Glycolysis induced by METTL14-related m6A methylation is essential for macrophage function and tumor progression in cervical cancer

Glycolysis induced by METTL14-related m6A methylation is essential for macrophage function and tumor progression in cervical cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Glycolysis induced by METTL14-related m6A methylation is essential for macrophage function and tumor progression in cervical cancer Bingyu Wang, Xinlin Jiao, Teng Zhang, Qingqing Liu, Jinwen Ye, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1949592/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cervical cancer is one of the most common tumors of women’s diseases. N6-methyladenosine (m6A) is an abundant RNA modification of mammalian mRNAs and plays a vital role in many diseases, especially tumors. In this study, we aimed to investigate the roles of m6A on macrophage function and tumor progression in cervical cancer. We measured the levels of m6A expression in cervical cancer tissues, and revealed biological functions of METTL14 (Methyltransferase14, N6-Adenosine-Methyltransferase Subunit) on tumor-associated macrophages (TAMs) and PD-1 expression. We found the METTL14-related methylation of m6A in cervical cancer was correlated with infiltration of TAMs, polarization status of infiltrated TAMs, and survival outcomes of cervical cancer patients. Mechanistically, lactate produced by tumor glycolysis in the acidic immunosuppressive tumor microenvironment (TME) has an important role as a proinflammatory and immunosuppressive mediator in this program. METTL14-related methylation of m6A may play a predictive role in the polarization and PD-1 expression of macrophages in TME. The study intends to reveal the effect of m6A on the immune microenvironment and macrophage differentiation of cervical cancer, which could be a viable therapeutic target for the treatment of advanced human cancers. Cervical Cancer m6A Glycolysis Macrophage Polarization PD-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cervical cancer is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer-associated mortality in women worldwide ( 1 ). In recent years, the acknowledgment of human papillomavirus (HPV) as a primary cause of cervical cancer led to further understanding of cervical cancer tumorigenesis. However, HPV infection is a necessary condition but not a sufficient condition ( 2 , 3 ). N6-methyladenosine (m6A) is the most abundant RNA modification of mammalian mRNAs and plays a vital role in many diseases, especially tumors. m6A modification is dynamically regulated by methyltransferases, demethylases and RNA-binding proteins ( 4 – 6 ). In cervical cancer, the transferase of m6A is at a high level. The increase of m6A methylation level can lead to the invasion and migration of tumor cells and determine a relatively poor clinical prognosis in human cancer patients( 7 – 9 ). M6A modification has been associated with immunoregulation. A recent article on a mouse model reports that m6A-modified mRNAs encoding lysosomal cathepsins are recognized by YTHDF1 in dendritic cells (DCs), subsequently enhancing the translation of cathepsins and facilitating tumor growth and shortening host survival ( 10 ). In CD4 + T cells, m6A levels are largely responsible for controlling naive T-cell homeostasis ( 11 ). However, our understanding of the regulation of m6A modification in macrophages is still in its infancy. Macrophages are prominent immune cells in TME that exert potent effects on tumorigenesis, which are associated with epigenetic reprogramming and are modified by epigenetic enzymes ( 12 ). Increasing evidence has demonstrated that tumor-associated macrophages (TAMs), characterized by distinctive M2-like macrophage properties, are critical players in the crosstalk between cancer cells and their microenvironment ( 13 – 15 ). Recently, studies have shown that a large number of M2 macrophages express PD-1. PD-1 expression of M2 macrophages negatively correlates with phagocytic potency against tumor cells, and blockade of PD-1/PD-L1 in vivo increases macrophage phagocytosis, reduces tumor growth and prolongs life survival ( 16 ). Here, we revealed that the METTL14 “writer” can be a critical regulator of tumor progression in cervical cancer. Besides, METTL14 in cervical cancer cells was found to contribute to the M2-like polarization and PD-1 expression of TAMs in the TME. In addition, studies have also shown that glycolysis in cancer cells leads to the accumulation of extracellular lactate, which in turn educates macrophages to become functional TAMs within the TME ( 17 , 18 ). Together, our data suggest an important interplay between METTL14-dependent m6A methylation and development of TME through the endogenous metabolic dysregulation, highlighting the possibility of identifying new targets and therapeutic strategies for cervical cancer treatment. Materials And Methods 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 × 10 6 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 × width 2 . 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. Result The expression level of m6A and METTL14 in cervical cancer In the initial research, we performed the m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq). Results demonstrated the m6A profile and motifs in the cervical cancer tissues compared with the normal tissues. The distribution situation of m6A modification included 3′ untranslated region (3′ UTR), CDS (coding sequence), 5′ UTR, and other regions. Remarkably, the m6A peaks were mainly located in the surrounding area of the stop codon and the end of the CDS (Fig. 1 A). In the cervical cancer tissue samples, the level of m6A was found to be upregulated compared to the normal control tissue. And the difference in KEGG suggests that the differential gene functions of the two groups of samples are mainly reflected in tumor-related pathways (Fig. 1 B). Furthermore, there are also differences in m6A methylation-related enzymes, including METTL14 (Fig. 1 C). The results of immunohistochemistry and western blot indicated that the protein expression of METTL14 was up-regulated in cervical cancer tissues compared with normal tissues (Fig. 1 D, 1 F). Simultaneously, there was an identical trend of the RNA expression of METTL14 using qPCR (Fig. 1 E). METTL14 indicates the poor prognosis of cervical cancer Survival analysis by Spearman’s rank correlation coefficient (GEPIA, http://gepia.cancer-pku.cn/ ) showed that higher METTL14 indicated the poor survival of cervical cancer both in overall survival and Disease Free Survival (RFS) (Fig. 1 G). Besides, the role of METTL14 was validated in cervical cancer cell lines. The expression of METTL14 was successively down-regulated in CASKI cells using shRNA (Fig. 1 H). Apoptosis, proliferation and cell migration were then evaluated. The results of CCK-8 showed that the proliferation ability of METTL14 was significantly decreased after downregulation of METTL14 (Fig. 1 I). Cell migration assays revealed that the number of cells that successfully penetrate the membrane decreased in the METTL14 shRNA group compared with the NC group (Fig. 1 J). When DNA damage, whether it is endogenous or exogenous, forms double-stranded breaks (DSBs), it is always followed by the phosphorylation of the histone, γ-H2AX. The detection of γ-H2AX by immunofluorescence allows the assessment of DNA damage, related DNA damage proteins and DNA repair. Representative immunofluorescence of γ-H2AX in METTL14 knockdown cells and NC are presented in Fig. 1 K. More apoptotic cells were present in the METTL14 shRNA group than in the NC group. This indicated that METTL14 assumes a crucial role in the regulation of the activity of tumor cells. M2 macrophages expressing PD-1 increased in cervical cancer We further investigated macrophages infiltration in the tumor lesions and normal lesions in patients with cervical cancer. Notably, the Immunofluorescence analysis revealed a higher percentage of PD-1 + M2 macrophages (PD-1 + CD206 + ) in the tumor lesions than normal lesions (Fig. 2 A). The appearance confirmed that the infiltration of macrophages in cervical cancer tend to be M2 type, while the expression of PD-1 was higher. METTL14 could promote the expression of PD-1 in M2 macrophages M2 macrophages, after co-cultured with the METTL14 shRNA group and control group, were collected. