SHMT2 arginine methylation by PRMT1 facilitates esophageal cancer progression by enhancing glycolysis and one-carbon metabolism

SHMT2 arginine methylation by PRMT1 facilitates esophageal cancer progression by enhancing glycolysis and one-carbon metabolism | 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 SHMT2 arginine methylation by PRMT1 facilitates esophageal cancer progression by enhancing glycolysis and one-carbon metabolism Zhengshui Xu, Changchun Ye, Yao Cheng, Feng Zhao, Jianzhong Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3291514/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background Protein arginine methyltransferase 1 (PRMT1) is the main enzyme that directly responsible for the production of asymmetric dimethylarginine (ADMA), and upregulation of PRMT1 is observed in a variety of malignancies, including esophageal cancer (ESCA). Dysregulation of arginine methylation caused by PRMT1 overexpression is a driver of poor cancer progression, and the detailed mechanism of modulation is currently unknown. Results The present study confirmed a novel oncogenic mechanism of PRMT1 in ESCA. PRMT1 levels were significantly upregulated in ESCA, and its high expression correlated with TNM stage and poor patient prognosis. We continued to find the mechanisms by which PRMT1 expression was more relevant to ESCA progression. RNA-seq and KEGG enrichment analyses revealed that differentially expressed genes after PRMT1 silencing in ESCA might modulate serine/one-carbon metabolism. Knockdown of PRMT1 in vitro resulted in a significant reduction in ESCA cell growth, and indicators related to serine/one-carbon metabolism and glycolysis, whereas its overexpression showed opposite results. The catalytic activity of PRMT1 was crucial in mediating these biological processes. We found that PRMT1 mediated the ADMA modification of serine hydroxymethyltransferase 2 (SHMT2) at arginine 415 (R415), which activated SHMT2 activity and enhanced serine/one-carbon metabolism and glycolysis. The R415K mutation largely eliminated the arginine methylation of SHMT2 by PRMT1, and weakened PRMT1-induced glycolysis and serine/one-carbon metabolism. Conclusion Our study further confirmed the link between the two proteins, PRMT1 and SHMT2, as well as arginine methylation and glycolysis. The study of deeper molecular mechanisms will reveal a broader role of arginine methylation in the regulation of glycolysis. Esophageal cancer PRMT1 SHMT2 glycolysis one-carbon metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Esophageal cancer (ESCA) has the seventh and sixth incidence and mortality worldwide, respectively [ 1 ]. The incidence of ESCA differs by region and gender, with 70% of ESCA occurring in men and the highest prevalence in East Asia [ 2 ]. ESCA represents a biologically aggressive disease with an unfavorable prognosis. The frequently reported short survival is mainly attributed to the limited benefit of systemic radiotherapy and the increased incidence of margin involvement in surgical specimens [ 3 ]. Notably, surgery combined with chemotherapy remains the preferred treatment for this specific population of patients with advanced ESCA, but more efforts should be devoted to elucidating the specific mechanisms of disease progression and establishing mechanism-based therapeutic strategies for patients with potential clinical consequences (recurrence or metastasis) [ 4 , 5 ]. Therefore, the study of tumor biomarkers is a promising approach to control disease progression. Dysregulation of arginine methylation is a driver of adverse cancer progression. Only the protein arginine methyltransferases (PRMTs) family catalyzes the methylation of protein arginine residues, and this post-transcriptional modification (PTM) functions a critical role in the subsequent biological consequences of their substrates [ 6 ]. Thus, an obvious future goal is to elucidate downstream pathways by identifying novel substrates of PRMTs, which will help us understand