Hospital. All cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO2.
Two small interfering RNA targeting LINC00342 (si-LINC00342–1 and − 2) and corresponding negative control (si-NC) were purchased from GenePharma Co. Ltd. (Shanghai, PR, China). MiR-545-5p mimics, NC mimics, miR-545-5p inhibitor and NC inhibitor were also purchased from GenePharma Co. Ltd. To overexpress LINC00342 and CNPY2, the full length of LINC00342 and CNPY2 were synthesized by Sangon Biotech (Shanghai, China) and cloned into pcDNA3.1 (GenePharma, Shanghai, China) vector to generate the overexpressing plasmids, which were named LINC00342 and CNPY2, respectively. And the empty vector pcDNA3.1 was considered as the negative control. Cell transfection for AGS cells was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). The sequences for si-LINC00342 were as follows: si-LINC00342–1: 5′-GAAGAUGCUAACUAGAAUAAC-3′, si-LINC00342–2: 5′-GGAUGAAUUGACAGAAGUAGG-3′ and si-NC: 5′-UUCUCCGAACGUGUCACGU-3′. After transfection for 48 h, the transfection efficiency was confirmed by qRT-PCR and cells were collected for the subsequent experiments.
The construction of stably knockdown AGS cells
To obtain the LINC00342 stably knockdown AGS cells, the short hairpin RNA against LINC00342 (sh-LINC00342) and negative control (sh-NC) were synthesized by Sangon Biotech (Shanghai, China) and transfected into AGS cells using Lipofectamine 3000. Cells were cultured in DMEM for 72 h and 2 μg/ml puromycin was added to select the stably transfected clones. The stably transfected AGS cells were used to establish the tumor model in vivo.
Cell proliferation was evaluated using cell counting kit-8 kit (CCK-8, Dojindo, Japan). Briefly, approximately 5 × 103 transfected or un-transfected cells AGS cells were seeded into 96-well plates and cultured for 24, 48, 72 and 96 h. At the indicted time, 10 μl of CCK-8 solution was added into each well and incubated for another 2 h. Then the absorbance at 450 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA).
Colony formation assay
Cell proliferation was evaluated using colony formation assay as previously described . In brief, 1 × 103 transfected or un-transfected AGS cells were seeded into 6-well plates. After culturing for 2 weeks, cells were fixed with 10% formaldehyde and then stained with 0.1% crystal violet. The colonies (≥ 40 cells) were photographed and counted under a microscope.
Wound healing assay
Cell migration was evaluated as previously described . Transfected or un-transfected AGS cells were seeded into 6-well plated and cultured for 24 h. When cell density reached approximately 90%, cell monolayers were scratched using a 100 μl micropipette tip. After culturing for another 48 h, cell migration (at 0 and 48 h) was observed and photographed by using a light microscope (Olympus, Tokyo, Japan).
Cell invasive potential was evaluated as previously reported using a modified 24 wells Boyden chamber (Corning, New York, USA), and the 8.0 μm pore polycarbonate filter inserts were coated with Matrigel (BD) . Briefly, FBS-free DMEM containing 1 × 103 transfected or un-transfected AGS cells were added into the upper chamber. The lower chamber was filled with 20% FBS contained DMEM. After 24 h of incubation, the invaded AGS cells in the lower chamber were fixed in 10% formaldehyde and stained with 0.1% crystal violet. The cells were counted and photographed using a light microscope (Olympus, Tokyo, Japan).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNAs were extracted from tissues or cultured cells using Trizol reagent (Invitrogen, CA, USA). qRT-PCR primers were as follows: LINC00342 F: 5′-CGTTCCAATGTGTTGGGT-3′, R: 5′-TGGGAGGAGGTTGAGATG-3′; miR-545-5p F: 5′-AGCGCGTCAGTAAATGTTTATT-3′, R: 5′-GTTGTTGGTTGGTTGGTTGT-3′; U6 F: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′, R: 5′-GTGCAGGGTCCGAGGT-3′; CNPY2 F: 5′-GACCATGCCCTGCACATATC-3′, R: 5′-TAAAAGGCATTGCCACCATT-3′; GAPDH F: 5′-GCACCGTCAAGGCTGAGAAC-3′, R: 5′-TGGTGAAGACGCCAGTGGA-3′. The relative fold changes of genes were calculated using the 2−ΔΔCt method. U6 and GAPDH were used as the internal control for miR-545-5p and LINC00342/CNPY2, respectively.