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1(PD-1 + CD206+) decreased in the macrophages co-cultured with METTL14 shRNA compared with macrophages co-cultured with control group (Fig. 2 B, C). The results of qPCR indicated that the expression of cytokines related to M2 macrophages had a lower level in the macrophages co-cultured with METTL14 shRNA (Fig. 2 D). We examined metabolites in tumor cell supernatants which may affect the function and polarization of macrophage and the results showed significant change of lactate acid in cell supernatants with reduced MEETL14 expression, while the fatty acid and NO content did not change significantly (Fig. 2 E). Lactic acid produced by glycolysis of tumor may be involved in PD-1 checkpoint expression of M2 macrophages Therefore, the function of METTL14 on the polarization of macrophages and PD-1 expression may be realized on regulating the secretion of lactate. The glycolysis inhibitor 2-deoxy-D-glucose (2-Deoxyglucose, 2-DG) was used to act on CASKI cervical cancer cells to build a glycolysis inhibition model in order to reduce the concentration of lactic acid. In order to prove that the effect of METTL14 on macrophages is dependent on lactic acid produced by tumor glycolysis, CASKI cells overexpressing METTL14 were treated with 2-DG. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1(PD-1 + CD206 + ) increased in the macrophages co-cultured with overexpression METTL14 CASKI cells (Fig. 3 C, E). However, a decreasing expression of CD206 and PD-1 was indicated after co-cultured with overexpression METTL14 CASKI cells treated with 2-DG compared with overexpression METTL14 CASKI cells (Fig. 3 C, E). The results of qPCR indicated the expression of cytokines related to M2 macrophages. As depicted in Fig. 3 D, the upregulation of cytokines related to M2 macrophages resulted from overexpression METTL14 treatment was significantly reversed in the cells co-cultured with overexpression METTL14 CASKI cells treated with 2-DG. Collectively, results suggested tumor glycolysis participates in the expression of PD1 in M2 macrophages. Lactic acid could act on M2 macrophages to promote PD-1 expression The mechanism of tumor glycolysis affecting macrophages is that cancer cell-derived lactate could induce the expression of PD-1 on M2 cancer-associated macrophages through activating GPR81 on the surface of macrophages. In order to prove this point, GPR81 siRNA was used to inhibit the expression of GPR81 on the surface of macrophages (Fig. 3 A). Besides, we cultured M2 macrophages treated with GPR81 siRNA. Flow cytometry results indicated that GPR81 siRNA had no significant effect on the polarization and PD-1 expression of M2 macrophages (Fig. 3 B). In this experiment, macrophages transfected with GPR81 siRNA were co-cultured with CASKI cells overexpressing METTL14. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1 (PD-1 + CD206+) decreased compared with the control group co-cultured with CASKI cells overexpressing METTL14 (Fig. 3 C, E). At the same time, cytokines associated with M2 macrophages were also reduced (Fig. 3 D). The results showed that lactic acid could act on M2 macrophages to promote PD-1 expression through GPR81. The level of glycolysis in cervical cancer M2-pyruvate kinase (PKM2) and hexokinase 2(HK2) are enzymes that play a crucial role in the progress of glycolysis. The results of immunohistochemistry and western blot indicated that the protein expression of PKM2 and HK2 was up-regulated in cervical cancer lesions compared with normal lesions (Fig. 4 A, B). qPCR also showed the mRNA level in cervical cancer lesions was significantly upregulated compared with the normal (Fig. 4 C). The above results suggested that the level of glycolysis in cervical cancer was increased. METTL14 promotes the glycolysis of cervical cancer cells The knockdown of METTL14 was constructed for the functional experiments. In the METTL14 shRNA cells, changes in protein levels of PKM2 and HK2 were suggested (Fig. 4 D). The results of targeted metabolomics sequencing showed that metabolites related to glucose metabolism including lactic acid changed significantly after the expression of METTL14 decreased (Fig. 4 E-H). Meanwhile, the content of energy carriers such as ATP and ADP decreased with the knockdown of METTL14 (Fig. 3 E-H). Overall, these findings reveal that METTL14 promotes the glycolysis of caski cells. AMPK plays a role in the regulation of glycolysis by METTL14 Nicotinamide adenine dinucleotide (NAD) is the main electron carrier coenzyme for the oxidation of all energy substrates, including glucose, fatty acids and ketones. AMPK signal pathway plays a role in regulating cellular NAD + levels. The results of targeted metabolomics sequencing showed a low expression of NAD + in METTL14 shRNA cells. Therefore, the AMPK signaling pathway could affect the regulation of METTLl14 on glycolysis (Fig. 5 A). Western blotting and immunochemistry were used to assess the expression of AMPK in the tumor lesions and normal lesions of patients with cervical cancer (Fig. 5 B, C). The protein level of AMPK was increased in cervical cancer. To further verify this, the expression of AMPK was detected in patients with cervical cancer using immunohistochemistry analysis. AMPK immunoreactivity was significantly higher in the tumor group than in the normal group. Altogether, these findings suggest that AMPK is abnormally expressed in adenomyosis, and the abnormal expression is likely to be associated with METTL14 and glycolysis. The overexpression of METTL14 was constructed for the functional experiments to confirm this statement. After the expression of METTLl14 increased, the protein and mRNA levels of AMPK, PKM2 and HK2 increased significantly (Fig. 5 D). Energy metabolism analysis indicated that METTL14 overexpression promoted lactate production (Fig. 5 F). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) assay showed that METTL14 markedly promoted glycolytic capacity (Fig. 5 E). Dorsomorphin (Compound C) is a selective and ATP-competitive AMPK inhibitor. In the CASKI cell line overexpressed METTL14, dorsomorphin successfully inhibited the expression of AMPK and had no significant effect on METTL14 (Fig. 5 D). After cells overexpressed METTL14 were added into dorsomorphin, the expression of PKM2 and HK2 were down-regulated compared with the group overexpressed METTL14 (Fig. 5 D). At the same time, lactate production and ECAR decreased (Fig. 5 E, F) while OCR was enhanced (Fig. 5 E). Collectively, these results suggest that AMPK participates in glycolysis depletion. The effect and mechanism of METTL14 detected by animal experiments The subcutaneous tumor-bearing model of cervical cancer was established in nude mice, and the effect of METTL14 on the proliferation of cervical cancer cells in animals was further verified. The results showed that the subcutaneous tumor volume in the METTL14 shRNA group grew more minor than the control group in 30 days (Fig. 6 A, B). Furthermore, TUNEL was used to detect the level of apoptosis in mouse subcutaneous tumors. The results showed a higher level of apoptosis in the subcutaneous tumor volume in the METTL14 shRNA group (Fig. 6 D). Besides, the results of immunohistochemistry suggest the protein expression of PKM2 and HK2 decreased in METTL14 shRNA tumor lesions compared to the control group (Fig. 6 C). At the same time, the expression of AMPK showed a low level in the subcutaneous tumor volume in the METTL14 shRNA group compared to the control group (Fig. 6 C). Animal experiments confirmed our previous results that METTL14 can promote tumor growth while affecting tumor glycolysis through the AMPK pathway in vivo. Discussion Previous studies have demonstrated that m6A RNA methylation regulators are associated with increased cancer aggressiveness and poor patient survival ( 19 ). In this study, we investigated the role of methyltransferase like 14 (METTL14), a major m6A “writer” ( 20 ). We detected a higher level of METTL14 expression in cervical cancer tissue than that in normal paracancer tissue. At the same time, with the knockdown of METTL14, the cell apoptosis level was decreased, while proliferation and migration were enhanced in vitro. Animal studies showed a similar trend. These data in the research indicated that METTL14 could act as the oncogenic element for cervical cancer tumorigenesis. Another major completion of our study showed a significant increase in the rate of PD-1-positive M2 macrophages in tumor tissues of patients with cervical cancer. Macrophages are typically divided into M1 and M2 phenotypes. M2-type macrophages reduce inflammatory responses and adaptive Th1 immunity, indicating that they are pro-tumorigenic. We have demonstrated that certain portion of M2 macrophages in cervical cancer tumor tissues express PD-1 in this study. The frequency of PD-1 + M2 macrophages was more abundant in cervical cancer tissue than in non-cancerous cervical tissue, suggesting the possibility that PD-1 + macrophages might play some important roles in the progression of cervical cancer. Increasing evidence suggested that targeting macrophage polarization benefits cervical cancer treatment ( 21 , 22 ). PD-1 was first discovered as a molecule expressed on T cells that induced apoptosis of T cells ( 23 ). Although most studies regarding immune evasion by PD-1 have focused on T cells, recent reports have demonstrated that other immune cells also express PD-1. Previous studies have showed that the phagocytotic ability of