the mechanisms of various diseases (including cancer) and establish therapeutic strategies. Importantly, over 90% of the activity of PRMTs in mammalian cells is accounted for by PRMT1, in other words, 85% of asymmetric dimethylarginine (ADMA) in mammals occurs in substrates of PRMT1 [ 7 , 8 ]. Evidence of a direct link between PRMT1 and disease progression has emerged. Several cancer types have shown overexpression or aberrant splicing of PRMT1, so the oncogenic involvement of PRMT1 in specific cancer types is not in question. This “oncogenic” effect of PRMT1 is essentially dependent on the methylation of EZH2 [ 9 ], Twist1 [ 10 ], FOXO1 [ 11 ], EGFR [ 12 ], C/EBPα [ 13 ], H4 [ 14 ] and other proteins. It interrupted E3 ligase TRAF6-mediated ubiquitination of EZH2 by methylating histone methyltransferase EZH2 to form meR342-EZH2, which led to enhanced levels of meR342-EZH2, and high levels of EMT were usually predictive of poor clinical outcomes in breast cancer patients [ 9 ]. The regulation of PRMTs is a complex process because in some cases the enzymatic activity of PRMTs can be altered by other PTMs (e.g., phosphorylation) [ 15 ]. Therefore, the mechanism of arginine methylation mediated by PRMT1 is a concept that must be explored. However, the role of PRMT1 in ESCA is very limitedly understood. Reprogramming and dysregulation of energy metabolism is a prevalent phenomenon in cancer [ 16 ]. Unlike normal cells, rapidly dividing cancer cells rely on aerobic glycolysis to take up and consume more glucose and convert it to lactate in the presence of sufficient oxygen. This process produces a large number of intermediate metabolites that support the rapid growth of cancer cells, and the accumulation of lactic acid contributes to the poor prognosis of human cancers [ 17 ]. Thus, the glycolytic switch in cancer cells greatly limits the development of anti-tumor therapeutic strategies, and useful ways to disrupt glycolysis in tumors need to be found. Several studies have identified the interaction between glycolysis and arginine methylation in cancer cells. For example, arginine methyltransferase 1 (CARM1)-mediated PKM2 methylation in breast cancer became a contributor to aerobic glycolysis in cancer cells [ 18 ]. However, whether PRMT1-mediated protein methylation regulates energy metabolism in ESCA cells remains unknown. Importantly, a branch source intermediate of glycolysis is imported into serine, which is subsequently converted to glycine to provide carbon units for one-carbon metabolism [ 19 ]. Serine hydroxymethyltransferases (SHMTs) are critical proteins that modulate one-carbon metabolism and include cytoplasmic isozymes (SHMT1) and mitochondrial isozymes (SHMT2) [ 20 ]. Available reports indicated that SHMT2 overexpressing cancer cells had strengthened glycolytic capacity under basal conditions [ 21 ]. Qi et al. also pointed that silencing SHMT2 was a key target to suppress the malignant phenotype and glycolysis of tumor cells [ 22 ]. However, few studies have pointed out the aberrant metabolic mechanisms by which SHMT2 modulated cancer glycolysis. Here, we demonstrated that PRMT1 was overexpressed in ESCA tissues, cells and provided direct evidence of its correlation with tumor stage and poor patient prognosis. Furthermore, silencing of PRMT1 restricted glucose uptake, ATP production and one-carbon metabolism (serine to glycine conversion) in cancer cells. Additionally, we found that PRMT1-SHMT2 interaction was a major contributor to the metabolic reprogramming of cancer. PRMT1 deposited ADMA at SHMT2-R415, and methylated SHMT2 accelerated aerobic glycolysis and one-carbon metabolism in tumor cells. Thus, we aimed to elucidate how PRMT1 regulated ESCA progression in a SHMT2-dependent mechanism, demonstrating the possibility of identifying novel targets for cancer therapy. Materials and Methods Clinical tissue samples and cell cultures Patients with ESCA who were diagnosed and treated at our institution were recruited for