Correlation and enrichment analysis
Profiles of gene expression in GC tissues were downloaded from The Cancer Genome Atlas database. Pearson correlation analysis was performed to screen the 600 genes that were most positively or most negatively associated with PGM1 expression. The 600 candidate genes were selected for enrichment analysis for identification of PGM1 functions. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted based on the clusterProfiler R software package R.
A total of 40 pairs of GC tissues and corresponding adjacent noncancerous tissues were collected from patients who underwent gastrectomy at Chinese PLA General Hospital from March 2020 to October 2020. Pathological examinations were performed by two independent pathologists. This study was approved by the Ethical Committee of Chinese PLA General Hospital. Informed consent was obtained from included patients.
GC cell lines (BGC-823, MKN-28, MGC-803, SGC-7901, HGC-27 and AGS), a human gastric epithelial cell line (GES-1) and a human embryonic kidney cell line (HEK-293T) were purchased from American Type Culture Collection (MD, USA). BGC-823 cells stably carrying luciferase (luc-BGC-823) were previously established and stored in our laboratory. Cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher, Waltham, MA, China) supplemented with 10% fetal bovine serum (FBS, Kangyuan Biotech, Tianjin, China) unless otherwise indicated. Cells were cultivated in an incubator at 5% CO2 atmosphere and 37℃. The low-glucose (LG) medium comprised of glucose-free DMEM (Thermo Fisher) supplemented with 10% FBS and 2.5 mM glucose. The normal-glucose (NG) medium contained glucose-free DMEM, 10% FBS and 10 mM glucose.
shRNA transfection and lentivirus infection
An shRNA against PGM1 was designed and synthesized by JTS Scientific (Wuhan, China). Transfection with PGM1 shRNA was performed using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s protocol. To establish cell lines with stable knockdown of PGM1, a lentiviral packaging kit (Yeasen, Shanghai, China) was used to produce lentivirus media for cell infection. BGC-823 and MKN-28 cells were seeded at the appropriate density and then treated with lentivirus for 24 h. 5 mg/mL puromycin was then added into medium to conduct chemical selection and exclude uninfected cells.
Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted from clinical specimens using TRIzol Reagent (Invitrogen, NY, USA). ExScript RT-PCR kit (TaKaRa, Japan) was used to perform reverse transcription. Then, cDNA was amplified using SYBR Premix Ex Taq II (TaKaRa) and Archimed X4 system (RocGene, Beijing, China). 2-ΔΔCt methods was employed to calculate relative expression of target genes. b-actin served as the internal control gene. The primers for PGM1 were 5’- CGACTCCTTTACGGAACTCA-3’ (forward) and 5’-TCCAGTGGTTTGGCGAAT-3’ (reverse). The primers for b-actin were 5’- TCGTGCGTGACATTAAGGAG-3’ (forward) and 5’- ATGCCAGGGTACATGGTGGT-3’ (reverse).
Western Blot (WB) analysis
For the preparation of WB samples, cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). Cell debris was removed by centrifugation. Protein quantification was conducted using BCA Protein Assay Kit (Thermo Fisher). SDS loading buffer (Solarbio) was added to the protein supernatants, which were then heated at 100°C for 15 min. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were blocked using 5% skim milk at room temperature for 1 h. The primary and secondary antibodies used in this study were purchased from Abcam (Cambridge, UK). The membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies at room temperature for 1 h. The blots were imaged using ECL Western Blotting Substrate (Solarbio) and a WB imaging system (Tanon, Beijing, China).
Cell counting kit-8 (CCK-8) assay
CCK-8 assay (Abmole Bioscience, Shanghai, China) was used to measure GC proliferation in vitro. At the indicated time, the complete medium used for cell cultivation was removed, and 10% CCK-8 solution diluted by the FBS-free medium was added into wells. The plates were incubated at 37℃ for 1 h protected from light. The absorbance of the CCK-8 solution at 450 nm was measured by a microplate reader (Biotek, VT, USA).
5-Ethynyl-2-deoxyuridine (EdU) assay
The EdU assay is another approach to measure the proliferative capability of cells. Cell Proliferation EdU Image Kit was purchased from Abbkine (Wuhan, China). The experiments were performed according to the manufacturer’s protocol. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Abbkine) to determine the total number of cells. Fluorescent images were observed under the fluorescence microscope.
To investigate glycolysis levels in GC, Lactate Colorimetric Assay Kit II, Pyruvate Colorimetric/Fluorometric Assay Kit, ATP Colorimetric/Fluorometric Assay Kit and Glucose Uptake Colorimetric Assay Kit (Biovision, CA, USA) were used according to the manufacturer’s protocols. Measurement of FASN activity was conducted using FASN Activity Kit (Solarbio). Absorbance was measured using the microplate reader as indicated by the corresponding protocols.
Cell apoptosis assay
Cells were harvested and washed with cold phosphate-buffered saline. FITC Annexin V Apoptosis Detection Kit 1 (BD Pharmingen, NJ, USA) was used to stain cells and detect apoptosis induced by orlistat. Apoptotic cells were detected by a FACSort Flow Cytometer (BD Pharmingen). Cells that were Annexin V- and propidium iodide (PI)- positive were regarded as late apoptotic cells. Annexin V-positive but PI-negative was the hallmark of early apoptosis. Living cells were negative for both Annexin V and PI.
In vivo experiments were conducted to further validate the efficacy of PGM1 knockdown and orlistat treatment. Four-week-old male nude mice were purchased from Charles River (Beijing, China) and housed under specific pathogen-free conditions. To generate mouse models with subcutaneous cancer, a total of 5×106 luc-BGC-823 cells were subcutaneously injected into the nude mice. The longest and shortest diameters were determined using a vernier caliper every 5 days. Tumor volume = (longest diameter × shortest diameter2)/2. For evaluation of metastatic capability, 2×106 luc-BGC-823 cells were suspended in phosphate buffer saline (PBS, Solarbio) and injected into tail veins of nude mice. One week after construction of the two models, mice were treated with 240 mg/g of orlistat or the corresponding solvent daily by intraperitoneal injection. After 30 days, the mice were intraperitoneally injected with 1.5 mg D-luciferin (Solarbio) dissolved in PBS and imaged using an in vivo imaging system (PerkinElmer, USA). To achieve euthanasia of the nude mice, they were placed in the chamber without CO2 addition. After 5 minutes of environment adaptation, CO2 was injected at the speed of 5 L/min, which was calculated as 20% volume of the chamber per minute. When cardiac arrest of all mice was observed, they were maintained in the chamber for additional 2 minutes and regarded as dead.
SPSS 25.2 and Prism 7.0 were used to conduct statistical analysis. The data was presented as means±SD. Two-sided Student’s t test and one-way ANOVA were used to compare variables between groups. Survival analysis was performed by Kaplan-Meier method. Chi-square test was used to examine the correlation between PGM1 expression and clinicopathological characteristics. The experiments were performed least in triplicate unless otherwise indicated. P ＜ 0.05 was regarded as the statistical significance.