PD-1 + macrophages were impaired compared with PD-1 − macrophages ( 24 ). Therefore, it is likely that the increasing rate of PD-1 + macrophages observed in the current study promotes tumor progression. A variety of factors may be involved in the regulation of macrophage polarization and function changes. Considering the high expression of METTL14 and the alterative of the trend of macrophages, the potential conjunction between them was explored. In our study, we also found that the expression of M2-associated genes (CD206) and PD-1 was significantly decreased after cocultured with METTL14-deficient cervical cancer cells. These results confirm the effective influence of METTL14 in cervical cancer, especially in the adjustment of macrophage polarization and the expression of PD-1 in macrophages, which suggests the research prospects of METTL14 in immunoregulation of cervical cancer. Therefore, we further illustrated the reasons for this phenomenon. Recent study has shown that cancer cells can harness metabolic byproducts to hijack the functions of tumor-infiltrating immune cells to their own benefit. It is now clear that metabolic fluctuations in tumor cells are intimately connected to the phenotype and function of immune system. In this context, several metabolic (such as NO, fatty acids and lactic acid) may be harnessed to mediate immunological effects that involve, at least partially, changes in TAM subsets ( 25 ). In our experiments, we detected abnormal lactate content in co-cultured cell supernatants. Lactic acid is a metabolic product produced from glucose through glycolysis and the conversion of pyruvate by lactate dehydrogenase (LDH) under oxygen deprivation conditions ( 26 , 27 ). In the past, lactic has been considered as an ineffective metabolite produced by glycolysis. Lactic acid represents a crucial microenvironmental stressor that controls a number of immunosuppressive phenomena related to tumor progression ( 28 ). There is growing evidence that M2 TAMs preferentially accumulate in high concentration lactic acid of TME, and this process that plays a crucial role in tumorigenesis. Our data are consistent with a previous report demonstrating a mechanism by which METTL14 potentiates the function of M2-like TAMs and the expression of PD-1 through the promotion of lactate secretion to drive the formation of a tumor-supportive TME. Indeed, acidification of the local microenvironment by tumor-derived lactic acid promoted the conversion of tumor-associated macrophages to the M2 phenotype. However, exactly how lactic acid governs M2 TAMs remains elusive. Studies have proven that tumor-derived lactic acid regulates macrophage polarization through the G protein-coupled receptors (GPRs) and the monocarboxylic acid transporters (MCTs) -mediated “lactate shuttle” ( 29 , 30 ). GPR81, a member of G protein-coupled receptors, exerts an essential function in M2-like TAM polarization during cervical cancer metastasis. This GPR81-dependent activity is primarily attributed to lactate. Indeed, inhibition of the GPR81 activity resulted in a significant blockade of M2-like TAM phenotypes and the expression of PD-1 on the surface of macrophages induced by lactate acid. Our study also focused on the mechanism of decreased lactate content in the supernatant of METTL14-deficiency cervical cancer cells. It has been demonstrated that glycolysis is the primary source of energy metabolism in tumor ( 31 , 32 ). This metabolic adaptation is critical for cancer cell proliferation, invasion, metastasis and particularly the response to therapeutic intervention ( 17 , 18 , 33 – 36 ). Therefore, the enhanced glycolysis in cancer cells leads to lactate accumulation within the tumor microenvironment (TME), which is correlated with malignant progression and a poor prognosis in various human cancers, including cervical cancer ( 37 – 40 ). This research illustrated metabolic enzymes and transporter, involved in glycolysis, are expressed at higher levels in tumor cells than that in normal cells. In cervical cancer cell lines, the content of lactic acid in cell supernatant changed with the abnormal of glycolysis level. In particular, our results revealed that METTL14 directly increased the expression of multiple glycolytic genes, thereby promoting glycolysis both in vitro and in vivo. Targeted metabolomics results suggested that the content of glucose metabolites in cells decreased significantly when the expression of METTL14 was at low levels. Therefore, the underlying mechanism of METTL14 might be the glycolysis pattern. Overall, studies showed that METTL14-dependent glycolysis in cancer cells leads to the accumulation of extracellular lactate, which in turn educates macrophages to become functional TAMs within the TME. Our results described the potential roles of m6A in cancer metabolism and created the possibility of developing therapeutic strategies against cancer progression by targeting m6A modification. AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes of intracellular AMP/ATP ratio. In eukaryotic cells, AMP-activated protein kinase (AMPK) plays a significant role in regulating cellular energy balance. Given its vital role in controlling energy homeostasis, AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases, including type 2 diabetes and, more recently, cancer( 41 – 43 ). Targeted metabolomics results showed that AMPK pathway-related metabolites were affected by METTL14. In this finding, we discovered that METTL14 positively enhanced the stability of AMPK mRNA to increase its protein expression, thereby accelerating the glycolysis process. These findings illustrated that AMPK might be a potential mechanism by which METTL14 regulates metabolism. Our data showed that lactate induction was specifically enriched with the overexpression of METTL14, these findings highlighted METTL14/AMPK signaling as a lactate induction pathway in glycolysis. In this study, we identified m6A methylation as a critical regulator of TME by directly targeting M2-like polarization and PD-1 expression of TAMs. Moreover, tumorigenesis-associated metabolic reprogramming from tumor cells was found to contribute to this phenomenon. Together, our data suggest an important interplay between the endogenous metabolic dysregulation and development of TME through a METTL14-dependent mechanism, highlighting the possibility of identifying new targets and therapeutic strategies for cervical cancer treatment. Declarations Conflicts of interest None declared. Data Availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Buskwofie A, David-West G, Clare CA (2020) A Review of Cervical Cancer: Incidence and Disparities. J Natl Med Assoc. 112: 229-32. doi: 10.1016/j.jnma.2020.03.002 Bhatla N, Singhal S (2020) Primary HPV screening for cervical cancer. 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(2022) Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments. Cancer Cell. 40: 201-18 e9. doi: 10.1016/j.ccell.2022.01.001 Hermans D, Gautam S, Garcia-Canaveras JC et al. (2020) Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8(+) T cell stemness and antitumor immunity. Proc Natl Acad Sci U S A. 117: 6047-55. doi: 10.1073/pnas.1920413117 Yang K, Xu J, Fan M, Tu F, Wang X, Ha T, Williams DL, Li C (2020) Lactate Suppresses Macrophage Pro-Inflammatory Response to LPS Stimulation by Inhibition of YAP and NF-kappaB Activation via GPR81-Mediated Signaling. Front Immunol. 11: 587913. doi: 10.3389/fimmu.2020.587913 de la Cruz-Lopez KG, Castro-Munoz LJ, Reyes-Hernandez DO, Garcia-Carranca A, Manzo-Merino J (2019) Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches. Front Oncol. 9: 1143. doi: 10.3389/fonc.2019.01143 Zhang D, Tang Z, Huang H et al. (2019) Metabolic regulation of gene expression by histone lactylation. Nature. 574: 575-80. doi: 10.1038/s41586-019-1678-1 Cheng A, Zhang P, Wang B et al. (2019) Aurora-A mediated phosphorylation of LDHB promotes glycolysis and tumor progression by relieving the substrate-inhibition effect. Nat Commun. 