this study. Fresh tumor tissue and paracancerous non-cancerous tissue specimens were surgically removed from 30 patients who had signed a written informed consent. Thirty patients with ESCA were identified as having clinical stage I-II (n = 15) or stage III-IV (n = 15). Patients receiving any preoperative anticancer therapy or other therapeutic measures affecting the level of gene expression in vivo were excluded. Complete clinical information was available for all cases. The collection and use of human tissues for the study was licensed and authorized by our ethics committee, in accordance with the Declaration of Helsinki . For immunohistochemical (IHC) staining, tissue sections were stained with anti-PRMT1 (Abcam, ab190892, 1:500). Four cell lines from esophageal cancer (KYSE150, hypofractionated squamous cell carcinoma; TE-10, hypofractionated squamous cell carcinoma; TE-1, highly differentiated squamous cell carcinoma; ECA109, highly differentiated squamous cell carcinoma) and human normal esophageal epithelial cells HEEC were purchased from the Shanghai Cell Bank (Shanghai, China) and maintained in a humid incubator at 37°C, 5% CO 2 . All five cell lines were grown in commercially available RPMI-1640 medium (Gibco™, 31870074) supplemented with 10% FBS (Gibco-BRL) and 100 U/ml penicillin/100 U/ml streptomycin (Sigma, USA). Cells were tested for mycoplasma contamination by PCR method at regular intervals (twice/month). Unless otherwise stated, all experiments were performed in medium containing serine and glycine. Immunofluorescence (IF) staining For IF analysis, cultured ESCA cells were spread on slides for growth, and the next day the cells were fixed with 4% paraformaldehyde solution for 10 min at room temperature (Beyotime, P0099). Subsequently, cells were permeabilized in permeabilization solution containing 0.2% Triton X-100 for 10 min at room temperature and then closed in 5% BSA for 1 h. Cells were detected with the following primary antibodies: anti-PRMT1 (Abcam, ab190892, 1:80), anti-SHMT2 (Abcam, ab180786, 1:100). After rinsing three times with PBS, Alexa Fluor-coupled secondary antibodies (Thermo Fisher Scientific) diluted in the blocking solution were then added to the slides and incubated for 1 h at room temperature. Nuclei were restained with DAPI (Sigma) at a concentration of 500 nM. Finally, images were observed using a confocal microscope (Zeiss). Bioinformatics analysis Differences in PRMT1 gene expression between 24 pan-cancer types and normal tissues in the TCGA dataset were analyzed by entering “PRMT1” in the “Gene_DE” module of the Tumor Immune Evaluation Resource (TIMER2.0) database. Download PRMT1 expression data from the TCGA database for 184 ESCA patients and 11 normal samples. The GSE189830 dataset was download from the GEO database, which included mRNA expression profiles of four pairs of ESCA tumor tissues and paired normal tissues. The identification of differentially expressed mRNAs was analyzed in the R software environment through the Limma package. Based on the KEGG database, pathway analysis was applied to screen important pathways of differentially expressed genes, and the enriched pathways were demonstrated using the R packages “tidyr” and “ggplot2”. Plasmid and cell transfection Independent lentiviral vectors encoding shPRMT1 and shSHMT2 were designed and constructed by Genechem (Shanghai, China), respectively. Serum-containing medium was supplemented with polyclathrin (5 µg/mL) and ESCA cells were subsequently infected with purchased lentiviral vectors and co-incubated with puromycin (5 µg/mL) to screen and obtain shPRMT1 or shSHMT2 stably expressed ECA109 and TE-1 cells. Specific small interfering RNAs (siPRMT1 1#, siPRMT1 2#) targeting specific regions of PRMT1 and a negative control siNC were transiently transfected into ESCA cells via Lipofectamine 3000 transfection reagent (Invitrogen, USA). For PRMT1 overexpression, human PRMT1 cDNA was cloned into the pcDNA3.1 vector provided by