Patients and tissue samples
A total of 20 cases of specimens, including paired cervical cancer tissues and para-carcinoma normal tissues, were collected from cervical cancer patients who underwent surgery at the Qilu Hospital of Shandong University from 2020 to 2022. The patients were diagnosed with cervical cancer on a pathological basis. Inclusion criteria were newly-treated patients with FIGO IB-IIA stage (regardless of histological type), the patient's age was greater than or equal to 18 years and the original treatment plan included radical resection of cervical cancer. Exclusion criteria is including history of pelvic lymph node resection or radiotherapy, abnormal liver and kidney function and other obvious contraindications to surgery. All experiments in this study were approved by the Ethics Committee of the Qilu Hospital of Shandong University. The patients and their families were informed of specimen collection, and the informed consent forms were signed.
Immunohistochemistry
Tissue sections were fixed with paraformaldehyde, and cut into 4.0mm-thick slices. Put the slices in the oven at 65°C for 65minutes. The slices were deparaffinized in xylene for 15 minutes twice, fixed with 100% ethanol for 10 minutes twice, dehydrated with 95%, 90%, 85%, 80% and 70% ethanol, 10 minutes each solution, and repaired in sodium citrate antigen retrieval solution heated to 96 °C for 15 minutes. Then endogenous peroxide activity was purged by 3% hydrogen peroxide in methanol for 15min at room temperature. The sections were washed thrice with phosphate-buffered saline (PBS) and blocked by the fetal bovine serum for 45 minutes and next, incubated with the anti-METTL14 antibody(Abcam, UK, 1:300), anti-PKM2 antibody(Abcam, UK, 1:500), anti-HK2 antibody(Abcam, UK, 1:500) and the anti-AMPK antibody (Abcam, UK, 1:200) overnight (about 19h) at 4°C. Then incubated with the secondary antibody for 60 minutes at room temperature (about 28°C). The tissue slices were washed with PBS and incubated with diaminobenzidine (DAB) for an appropriate time. Finally, the slices were stained with hematoxylin for several seconds, transparent and dehydrated by gradient alcohol solution and xylene solution, and finalized with the neutral balsam. On the third day, captured images under the microscope. Repeated every experiment 3 times at least.
MeRIP-seq assays
For MeRIP-seq, total RNA was isolated using TRIzol reagent. The obtained mRNA was further purified using the Dynabeads mRNA DIRECT Kit (Thermo Fisher) and fragmented by sonication. MeRIP-seq and library preparation were performed as per the reported protocol with some modifications. In brief, sonicated mRNA was mixed with m6A antibody (Synaptic Systems, 202003) in IP buffer and incubated under head-to-tail mixing at 4°C for 2 h. The mixture was supplemented with protein A magnetic beads (Thermo Fisher) and incubated under head-to-tail mixing at 4°C for another 2h. The beads were then washed with IP buffer three times before elution with m6A elution buffer twice. The eluates were combined and purified by an RNA Clean and Concentrator (Zymo, Orange, CA). The purified mRNA fragments were used to construct libraries with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was carried out on the Illumina HiSeq 2000 system with a pair-end 150-bp read length. Reads were aligned to human genome version 38 (GRCh38) with TopHat. The longest isoform was retained if a gene had more than one isoform. Differential m6A-modified peaks between IP and input samples were identified using exomePeak (p < 0.01).