10: 5566. doi: 10.1038/s41467-019-13485-8 Carling D (2017) AMPK signalling in health and disease. Curr Opin Cell Biol. 45: 31-7. doi: 10.1016/j.ceb.2017.01.005 Herzig S, Shaw RJ (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 19: 121-35. doi: 10.1038/nrm.2017.95 Li Y, Chen Y (2019) AMPK and Autophagy. Adv Exp Med Biol. 1206: 85-108. doi: 10.1007/978-981-15-0602-4_4 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1949592","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128215622,"identity":"3abfd767-d23e-41bf-a729-051c2b50ac88","order_by":0,"name":"Bingyu Wang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bingyu","middleName":"","lastName":"Wang","suffix":""},{"id":128215623,"identity":"ec720bec-4f1a-49a1-abef-2eebf4fa081f","order_by":1,"name":"Xinlin Jiao","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinlin","middleName":"","lastName":"Jiao","suffix":""},{"id":128215624,"identity":"fdfb455b-a1de-4649-baa5-1d9c37dd8a2b","order_by":2,"name":"Teng Zhang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Teng","middleName":"","lastName":"Zhang","suffix":""},{"id":128215625,"identity":"05b5e936-31ff-4c30-9914-d823934d1c6f","order_by":3,"name":"Qingqing Liu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Liu","suffix":""},{"id":128215626,"identity":"dc15506a-9b6e-4410-9b27-3b3382491482","order_by":4,"name":"Jinwen Ye","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinwen","middleName":"","lastName":"Ye","suffix":""},{"id":128215627,"identity":"40619256-c148-4727-ad93-16258f757ec4","order_by":5,"name":"Yuan Feng","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Feng","suffix":""},{"id":128215628,"identity":"79e28176-d295-4c66-814e-d046a7ea5ca8","order_by":6,"name":"Baoxia Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACxjYQycPA3sAgAeI2EK+F5wDRWhjYIJREApFa+Gcktz34UXFYhn/m84e3eRhsZDccYH72AJ8WiRuJ7YY9Z9J4JG7nGFvzMKQZbzjAZm6AT4uBRGKbBG+bDQ/D7Rw2aR6Gw4kbDvCwSRDSIvm3TYJH/ubxZ0At/4nTIg2yxeAGgxlQywHCWiTOPGw3lgH6xfBMjrHlHINk45mH2czwauFvT3/28E3FYXu548cf3nhTYSfbd7z5GV4t6O4EYmYS1I+CUTAKRsEowA4A5v9D8n4IkvIAAAAASUVORK5CYII=","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Baoxia","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2022-08-10 14:44:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1949592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1949592/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25274548,"identity":"ced76a04-19fa-4362-8ef0-27303047c33b","added_by":"auto","created_at":"2022-08-16 16:15:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1179127,"visible":true,"origin":"","legend":"\u003cp\u003e\tIncreased m6A methylation level in cervical cancer tissue and poor prognosis caused by METTL14\u003c/p\u003e\u003cp\u003e\t(A). MeRIP-seq assays revealed the m6A peaks were mainly located in the surrounding area of stop codon and the end of the CDS. (B). KEGG analysis showed that the difference between cervical cancer and para-cancerous tissue tissues is mainly reflected in tumor-related pathways. (C). Expression of m6A regulatory factors. (D). Representative micrographs of METTL14 immunostaining in cervical cancer and paracancerous tissues. Quantitative analysis of the percent of the positive cells of METTL14 immunostaining in tumor lesions or control subjects. (E). mRNA level of METTL14 in cervical cancer (n=10) and paracancerous tissue (n=10). (F). The protein level of METTL14 in cervical cancer (n=10) and paracancerous tissue (n=10). GAPDH served as a loading control. \u0026nbsp;(G). Survival analysis by Spearman’s rank correlation coefficient (GEPIA, http://gepia.cancer-pku.cn/) showed that higher METTL14 indicated the poor survival of cervical cancer both in overall survival and Disease Free Survival (RFS)​. (H). The protein expression of METTL14 after transfecting control shRNA by western blot. GAPDH served as a loading control. (I). Cell proliferation was detected by CCK8 assay. (J). Microscopic observation of results of a transwell assay. Cells stained with crystal violet are successfully migrated cells. The number of cells that migrated successfully decreased, accompanied with the decrease of METTL14 expression. (K). Cell immunofluorescence results of γ-H2AX. The strong fluorescence of γ-H2AX indicated an increasing level of cell apoptosis. The results represent the mean ± SD. *P＜0.05, **P \u0026lt; 0.01, ***P\u0026lt; 0.001, scale bar=50um.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/22dc41897b58c66507907b34.png"},{"id":25272965,"identity":"74a45f7e-1c05-4e8c-b9b0-655122299098","added_by":"auto","created_at":"2022-08-16 16:05:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":514269,"visible":true,"origin":"","legend":"\u003cp\u003e\tAbnormal aggregation of PD-1\u003csup\u003e+\u003c/sup\u003e M2 macrophages in cervical cancer and the effect of METTL14 on PD-1\u003csup\u003e+\u003c/sup\u003e M2 macrophages\u003c/p\u003e\u003cp\u003e\t(A). Tissue immunofluorescence indicated the aggregation of PD-1\u003csup\u003e+ \u003c/sup\u003eM2 macrophages. Red fluorescence represents CD206 (a marker for M2 macrophages), while green fluorescence represents PD-1. The cells circled by the white box are cells that collectively express CD206 and PD-1, that is, PD-1\u003csup\u003e+\u003c/sup\u003e M2 macrophages. (B). Representative and quantitative flow cytometry results for CD206 (M2 marker) and PD-1 expression in THP-1 cell line after co-cultured with CASKI cells with or without METTL14 shRNA. (C). Immunofluorescence indicated the PD-1 expression of M2 macrophages after co-cultured with CASKI cells with or without METTL14 shRNA. Red fluorescence represented CD206 and green fluorescence represented PD-1. (D). mRNA level of IL-10, TGF-β and ARG-1 in THP-1 cell line after co-cultured with CASKI cells with or without METTL14 shRNA. (E) Lactic acid, fatty acid and NO concentration in CASKI cell supernatant. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt; 0.001, scale bar(A)=20um, scale bar(C)=50um.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/553b001e3c02a8559392e143.png"},{"id":25272966,"identity":"0996b094-fac0-49e4-be6d-fccc5bb8c7dc","added_by":"auto","created_at":"2022-08-16 16:05:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":531391,"visible":true,"origin":"","legend":"\u003cp\u003e\tLactate acid from tumor glycolysis could activate GPR81 on the surface of macrophages, leading to macrophage polarization and PD-1 expression.\u003c/p\u003e\u003cp\u003e\t(A). The protein expression of GPR81 in macrophages after transfecting siRNA by western blot. GAPDH served as a loading control. (B). Influence of GPR81 deletion in macrophages on the expression of CD206 and PD-1 in macrophages. (C-E). Cells were divided into four following groups: macrophages co-cultured with CASKI cells; macrophages co-cultured with CASKI cells overexpressing METTL14; macrophages co-cultured with glycolysis inhibited CASKI cells overexpressing METTL14; GPR81 siRNA macrophages co-cultured with CASKI cells overexpressing METTL14. (C) Representative and quantitative flow cytometry results for CD206 (M2 marker) and PD-1 expression in each group. (D). mRNA level of IL-10, TGF-β and ARG-1 in each group. (E). Immunofluorescence indicated the PD-1 expression of M2 macrophages in each group. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt; 0.001, scale bar=50um.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/e75d99fee5555d20c65ec6e9.png"},{"id":25273696,"identity":"86a34866-1bba-411b-b899-c50ee8609831","added_by":"auto","created_at":"2022-08-16 16:10:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1359764,"visible":true,"origin":"","legend":"\u003cp\u003e\tIncreased glycolysis level in cervical cancer tissue and downregulation of METTL14 inhibited glycolysis in CASKI cell lines.\u003c/p\u003e\u003cp\u003e\t\u003c/p\u003e\u003cp\u003e\t(A). Representative micrographs of PKM2 and HK2 immunostaining in cervical cancer (n=10) and paracancerous tissues (n=10). Quantitative analysis of the percent of the positive cells of PKM2 and HK2 immunostaining in tumor lesions or control subjects. (B). The protein level of PKM2 and HK2 in cervical cancer (n=10) and paracancerous tissue (n=10). GAPDH served as a loading control.\u0026nbsp;(C). mRNA level of PKM2 and HK2 in cervical cancer (n=10) and paracancerous tissue (n=10). (D). Protein level of HK2 and PKM2 in CASKI cell lines while METTL14 was downregulated by shRNA. GAPDH served as a loading control. (E). Quantitative analysis results of metabolites related to glucose metabolism. (F). The heatmap of hierarchical clustering analysis displays the metabolite signal intensity averaged of the shMETTL14 group (n=10) and the NC group (n=10), respectively. The Euclidean distance matrix was used to analyze the quantitative values of metabolites, and the metabolites were clustered by a complete linkage method. Colors represent standardized mean difference: blue, metabolite with increased levels; red, metabolite with decreased levels. (G). Z-score normalization of metabolomics data. (H). correlation heatmap diagram of shMETTL14 group (n=10) vs NC group. Color intensity is proportional to the correlation coefficients of different metabolites. The darker the color, the stronger the correlation. Red circles indicate a positive correlation, and the blue ones indicate a negative correlation. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt; 0.001, scale bar=50um.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/1871c4a0e51a071517a91e77.png"},{"id":25272970,"identity":"5cc5f9d7-b91b-4925-8351-81815930e055","added_by":"auto","created_at":"2022-08-16 16:05:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179925,"visible":true,"origin":"","legend":"\u003cp\u003e\tThe role of AMPK in the regulation of glycolysis by METTL14 and aberrant expression of AMPK in cervical cancer.