Genechem (Shanghai, China), labeled as oePRMT1 or PRMT1. HA-tagged SHMT2 was also cloned into the pcDNA3.1 vector, labeled as SHMT2-WT, and the plasmid (wild type) was constructed to generate Mutants of HA-tagged SHMT2, including the following point mutations: only Arg-415 was mutated to Lys (R415K). Flag-SHMT2 was cloned into pCDH-CMV, labeled as Flag-SHMT2-WT, and Flag-SHMT2 R-to-K mutants (R41K, R273K, R358K, R415K) were next developed using a targeted mutagenesis kit (Agilent, 200515). Transfection of specific plasmids was achieved in ECA109 and TE-1 cells with the aid of Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's requirements. MTT, EdU and colony formation assay The viability of ECA109 and TE-1 cells was monitored using MTT assay. 1×10 3 ESCA cells with different treatments were grown in 96-well plates (100 µL) maintained for different times (0h, 24h, 48h, 72h). Each group of cells was set with 5 replicate wells. Next, MTT working solution (Beyotime, 10 µL) was added to the wells at the set time points (0h, 24h, 48h, 72h) and incubation was continued for 4 h at room temperature. Finally, the optical density (OD) values of each well were quantified at 490 nm using an enzyme marker (BioTek Instruments). ECA109 and TE-1 cell proliferation capacity analysis was done by colony formation assay and EdU assay. For colony formation analysis, equal amounts of ESCA cells (ECA109 and TE-1, 600 cells/well) with different treatments were spread in six-well plates. Colonies were clearly visualized by maintaining them in an incubator (37°C, 5% CO 2 ) for 14 days, followed by fixing and staining of target cell colonies with paraformaldehyde fixative (4%, Beyotime, P0099) and crystalline violet staining solution (Beyotime, C0121). The staining solution was discarded, the cells were rinsed with PBS, and representative pictures were captured under a microscope (Olympus Inc.) EdU analysis was completed with the aid of the BeyoClick™ EdU-488 Cell Proliferation Assay Kit (Beyotime, C0071S). Briefly, cells were incubated in 10 µM EdU working solution (obtained by dilution of complete medium) for 2 h, fixed with immunostaining fixative (Beyotime, P0098,1 mL) for 15 min at room temperature, and permeabilized with 1 mL of permeabilization solution (0.3% Triton X-100) for 15 min. EdU was then detected by Click reaction mixture at room temperature. To quantify the proportion of cell proliferation, EdU-positive cells were visualized using HOECHST 33342 staining of cell nuclei and the ratio of EDU-positive cells was subsequently calculated (Image-Pro Plus 6.0, USA). Measurement of glycolytic activity According to the manufacturer's instructions, glucose uptake, lactate production, and ATP levels were monitored by the Glucose Uptake Assay Kit (ab136955, Abcam, USA), the Lactate Assay Analysis Kit (K607-100, Biovision, USA), and the Luminescent ATP Assay Kit (ab113849, Abcam, USA), respectively. Western blotting and Co-IP Analysis Experiments were performed according to standard western blotting protocols. Briefly, cultured ESCA cell or tumor tissue protein lysates were obtained from RIPA lysis buffer (P0013E, Beyotime, China). After quantification of the proteins, aliquots of protein samples were loaded and separated on SDS-PAGE and then transferred to PVDF membranes. After incubation with the corresponding antibodies, blots were visualized using the ECL chemiluminescence kit (Millipore). Immunoprecipitation assays were performed according to the previously described steps [ 23 ]. Briefly, collected ECA109 and TE-1 cells were lysed (on ice, 30 min) in RIPA lysis buffer (P0013E, Beyotime, China) as indicated, requiring the addition of a protease inhibitor (PMSF, Beyotime) to this lysate. After removal of cell debris by centrifugation (13,000g, 15min, 4°C), protein concentration in cell lysates was determined by the BCA method. For IP of HA-tagged/Flag-tagged proteins, lysates were incubated with Anti-HA magnetic beads (P2185S, Beyotime) or Flag