Total proteins of tissues or cultured cells were extracted using RIPA lysis buffer. Equal amount of protein samples were separated by 10% SDS-PAGE and transferred into PVDF membranes. Then primary antibody against CNPY2 (Abcam, ab233136, 1:500) and internal reference GAPDH (Abcam, ab9485, 1:1000) were added for incubation with membranes and incubated at 4 °C overnight. Then the membranes were exposed to horseradish peroxidase (HRP)-conjugated IgG for 2 h. The protein bands were visualized using the enhanced chemiluminescence detection kit (ECL, thermo scientific, USA).
Luciferase reporter assay
The putative binding sites between LINC00342 and miR-545-5p, as well as miR-545-5p and CNPY2 were predicted by StarBase (http://starbase.sysu.edu.cn/index.php). The wild type (WT) and mutant type (MT) 3′-UTR of LINC00342 and CNPY2 containing the putative binding site with miR-545-5p were synthesized by Sangon Biotech (Shanghai, China) and cloned into pmirGLO luciferase vector to generate LINC00342 3′-UTR WT/MT and CNPY2 3′-UTR WT/MT. Then the luciferase reporter vector was co-transfected with miR-545-5p mimics or NC mimics into AGS cells by using Lipofectamine 3000. After 48 h for transfection, the relative luciferase reporter activity was measured using a dual luciferase reporter system.
Transfected or un-transfected AGS cells were permeabilized in 0.1% Triton X-100 and incubated with anti-CNPY2 antibody (Abcam, ab233136, 1:500) at 4 °C overnight. After washing with PBS, cells were incubated with FITC-conjugated goat anti-rabbit secondary antibody (Abcam, ab6717, 1:1000) for 2 h. Then cell nuclei were stained using DAPI solution (Sangon Biotech, Shanghai, China). Images were captured using a fluorescence microscope.
In situ hybridization (ISH)
Gastric cancer tissues and adjacent normal tissues were fixed in 4% formaldehyde, dehydrated in gradient ethanol, embedded in paraffin, and cut into 5 μm sections. The Digoxin labeled LINC00342 probe used for ISH was synthesized by Sangon Biotech (Shanghai, China). The slides were processed using the Enhanced Sensitive ISH Detection Kit (POD) (Boster Biotechnology, California, USA), and counterstained in haematoxylin for 90 s. After drying, the slides were photographed using a fluorescence microscope.
In vivo tumorigenesis
BALB/c nude mice (male, 6-week-old) were used to establish the in vivo model as previously described . In brief, 5 × 106 AGS cells stably transfected with sh-LINC00342 or sh-NC were subcutaneously inoculated into BALB/c nude mice (n = 3 per group). The tumor volume was measured using the formula: length × width2/2 every 1 week for a total of 5 weeks. Tumors from the two groups were collected and photographed using a digital camera. All animal experiments were approved by the Animal Ethics Committee of the Shijiazhuang People’s Hospital in accordance with the Use Committee for Animal Care.
Immunohistochemistry (IHC) analysis
The tumors of mice from two groups were cut into 3-μm slices. The antibody against Ki-67 (Abcam, ab15580, 1:500) were used for IHC as previously described . Subsequently, a DAB Substrate Kit (Invitrogen, California, USA) was used to stain the tumor tissues to detect the expression of Ki-67.
All data were presented as means ± standard deviation (SD). Statistical analyses were performed using SPSS 21.0. The diagnostic value of LINC00342 was evaluated by analyzing the ROC curve. The overall survival of GC patients was evaluated using Kaplan-Meier curve followed by log-rank test. Spearman Pearson correlation analysis was used to assess the relationship between the expression levels of LINC00342 and miR-545-5p. Difference between two groups was explored by Student’s t-test. Differences among multiple groups were explored by one-way ANOVA. Correlation between LINC00342 and CNPY2 was analyzed by Linear regression analysis. P < 0.05 was considered as the significant threshold.