\u003c/p\u003e\u003cp\u003e\t(A). KEGG pathway of AMPK, blue points indicate decreased expression while METTL14 was downregulated by shRNA. (B). Western blot and qPCR analysis of AMPK expression in cervical cancer and control subjects. GAPDH was used as the sample-loading control. (C). Immunochemistry analysis of AMPK expression in cervical cancer (n=10) and control subjects (n=10). (D). The protein level of AMPK after METTL14 was downregulated by shRNA was detected using western blot. Results of western blot indicated that AMPK was successfully inhibited by dorsomorphin and the protein expression of PKM2 and HK2 changed correspondingly. (E). The cellular ECAR and OCR were measured by sea-horse. (F). Lactate production, scale bar=50um.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/1176d6595ccae727b44b3ba0.png"},{"id":25272969,"identity":"95760b6c-a107-40b9-a128-a09f95ead7b3","added_by":"auto","created_at":"2022-08-16 16:05:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4163848,"visible":true,"origin":"","legend":"\u003cp\u003e\tMETTL14 decreases glycolysis and tumor growth in vivo\u003c/p\u003e\u003cp\u003e\t(A). CASKI cells with or without METTL14 shRNA were subcutaneously inoculated into nude mice. Each group contained 6 mice. (B). The tumor size. (C). Representative micrographs of PKM2 and HK2 immunostaining in the shMETTL14 group and control subjects and quantitative analysis of the percent of the positive cells. (D). Representative micrographs of TUNEL staining for apoptosis in the shMETTL14 group and control subjects. A TUNEL-positive apoptotic cell is indicated by a black arrowhead, and the apoptotic nuclei that show DNA fragmentation appear dark brown. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt; 0.001, scale bar=50um.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/f8a904a0966b1204df58d572.png"},{"id":25366536,"identity":"23f631a4-7d96-412d-81c3-4cdd2d1a15b9","added_by":"auto","created_at":"2022-08-18 13:14:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4233284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/69080ed2-4bf5-4cab-a23f-968563a48196.pdf"},{"id":25272964,"identity":"4624a0cf-5e8f-424d-996c-fd926ae9a2e8","added_by":"auto","created_at":"2022-08-16 16:05:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20558,"visible":true,"origin":"","legend":"","description":"","filename":"siRNAshRNAsequenceandPCRprimers.docx","url":"https://assets-eu.researchsquare.com/files/rs-1949592/v1/da8f95dbee17a27cc37079a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Glycolysis induced by METTL14-related m6A methylation is essential for macrophage function and tumor progression in cervical cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical cancer is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer-associated mortality in women worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In recent years, the acknowledgment of human papillomavirus (HPV) as a primary cause of cervical cancer led to further understanding of cervical cancer tumorigenesis. However, HPV infection is a necessary condition but not a sufficient condition (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). N6-methyladenosine (m6A) is the most abundant RNA modification of mammalian mRNAs and plays a vital role in many diseases, especially tumors. m6A modification is dynamically regulated by methyltransferases, demethylases and RNA-binding proteins (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In cervical cancer, the transferase of m6A is at a high level. The increase of m6A methylation level can lead to the invasion and migration of tumor cells and determine a relatively poor clinical prognosis in human cancer patients(\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eM6A modification has been associated with immunoregulation. A recent article on a mouse model reports that m6A-modified mRNAs encoding lysosomal cathepsins are recognized by YTHDF1 in dendritic cells (DCs), subsequently enhancing the translation of cathepsins and facilitating tumor growth and shortening host survival (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In CD4\u0026thinsp;+\u0026thinsp;T cells, m6A levels are largely responsible for controlling naive T-cell homeostasis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, our understanding of the regulation of m6A modification in macrophages is still in its infancy. Macrophages are prominent immune cells in TME that exert potent effects on tumorigenesis, which are associated with epigenetic reprogramming and are modified by epigenetic enzymes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Increasing evidence has demonstrated that tumor-associated macrophages (TAMs), characterized by distinctive M2-like macrophage properties, are critical players in the crosstalk between cancer cells and their microenvironment (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Recently, studies have shown that a large number of M2 macrophages express PD-1. PD-1 expression of M2 macrophages negatively correlates with phagocytic potency against tumor cells, and blockade of PD-1/PD-L1 in vivo increases macrophage phagocytosis, reduces tumor growth and prolongs life survival (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we revealed that the METTL14 \u0026ldquo;writer\u0026rdquo; can be a critical regulator of tumor progression in cervical cancer. Besides, METTL14 in cervical cancer cells was found to contribute to the M2-like polarization and PD-1 expression of TAMs in the TME. In addition, studies have also shown that glycolysis in cancer cells leads to the accumulation of extracellular lactate, which in turn educates macrophages to become functional TAMs within the TME (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Together, our data suggest an important interplay between METTL14-dependent m6A methylation and development of TME through the endogenous metabolic dysregulation, highlighting the possibility of identifying new targets and therapeutic strategies for cervical cancer treatment.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and tissue samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 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\u0026apos;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue sections were fixed with paraformaldehyde, and cut into 4.0mm-thick slices. Put the slices in the oven at 65\u0026deg;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 \u0026deg;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\u0026deg;C. Then incubated with the secondary antibody for 60 minutes at room temperature (about 28\u0026deg;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeRIP-seq assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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\u0026deg;C for 2 h. The mixture was supplemented with protein A magnetic beads (Thermo Fisher) and incubated under head-to-tail mixing at 4\u0026deg;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 \u0026lt; 0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor PD-1, CD206 and \u0026gamma;-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 \u0026gamma;-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\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal protein extraction and western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected tissues and cells were melted into liquid , added 500\u0026mu;l 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\u0026deg;C. Then tested the concentration of proteins (40\u0026mu;g per sample) with the BCA protein assay kit, and warmed the proteins supernatant with SDS-loading buffer by bainmarie at 100\u0026deg;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 (0.22\u0026mu;m or 0.45\u0026mu;m) membranes for 60-90 minutes, blocked with 5% skimmed milk powders which dissolved in TBST for 90 minutes at room temperature (about 28\u0026deg;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\u0026deg;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative RT-PCR (real-time PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing 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\u003csup\u003e\u0026minus;∆∆\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and Caski cells cocultured with macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaski 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\u0026times;) (Solarbio, US) at 37\u0026deg;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\u0026deg;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\u0026amp;D, USA) and 10ng/ml of IL-13 (R\u0026amp;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\u0026times;105 cells) were added to the wells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShort Hairpin RNAs (shRNA), Small Interfering RNA (siRNA) and Genetic Knockout\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell 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 500\u0026mu;l of MEM. After 24\u0026ndash;48\u0026thinsp;h of incubation at 37\u0026thinsp;\u0026deg;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\u0026thinsp;min. Invaded cells on the lower side of the filter were stained with 0.5% crystal violet for 20\u0026thinsp;min, and images were captured using a microscope. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLactic acid production, ECAR and OCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular 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\u0026thinsp;mM pyruvate, 10\u0026thinsp;mM glucose, and 2\u0026thinsp;mM glutamine for 1.5h at 37\u0026thinsp;\u0026deg;C, then measured by the XF Cell Mito Stress Kit (Seahorse Bioscience). The concentrations of ROT/AA and 2-DG were 1.0\u0026thinsp;uM and 0.5\u0026thinsp;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\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHP-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\u0026amp;D, USA) and anti-PD-1(B\u0026amp;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\u0026rsquo;s protocols. Isotype controls were used to confirm antibody specificity. Single color stain controls were used to enable correct compensation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies were performed according to institutional guidelines. Caski cells were stably transfected with NC or shMETTL14 vectors. A total of 5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e viable cells were injected into the right flanks of nude mice. Then after 12\u0026thinsp;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\u0026thinsp;=\u0026thinsp;1/2\u0026thinsp;\u0026times;\u0026thinsp;length\u0026thinsp;\u0026times;\u0026thinsp;width\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical software Prism 7 (GraphPad) was used for data analyses. Statistical significance was determined by Student\u0026rsquo;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 \u0026lt; 0.05. Statistically significant results are expressed using asterisks, where *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe expression level of m6A and METTL14 in cervical cancer\u003c/h2\u003e \u003cp\u003eIn the initial research, we performed the m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq). Results demonstrated the m6A profile and motifs in the cervical cancer tissues compared with the normal tissues. The distribution situation of m6A modification included 3\u0026prime; untranslated region (3\u0026prime; UTR), CDS (coding sequence), 5\u0026prime; UTR, and other regions. Remarkably, the m6A peaks were mainly located in the surrounding area of the stop codon and the end of the CDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the cervical cancer tissue samples, the level of m6A was found to be upregulated compared to the normal control tissue. And the difference in KEGG suggests that the differential gene functions of the two groups of samples are mainly reflected in tumor-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, there are also differences in m6A methylation-related enzymes, including METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results of immunohistochemistry and western blot indicated that the protein expression of METTL14 was up-regulated in cervical cancer tissues compared with normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Simultaneously, there was an identical trend of the RNA expression of METTL14 using qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMETTL14 indicates the poor prognosis of cervical cancer\u003c/h2\u003e \u003cp\u003eSurvival analysis by Spearman\u0026rsquo;s rank correlation coefficient (GEPIA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) showed that higher METTL14 indicated the poor survival of cervical cancer both in overall survival and Disease Free Survival (RFS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Besides, the role of METTL14 was validated in cervical cancer cell lines. The expression of METTL14 was successively down-regulated in CASKI cells using shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Apoptosis, proliferation and cell migration were then evaluated. The results of CCK-8 showed that the proliferation ability of METTL14 was significantly decreased after downregulation of METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Cell migration assays revealed that the number of cells that successfully penetrate the membrane decreased in the METTL14 shRNA group compared with the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). When DNA damage, whether it is endogenous or exogenous, forms double-stranded breaks (DSBs), it is always followed by the phosphorylation of the histone, γ-H2AX. The detection of γ-H2AX by immunofluorescence allows the assessment of DNA damage, related DNA damage proteins and DNA repair. Representative immunofluorescence of γ-H2AX in METTL14 knockdown cells and NC are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK. More apoptotic cells were present in the METTL14 shRNA group than in the NC group. This indicated that METTL14 assumes a crucial role in the regulation of the activity of tumor cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eM2 macrophages expressing PD-1 increased in cervical cancer\u003c/h2\u003e \u003cp\u003eWe further investigated macrophages infiltration in the tumor lesions and normal lesions in patients with cervical cancer. Notably, the Immunofluorescence analysis revealed a higher percentage of PD-1\u003csup\u003e+\u003c/sup\u003e M2 macrophages (PD-1\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e) in the tumor lesions than normal lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The appearance confirmed that the infiltration of macrophages in cervical cancer tend to be M2 type, while the expression of PD-1 was higher.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMETTL14 could promote the expression of PD-1 in M2 macrophages\u003c/h2\u003e \u003cp\u003eM2 macrophages, after co-cultured with the METTL14 shRNA group and control group, were collected. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1(PD-1\u0026thinsp;+\u0026thinsp;CD206+) decreased in the macrophages co-cultured with METTL14 shRNA compared with macrophages co-cultured with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). The results of qPCR indicated that the expression of cytokines related to M2 macrophages had a lower level in the macrophages co-cultured with METTL14 shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We examined metabolites in tumor cell supernatants which may affect the function and polarization of macrophage and the results showed significant change of lactate acid in cell supernatants with reduced MEETL14 expression, while the fatty acid and NO content did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactic acid produced by glycolysis of tumor may be involved in PD-1 checkpoint expression of M2 macrophages\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTherefore, the function of METTL14 on the polarization of macrophages and PD-1 expression may be realized on regulating the secretion of lactate. The glycolysis inhibitor 2-deoxy-D-glucose (2-Deoxyglucose, 2-DG) was used to act on CASKI cervical cancer cells to build a glycolysis inhibition model in order to reduce the concentration of lactic acid. In order to prove that the effect of METTL14 on macrophages is dependent on lactic acid produced by tumor glycolysis, CASKI cells overexpressing METTL14 were treated with 2-DG. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1(PD-1\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e) increased in the macrophages co-cultured with overexpression METTL14 CASKI cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). However, a decreasing expression of CD206 and PD-1 was indicated after co-cultured with overexpression METTL14 CASKI cells treated with 2-DG compared with overexpression METTL14 CASKI cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). The results of qPCR indicated the expression of cytokines related to M2 macrophages. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the upregulation of cytokines related to M2 macrophages resulted from overexpression METTL14 treatment was significantly reversed in the cells co-cultured with overexpression METTL14 CASKI cells treated with 2-DG. Collectively, results suggested tumor glycolysis participates in the expression of PD1 in M2 macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eLactic acid could act on M2 macrophages to promote PD-1 expression\u003c/h2\u003e \u003cp\u003eThe mechanism of tumor glycolysis affecting macrophages is that cancer cell-derived lactate could induce the expression of PD-1 on M2 cancer-associated macrophages through activating GPR81 on the surface of macrophages. In order to prove this point, GPR81 siRNA was used to inhibit the expression of GPR81 on the surface of macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Besides, we cultured M2 macrophages treated with GPR81 siRNA. Flow cytometry results indicated that GPR81 siRNA had no significant effect on the polarization and PD-1 expression of M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In this experiment, macrophages transfected with GPR81 siRNA were co-cultured with CASKI cells overexpressing METTL14. Flow cytometry and immunofluorescence showed that M2 macrophages expressing PD-1 (PD-1\u0026thinsp;+\u0026thinsp;CD206+) decreased compared with the control group co-cultured with CASKI cells overexpressing METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). At the same time, cytokines associated with M2 macrophages were also reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The results showed that lactic acid could act on M2 macrophages to promote PD-1 expression through GPR81.