Study patients
By analyzing the sequencing results from the GEO gastric cancer public database (GSE96856), the metabolism-related long-chain noncoding RNA (Linc-00475) was screened to predict its downstream gene (p53). A total of 107 tissue specimens were selected for radical gastric cancer surgery in the Department of Gastrointestinal Nutrition and Hernia Surgery, The Second Hospital of Jilin University between June 2014 and June 2015. Among the 107 patients, 52 were aged <60 years and 55 were aged ≥60 years. There were 27 males and 80 females. The cohort included 42 cases with a tumor diameter of <5 cm and 65 cases with a tumor diameter of ≥5 cm. The degree of differentiation was high/moderate in 30 cases and low in 77 cases. Tumors were located in the upper region in 16 cases and middle and lower in 91 cases. A total of 83 cases were of Borrmann types Ⅰ, Ⅱ, and Ⅲ, and 24 cases were of Borrmann type Ⅳ. The TNM stage was identified based on the Eighth Edition of AJCC, where 22 cases were in stage T1–2 and 85 cases were in stage T3–4. There were 32 cases of N0 and 75 cases of N1–3. All patients were single-focus. The patients did not receive radiotherapy and chemotherapy before the operation, and their postoperative follow-up data were complete. The study protocol was approved by the hospital ethics committee, and all patients signed the informed consent form.
Immunohistochemical standing and in situ hybridization (ISH)
Paraffin specimens were kept in the pathology department of our hospital. Tissue specimens were cut into 4 μm-thick sections via continuous sectioning. After dewaxing and hydration, tissue sections were soaked in a 3% H2O2 solution at room temperature for 10 min and then repaired in a sodium citrate antigen repair solution (pH 6.0) at 121℃ for 2 min. The slices were then incubated with a primary antibody for p53 (1:1000, Abcam, Cambridge, MA, USA) at 4℃ overnight, followed by incubation with a secondary antibody at 37℃ for 30 min, DAB staining, hematoxylin re-staining for 1 min, and 1% hydrochloric acid alcohol differentiation. After washing with tap water and blue returning for 10 min, the samples were dehydrated in gradient solutions of ethanol, treated with xylene, and air dried to create transparent tissue sections. They were then sealed and prepared for microscopic observation.
ISH, dewaxing, and hydration methods were the same as described above. The sections were treated with protease, diluted with citric acid for 30 min, and fixed with 1% paraformaldehyde for 10 min at room temperature. A total of 20 µL of pre-hybridization solution were added to each slice for 2–4 h of hybridization, followed by 20 µL of Linc-00475 probe hybridization solution, and kept constant at 37°C overnight. Blocking solution was added at room temperature for 1 h on the next day. Then, biotinylated mouse anti-human digoxin antibody was added dropwise and incubated at room temperature for 60 min, followed by dropwise addition of the streptavidin-biotin complex and incubation at room temperature for 30 min, and finally biotinylated peroxidase and incubation at room temperature for 30 min. The samples were then stained with DAB, counterstained with hematoxylin, dehydrated in gradient ethanol, treated with xylene, air dried, sealed, and prepared for microscopic observation.
Immunoreactivity scoring
Staining intensity grading: 0 points for colorlessness, 1 point for yellow, 2 points for brown and 3 points for dark brown; ratio of positive cells: <5% for 0 points, 5%-25% for 1 point, 25%-50% for 2 points, 50%-75% for 3 points, and >75% is 4 points. The staining intensity score is multiplied by the score of the proportion of positive cells as the final score; ≥6 is considered as positive expression, and ≤4 is considered as negative expression [14].
Cell culture and virus transfection
GSE-1, MKN-28, BGC-823, AGS, MKN-45 were cultured in DMEM and F-12 medium (Gibco, Thermo Fisher, China) containing 10% fetal bovine serum serum (Gibco, Thermo Fisher, China). After digestion, centrifugation, and resuspension, the cells are counted. Add 1×105 cells to each well, and wait until the cells are fused 50%-70% for use the next day; add the calculated virus to the 12-well plate, Change the medium after 14 hours. After the cells are fused to about 80%, they will be transferred to the culture dish and continue to be cultured. Puromycin is added for selection until stable. The transfection efficiency is verified by Western blot and qRT-PCR, and subsequent experiments are performed.
qRT-PCR
The qRT-PCR analysis of Linc-00475 expression in 38 fresh gastric cancer pathological tissue samples and cell lines obtained between May and August 2020 at our center was performed as described previously [14]. The primer sequences for qRT-PCR are as follows. GAPDH mRNA was used as the internal reference gene. Data assessment was determined by the −ΔΔCt method.
Linc-00475 forward: 5′-GAGGGTTCACAAACGAGCCT-3′
Linc-00475 reverse: 5′-CCCAGGCTCCTAACCATGTC -3′
Extracellular acidification rate (ECAR)
Cells were divided into blank and experimental groups and inoculated into 96-micro-well plates for ECAR testing. A total of 200 μL of hydration solution were added to each well in addition to glutamine detection and stock solutions from the glycolysis kit prior to testing.
Oxygen consumption rate (OCR)
OCR testing methods were the same as those for ECAR. The glycolysis pressure kit (Abcam, Britain)was configured using stock compounds, which were successively added into the hole and tested on a computer.
Lactic acid, glucose, and ATP level detection
Cells in the blank and experimental groups were inoculated into six-well plates at a density of 2 × 105 cells per well. After reaching 90% confluence, the cells were collected and centrifuged at room temperature and 500 rpm for 5 min. Culture medium supernatant was then collected into an Eppendorf tube and analyzed according to the instructions for the glucose, lactic acid, and ATP testing kit (BioVision, USA) to determine the optical density (OD) value of each group using a microplate reader. Glucose consumption and lactic acid level of the supernatant in each group were calculated according to the formula provided in the manufacturer instructions, such that the higher the OD value, the higher the ATP content.
Statistical analysis
SPSS 22.0 and GraphPad Prism 7.0 software were used for statistical analysis. The measurement data were compared between the two groups via a non-paired t-test. Chi-square test was used to analyze the relationship between Linc-00475 and p53 expression and clinicopathological factors. Pearson test was used to analyze the correlation between Linc-00475 and p53 expression. Kaplan-Meier method was used for survival analysis, while log rank test was utilized to calculate survival differences between different groups. Univariate and multivariate Cox risk ratio models were fitted to screen out independent prognostic factors. P < 0.05 indicated statistically significant differences.