Immunofluorescence Staining
For PD-1, CD206 and γ-H2AX staining, histological section and cells were blocked with 10% fetal bovine serum (GIBCO, US) and were stained with anti-PD-1 antibody (Abcam, UK, 1:250), anti-CD206 antibody (Abcam, UK, 1:200 and the γ-H2AX antibody (Abcam, UK, 1:300). Dylight 488 Goat Anti-Rabbit IgG (Abbkine, China) and Dylight 594 Goat Anti-Mouse IgG were used as secondary antibodies. A fluorescence microscope is used to observe experimental results
Total protein extraction and western blot
The collected tissues and cells were melted into liquid , added 500μl RIPA buffer with 1%PMSF, then pipetted the mixture into a 1.5ml ep-tube leave 30 minutes on the ice before centrifuged 12000rpm at 4°C. Then tested the concentration of proteins (40μg per sample) with the BCA protein assay kit, and warmed the proteins supernatant with SDS-loading buffer by bainmarie at 100°C for 10 minutes. Each protein sample was separated by 10% SDS-PAGE gel electrophoresis for about 2 hours and then transferred the proteins on the gel electrophoresis into PVDF (0.22μm or 0.45μm) membranes for 60-90 minutes, blocked with 5% skimmed milk powders which dissolved in TBST for 90 minutes at room temperature (about 28°C), after that, put the PVDF membranes into diluted primary antibodies (diluted by Western Primary Antibody Dilution Buffer, Beyotime, China) METTL14(Abcam, UK, 1:1000), AMPK (Abcam, UK, 1:1000), HK2(Abcam, UK, 1:1000), PKM2(Abcam, UK, 1:1000) and GAPDH (GoodHere, China, 1:5000) at 4°C overnight, incubated with diluted secondary antibodies (diluted by TBST) HRP-labelled Goat Anti-Mouse IgG(H+L) (Beyotime, China, A0216, 1:1000), HRP-labelled Goat Anti-Rabbit IgG(H+L) (Beyotime, China, A0208,1:2000) for 90 minutes and detected the expression by HRP chemiluminescence detection kit (MILLIPORE ImmobilonTM Western Chemiluminescent HRP Substrate, USA, WBKLS0100). Repeated every experiment 3 times at least.
RNA extraction and quantitative RT-PCR (real-time PCR)
Using Trizol (LIFE ambion Trizol, USA) regent to extract the total RNA of tissues and cells and test the concentration of each RNA sample. Reversed the RNA into cDNA by the reverse transcription kit. Then amplified aimed gene fragment and detected it with SYBR Green qPCR kit (TOYOBO, Japan), quantitative real-time PCR was performed for 40 cycles. All experiments were performed 3 times at least for each sample. Relative gene expression levels were analyzed by 2−∆∆CT method.
Cell culture and Caski cells cocultured with macrophages
Caski cells (cervical cancer cells) and THP-1 cells (acute monocytic leukemia) were cultured in 1640 Medium (GIBICO, US) supplemented with 10% fetal bovine serum (GIBCO, US) and 1% Penicillin-Streptomycin(100×) (Solarbio, US) at 37°C in a humidified atmosphere containing 5% CO2. THP-1 was cultivated in 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, Australia) and 1% antibiotics at 37°C in a humidified 5% CO2 incubator. The glycolysis inhibitor 2-deoxy-D-glucose (2-Deoxyglucose, 2-DG) (8mM, 12h, MedChemExpress, China) was used to act on Caski cervical cancer cells to build a glycolysis inhibition model. Dorsomorphin (Compound C) is a selective and ATP-competitive AMPK inhibitor. Dorsomorphin (compound C) (10 uM, 18h, MedChemExpress, China) reduced AMPK phosphorylation levels in Caski cells. THP-1-derived macrophages were obtained after treated with phorbol ester (PMA, Sigma, USA) (50ng/ml) for 48h. Then cells were stimulated with 10ng/ml of IL-4 (R&D, USA) and 10ng/ml of IL-13 (R&D, USA) to M2 polarization. Caski cells are cocultured with THP-1-derived M2 macrophages using a standard Transwell insert (0.4 um; Corning, USA). Each well of plates was plated with approximately10,000 Caski cells. After incubation for 24 h with a 10% FBS medium, the cells were washed, and the inserts with induced macrophages (7.5×105 cells) were added to the wells.
Metabolomics
The cells of the shMETTL14 group(n=10) and the NC group(n=10) were mixed with 80% acetonitrile, ground and centrifuged, and the supernatant was taken out for further experiment. 100ul 3-NPH (200 mM, containing internal standard 40 ng/mL malic acid-d3) and 100ul EDC (120 mM; containing 6% pyridine) were added to the supernatant, vortexed for 1 min and 40 ℃ for 1 hour. Next, centrifuge for 15 min, take the supernatant,pass it through a 0.22 um filter membrane, dilute 3 times with 80% acetonitrile water (including 10 ng/mL of the internal standard after derivatization), and inject it into the machine for LC-MS/MS analyze.