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eThe level of glycolysis in cervical cancer\u003c/h2\u003e \u003cp\u003eM2-pyruvate kinase (PKM2) and hexokinase 2(HK2) are enzymes that play a crucial role in the progress of glycolysis. The results of immunohistochemistry and western blot indicated that the protein expression of PKM2 and HK2 was up-regulated in cervical cancer lesions compared with normal lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). qPCR also showed the mRNA level in cervical cancer lesions was significantly upregulated compared with the normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The above results suggested that the level of glycolysis in cervical cancer was increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eMETTL14 promotes the glycolysis of cervical cancer cells\u003c/h2\u003e \u003cp\u003eThe knockdown of METTL14 was constructed for the functional experiments. In the METTL14 shRNA cells, changes in protein levels of PKM2 and HK2 were suggested (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The results of targeted metabolomics sequencing showed that metabolites related to glucose metabolism including lactic acid changed significantly after the expression of METTL14 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). Meanwhile, the content of energy carriers such as ATP and ADP decreased with the knockdown of METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). Overall, these findings reveal that METTL14 promotes the glycolysis of caski cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eAMPK plays a role in the regulation of glycolysis by METTL14\u003c/h2\u003e \u003cp\u003eNicotinamide adenine dinucleotide (NAD) is the main electron carrier coenzyme for the oxidation of all energy substrates, including glucose, fatty acids and ketones. AMPK signal pathway plays a role in regulating cellular NAD\u0026thinsp;+\u0026thinsp;levels. The results of targeted metabolomics sequencing showed a low expression of NAD\u0026thinsp;+\u0026thinsp;in METTL14 shRNA cells. Therefore, the AMPK signaling pathway could affect the regulation of METTLl14 on glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blotting and immunochemistry were used to assess the expression of AMPK in the tumor lesions and normal lesions of patients with cervical cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). The protein level of AMPK was increased in cervical cancer. To further verify this, the expression of AMPK was detected in patients with cervical cancer using immunohistochemistry analysis. AMPK immunoreactivity was significantly higher in the tumor group than in the normal group. Altogether, these findings suggest that AMPK is abnormally expressed in adenomyosis, and the abnormal expression is likely to be associated with METTL14 and glycolysis. The overexpression of METTL14 was constructed for the functional experiments to confirm this statement. After the expression of METTLl14 increased, the protein and mRNA levels of AMPK, PKM2 and HK2 increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Energy metabolism analysis indicated that METTL14 overexpression promoted lactate production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) assay showed that METTL14 markedly promoted glycolytic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Dorsomorphin (Compound C) is a selective and ATP-competitive AMPK inhibitor. In the CASKI cell line overexpressed METTL14, dorsomorphin successfully inhibited the expression of AMPK and had no significant effect on METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). After cells overexpressed METTL14 were added into dorsomorphin, the expression of PKM2 and HK2 were down-regulated compared with the group overexpressed METTL14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). At the same time, lactate production and ECAR decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F) while OCR was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Collectively, these results suggest that AMPK participates in glycolysis depletion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eThe effect and mechanism of METTL14 detected by animal experiments\u003c/h2\u003e \u003cp\u003eThe subcutaneous tumor-bearing model of cervical cancer was established in nude mice, and the effect of METTL14 on the proliferation of cervical cancer cells in animals was further verified. The results showed that the subcutaneous tumor volume in the METTL14 shRNA group grew more minor than the control group in 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Furthermore, TUNEL was used to detect the level of apoptosis in mouse subcutaneous tumors. The results showed a higher level of apoptosis in the subcutaneous tumor volume in the METTL14 shRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Besides, the results of immunohistochemistry suggest the protein expression of PKM2 and HK2 decreased in METTL14 shRNA tumor lesions compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). At the same time, the expression of AMPK showed a low level in the subcutaneous tumor volume in the METTL14 shRNA group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Animal experiments confirmed our previous results that METTL14 can promote tumor growth while affecting tumor glycolysis through the AMPK pathway in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have demonstrated that m6A RNA methylation regulators are associated with increased cancer aggressiveness and poor patient survival (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In this study, we investigated the role of methyltransferase like 14 (METTL14), a major m6A \u0026ldquo;writer\u0026rdquo; (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). We detected a higher level of METTL14 expression in cervical cancer tissue than that in normal paracancer tissue. At the same time, with the knockdown of METTL14, the cell apoptosis level was decreased, while proliferation and migration were enhanced in vitro. Animal studies showed a similar trend. These data in the research indicated that METTL14 could act as the oncogenic element for cervical cancer tumorigenesis.\u003c/p\u003e \u003cp\u003eAnother major completion of our study showed a significant increase in the rate of PD-1-positive M2 macrophages in tumor tissues of patients with cervical cancer. Macrophages are typically divided into M1 and M2 phenotypes. M2-type macrophages reduce inflammatory responses and adaptive Th1 immunity, indicating that they are pro-tumorigenic. We have demonstrated that certain portion of M2 macrophages in cervical cancer tumor tissues express PD-1 in this study. The frequency of PD-1\u003csup\u003e+\u003c/sup\u003e M2 macrophages was more abundant in cervical cancer tissue than in non-cancerous cervical tissue, suggesting the possibility that PD-1\u003csup\u003e+\u003c/sup\u003e macrophages might play some important roles in the progression of cervical cancer. Increasing evidence suggested that targeting macrophage polarization benefits cervical cancer treatment (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). PD-1 was first discovered as a molecule expressed on T cells that induced apoptosis of T cells (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Although most studies regarding immune evasion by PD-1 have focused on T cells, recent reports have demonstrated that other immune cells also express PD-1. Previous studies have showed that the phagocytotic ability of PD-1\u003csup\u003e+\u003c/sup\u003e macrophages were impaired compared with PD-1\u003csup\u003e\u0026minus;\u003c/sup\u003e macrophages (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Therefore, it is likely that the increasing rate of PD-1\u003csup\u003e+\u003c/sup\u003e macrophages observed in the current study promotes tumor progression. A variety of factors may be involved in the regulation of macrophage polarization and function changes. Considering the high expression of METTL14 and the alterative of the trend of macrophages, the potential conjunction between them was explored. In our study, we also found that the expression of M2-associated genes (CD206) and PD-1 was significantly decreased after cocultured with METTL14-deficient cervical cancer cells. These results confirm the effective influence of METTL14 in cervical cancer, especially in the adjustment of macrophage polarization and the expression of PD-1 in macrophages, which suggests the research prospects of METTL14 in immunoregulation of cervical cancer.