microspheres (MilliporeSigma, A2220) overnight at 4°C. For IP of endogenous PRMT1 and SHMT2 proteins, clarified lysates were co-incubated (4°C) with primary antibodies to the indicated proteins (anti-PRMT1, Abcam, ab190892; anti-SHMT2, Abcam, ab240606) or isotype-matched IgG. Protein A/G agarose beads (Abcam, ab286842) were then added and incubated overnight (4°C). Beads were collected to release immunoprecipitated protein samples and used for subsequent western blotting analysis. Amino acid and enzyme activity analysis The concentration of serine in ESCA cell lysates (1 × 10 6 ) was measured via the DL-Serine Assay Kit (ab241027, Abcam). Cell supernatant samples were pretreated and deproteinated with the sample cleanup mixture provided in the kit according to the manufacturer's protocol, and then assayed. Glycine in ESCA cell lysates was quantified using a fluorometric assay (Glycine Assay Kit, Biovision). SHMT2 activity analysis was done by Serine Hydroxymethyltransferase ELISA Assay Kit (JianglaiBio, Shanghai, China). RNA isolation, qPCR and RNA sequencing Total RNA from ESCA tissues, paired normal tissue samples and ESCA cell lines was isolated by TRIzol (Invitrogen, USA). The purity of the total RNA obtained was determined by a NanoDrop 2000C ultra-micro spectrophotometer (Thermo Scientific). RNA was synthesized into cDNA using a reverse transcription kit (PrimeScript RT Master Mix, Takara). SYBR Premix Ex Taq II kit (Takara) and an ABI Prism® 7500 fluorescent quantitative PCR system (Applied Biosystems, USA) were performed to quantify the PCR amplification of PRMT1, SHMT2 and GAPDH. The relative transcript levels of the target RNAs were calculated using the 2 −ΔΔCt method, and their expression levels were normalized to the expression of GAPDH. The primers used in this study were as follows. PRMT1, F, 5'-CTTTGACTCCTACGCACACTT-3' and R, 5'-GTGCCGGTTATGAAACATGGA-3'. SHMT2, F, 5'-TGATTCCCTCGCCTTTCAAGC-3' and R, 5'-TTTCCGGTAGAAGATGAGCCC-3'. '-TGATTCCCTCGCCTTTCAAGC-3' and R, 5'-TTTCCGGTAGAAGATGAGCCC-3'. RNA-seq was completed in three biological replicates. We compared the differential gene expression between shNC and shPRMT1 cohorts. A cDNA library for each RNA sample was constructed using the RNA Library Preparation Kit (Illumina, USA). RNA sequencing was then performed with the Illumina NovaSeq 6000 sequencing platform and analysis was executed using the OmicStudio tool. The criteria for differential genes were set at a P value 1.5. In vivo research Twenty 6–8 week female BALB/c nude mice (weight 18–20 g, SLAC Laboratory Animals Ltd., Shanghai, China) were employed for the in vivo xenograft tumor assay. The use of animals and experimental procedures were in strict accordance with institutional guidelines and were approved by the Second Affiliated Hospital of Xi'an Jiaotong University Ethics Committee. For in vivo tumor formation, ECA109 cells (5 × 10 6 ) transfected with Vector + SHMT2-WT (Vector + WT group), PRMT1 + SHMT2-WT (PRMT1 + WT group), Vector + SHMT2-R415K (Vector + R415K group) and PRMT1 + SHMT2-R415K (PRMT1 + R415K group) were injected subcutaneously into the right side of nude mice (n = 5). All mice were monitored, and the tumor volume and size were estimated every four days starting from the fourth day after inoculation, and were calculated as 0.5 × (length × width 2 ). After four weeks, all nude mice were executed and tumor tissues were immediately excised for photographing and weighing. Additional, acquired tumor tissues were also executed with hematoxylin-eosin (H&E) staining. Briefly, 5 µm paraffin sections of tissue were prepared and sections were processed using the H&E staining kit (C0105S, Beyotime). Sections were captured under the microscope after blocker sealing. Statistical analysis GraphPad Prism 8.0 software was employed in analysis of the data in this study. All experiments were performed at least three times independently, and the data obtained were reported as mean ± SD. Statistical differences between