Short Hairpin RNAs (shRNA), Small Interfering RNA (siRNA) and Genetic Knockout
Plasmids expressing short hairpin RNA targeting METTL14 or scramble sequences were purchased from Genechem. ShRNA sequences were packed into a lentivirus packaging construct and transfected into Cakli cells with lipofectamine 2000 (Invitrogen). Caski cells were infected with shRNA expressing lentiviruses and selected with 10 mg/ml puromycin. siRNA targeting GPR81 were transfected into THP-1 with lipofectamine 2000.
Cell migration assay
Cell migration was detected using a standard Transwell insert (0.8um; Corning, USA). Caski cells were applied in the upper compartment with 500ul of serum-free medium, and the lower compartment was filled with 500μl of MEM. After 24–48 h of incubation at 37 °C, noninvaded cells on the upper surface of the filter were removed carefully with a cotton swab, and cells were fixed with 100% methanol for 2 min. Invaded cells on the lower side of the filter were stained with 0.5% crystal violet for 20 min, and images were captured using a microscope.
CCK-8 assay
Cell proliferation was evaluated using the CCK-8 (Cell Counting Kit-8) assay kit (Beyotimey, CHINA). Briefly, 100l of the Cell Counting Kit solution was added to the culture medium and incubated for an additional 3 h. The absorbance was determined at 450 nm wavelength with a reference wavelength of 630 nm.
Lactic acid production, ECAR and OCR
Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed using the Seahorse XF96 instruments (Seahorse Bioscience, USA). For the OCR and ECAR test, the cell medium was replaced by an assay medium (Seahorse Bioscience) supplemented with 1 mM pyruvate, 10 mM glucose, and 2 mM glutamine for 1.5h at 37 °C, then measured by the XF Cell Mito Stress Kit (Seahorse Bioscience). The concentrations of ROT/AA and 2-DG were 1.0 uM and 0.5 uM respectively, then measured by the Glycolytic Stress Test Kit (Seahorse Bioscience). The OCR and ECAR results were adjusted to the Seahorse XF96 Wave software. The lactate concentration in cultured media was measured using LA Assay Kit (Solarbio, China), following the manufacturer’s instructions.
Flow cytometry
THP-1-derived macrophages were obtained and evaluated by flow cytometry. To avoid the adherence of macrophages to the tube wall, macrophages were incubated in 2% paraformaldehyde for 30 min on ice for antibody staining. THP-1-derived macrophages were stained with the following fluorochrome-labeled antibody for 30min at 4℃: anti-CD206(B&D, USA) and anti-PD-1(B&D, USA). After the surface staining, these antibodies were detected using Guava easyCyte 6HT-2L (Millipore, USA), and the data were analyzed using Guava Soft 3.1.1 software (Millipore, USA). All staining was performed according to the manufacturer’s protocols. Isotype controls were used to confirm antibody specificity. Single color stain controls were used to enable correct compensation.
In vivo studies
Animal studies were performed according to institutional guidelines. Caski cells were stably transfected with NC or shMETTL14 vectors. A total of 5 × 106 viable cells were injected into the right flanks of nude mice. Then after 12 days, they were sacrificed, the tumors were dissected, and tumor weights were measured. Tumor sizes were measured using a Vernier caliper, and the tumor volume was calculated using the following formula: volume = 1/2 × length × width2.
Statistical analysis
The statistical software Prism 7 (GraphPad) was used for data analyses. Statistical significance was determined by Student’s t-test. Multiple means were compared by one-way analysis of variance (ANOVA). Error bars in the figures indicate the SEM. Statistical significance was set at P < 0.05. Statistically significant results are expressed using asterisks, where *P < 0.05, **P < 0.01, and ***P < 0.001.