\u003c/p\u003e \u003cp\u003eTherefore, we further illustrated the reasons for this phenomenon. Recent study has shown that cancer cells can harness metabolic byproducts to hijack the functions of tumor-infiltrating immune cells to their own benefit. It is now clear that metabolic fluctuations in tumor cells are intimately connected to the phenotype and function of immune system. In this context, several metabolic (such as NO, fatty acids and lactic acid) may be harnessed to mediate immunological effects that involve, at least partially, changes in TAM subsets (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In our experiments, we detected abnormal lactate content in co-cultured cell supernatants. Lactic acid is a metabolic product produced from glucose through glycolysis and the conversion of pyruvate by lactate dehydrogenase (LDH) under oxygen deprivation conditions (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In the past, lactic has been considered as an ineffective metabolite produced by glycolysis. Lactic acid represents a crucial microenvironmental stressor that controls a number of immunosuppressive phenomena related to tumor progression (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). There is growing evidence that M2 TAMs preferentially accumulate in high concentration lactic acid of TME, and this process that plays a crucial role in tumorigenesis. Our data are consistent with a previous report demonstrating a mechanism by which METTL14 potentiates the function of M2-like TAMs and the expression of PD-1 through the promotion of lactate secretion to drive the formation of a tumor-supportive TME. Indeed, acidification of the local microenvironment by tumor-derived lactic acid promoted the conversion of tumor-associated macrophages to the M2 phenotype.\u003c/p\u003e \u003cp\u003eHowever, exactly how lactic acid governs M2 TAMs remains elusive. Studies have proven that tumor-derived lactic acid regulates macrophage polarization through the G protein-coupled receptors (GPRs) and the monocarboxylic acid transporters (MCTs) -mediated \u0026ldquo;lactate shuttle\u0026rdquo; (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). GPR81, a member of G protein-coupled receptors, exerts an essential function in M2-like TAM polarization during cervical cancer metastasis. This GPR81-dependent activity is primarily attributed to lactate. Indeed, inhibition of the GPR81 activity resulted in a significant blockade of M2-like TAM phenotypes and the expression of PD-1 on the surface of macrophages induced by lactate acid.\u003c/p\u003e \u003cp\u003eOur study also focused on the mechanism of decreased lactate content in the supernatant of METTL14-deficiency cervical cancer cells. It has been demonstrated that glycolysis is the primary source of energy metabolism in tumor (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). This metabolic adaptation is critical for cancer cell proliferation, invasion, metastasis and particularly the response to therapeutic intervention (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Therefore, the enhanced glycolysis in cancer cells leads to lactate accumulation within the tumor microenvironment (TME), which is correlated with malignant progression and a poor prognosis in various human cancers, including cervical cancer (\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). This research illustrated metabolic enzymes and transporter, involved in glycolysis, are expressed at higher levels in tumor cells than that in normal cells. In cervical cancer cell lines, the content of lactic acid in cell supernatant changed with the abnormal of glycolysis level. In particular, our results revealed that METTL14 directly increased the expression of multiple glycolytic genes, thereby promoting glycolysis both in vitro and in vivo. Targeted metabolomics results suggested that the content of glucose metabolites in cells decreased significantly when the expression of METTL14 was at low levels. Therefore, the underlying mechanism of METTL14 might be the glycolysis pattern. Overall, studies showed that METTL14-dependent glycolysis in cancer cells leads to the accumulation of extracellular lactate, which in turn educates macrophages to become functional TAMs within the TME. Our results described the potential roles of m6A in cancer metabolism and created the possibility of developing therapeutic strategies against cancer progression by targeting m6A modification.\u003c/p\u003e \u003cp\u003eAMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes of intracellular AMP/ATP ratio. In eukaryotic cells, AMP-activated protein kinase (AMPK) plays a significant role in regulating cellular energy balance. Given its vital role in controlling energy homeostasis, AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases, including type 2 diabetes and, more recently, cancer(\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Targeted metabolomics results showed that AMPK pathway-related metabolites were affected by METTL14. In this finding, we discovered that METTL14 positively enhanced the stability of AMPK mRNA to increase its protein expression, thereby accelerating the glycolysis process. These findings illustrated that AMPK might be a potential mechanism by which METTL14 regulates metabolism. Our data showed that lactate induction was specifically enriched with the overexpression of METTL14, these findings highlighted METTL14/AMPK signaling as a lactate induction pathway in glycolysis.\u003c/p\u003e \u003cp\u003eIn this study, we identified m6A methylation as a critical regulator of TME by directly targeting M2-like polarization and PD-1 expression of TAMs. Moreover, tumorigenesis-associated metabolic reprogramming from tumor cells was found to contribute to this phenomenon. Together, our data suggest an important interplay between the endogenous metabolic dysregulation and development of TME through a METTL14-dependent mechanism, highlighting the possibility of identifying new targets and therapeutic strategies for cervical cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBuskwofie A, David-West G, Clare CA (2020) A Review of Cervical Cancer: Incidence and Disparities. 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Adv Exp Med Biol. 1206: 85-108. doi: 10.1007/978-981-15-0602-4_4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cervical Cancer, m6A, Glycolysis, Macrophage Polarization, PD-1","lastPublishedDoi":"10.21203/rs.3.rs-1949592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1949592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\tCervical cancer is one of the most common tumors of women’s diseases. N6-methyladenosine (m6A) is an abundant RNA modification of mammalian mRNAs and plays a vital role in many diseases, especially tumors. In this study, we aimed to investigate the roles of m6A on macrophage function and tumor progression in cervical cancer. We measured the levels of m6A expression in cervical cancer tissues, and revealed biological functions of METTL14 (Methyltransferase14, N6-Adenosine-Methyltransferase Subunit) on tumor-associated macrophages (TAMs) and PD-1 expression. We found the METTL14-related methylation of m6A in cervical cancer was correlated with infiltration of TAMs, polarization status of infiltrated TAMs, and survival outcomes of cervical cancer patients. Mechanistically, lactate produced by tumor glycolysis in the acidic immunosuppressive tumor microenvironment (TME) has an important role as a proinflammatory and immunosuppressive mediator in this program. METTL14-related methylation of m6A may play a predictive role in the polarization and PD-1 expression of macrophages in TME. The study intends to reveal the effect of m6A on the immune microenvironment and macrophage differentiation of cervical cancer, which could be a viable therapeutic target for the treatment of advanced human cancers.\u003c/p\u003e","manuscriptTitle":"Glycolysis induced by METTL14-related m6A methylation is essential for macrophage function and tumor progression in cervical cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-16 16:04:59","doi":"10.21203/rs.3.rs-1949592/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48e5032f-917e-4788-ad14-93e9e23675d8","owner":[],"postedDate":"August 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-18T13:14:29+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-16 16:04:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1949592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1949592","identity":"rs-1949592","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}