two consecutive data sets were compared using Student's t -test. Kaplan-Meier curves were plotted to demonstrate the correlation between PRMT1 expression and patient survival. P -values less than 0.05 were considered statistically significant. Results PRMT1 overexpression is associated with poor clinical outcomes in patients with esophageal cancer Abnormally high expression of the PRMT1 gene has been seen in multiple cancer types. Existing studies on PRMT1 expression in cancer have applied inconsistent study protocols, and most have focused on only a single cancer type. In the current study, we comprehensively analyzed the differential expression of PRMT1 in 24 cancers (BLCA、BRCA、CESC、COAD、ESCA、GBM、HNSC-HPV+、HNSC、KICH、KIRC、KIRP、LIHC、LUAD、LUSC、PAAD、PCPG、PRAD、READ、SKCM、STAD、THCA、UCEC) using the TIMER2.0 database. We could observe from Fig. 1 A that PRMT1 was significantly differentially expressed ( P < 0.05) in all 17 cancers including ESCA, among which it was significantly elevated in 16 cancer types (BLCA, BRCA, CHOL, COAD, ESCA, GBM, HNSC-HPV+, HNSC, KIRC, LIHC, LUAD, LUSC READ, STAD, THCA, UCEC) and significantly low expressed in only one cancer type (KICH). To perform further validation of differentially expressed genes (DEGs) in human ESCA, we performed analysis on the obtained publicly available GSE189830 dataset to identify the important drivers involved in ESCA. The volcano map revealed multiple DEGs between ESCA (n = 4) and normal samples (n = 4) (Fig. 1 C). Subsequently, 100 differentially expressed genes, including 50 up-regulated and 50 down-regulated, were visualized in the heat map (Fig. 1 B). Further analysis of PRMT1 expression in the ESCA cohort based on sample types in the TCGA database showed that PRMT1 levels were increased in tissues from patients with esophageal cancer (n = 184) compared to normal samples (n = 11) (Fig. 1 D). Thereafter, PRMT1 expression was examined in 30 pairs of collected clinical specimens, and the results demonstrated that the level of PRMT1 mRNA was higher in all ESCA tissues than in paired non-tumor tissue samples (Fig. 1 E). We also analyzed the correlation between PRMT1 expression and tumor stage in 30 ESCA tissue specimens, and the results presented that higher PRMT1 expression was more likely to be found in advanced ESCA samples (clinical stage III-IV) (Fig. 1 F). In addition, patients with lower PMRT1 levels had significantly higher survival rates (Fig. 1 G). In addition, western blotting (containing 5 pairs of well-annotated patients) and IHC analyses demonstrated consistently elevated PRMT1 expression in ESCA tissue samples (Fig. 1 H and 1 I). We also determined PMRT1 expression in acquired ESCA cells (KYSE150, TE-10, TE-1, ECA109). Enhanced PMRT1 expression was measured in all ESCA cells relative to HEEC cells (Fig. 1 J), and the cells with the most upregulated expression (TE-1, ECA109) were chosen for subsequent experiments. The above data supported the correlation between high PRMT1 expression and poor prognosis of esophageal cancer. Silencing of PRMT1 hinders ESCA cell growth and glycolysis Next, we planned to determine the PRMT1-mediated oncogenic properties in esophageal cancer. The two cell lines with the highest PRMT1 expression (TE-1, ECA109) were selected to generate PRMT1 knockdown cells to carry out the next step of this study (Fig. 2 A). Then, we found that the presence of siPRMT1 effectively attenuated the viability of TE-1 and ECA109 cells (Fig. 2 B). Similarly, unlike siNC treatment, silencing of PRMT1 reduced the number of colony formation in ESCA cells (Fig. 2 C). EdU assay showed that EdU incorporation was reduced in TE-1 and ECA109 cells after transfection with siPRMT1, and the proportion of EdU-positive cells decreased dramatically (Fig. 2 D). The enhanced glycolysis of tumor cells conferred a proliferative advantage to achieve tumor cell overgrowth. Furthermore, the activation of glycolytic pathway in tumor cells was characterized by more glycine production and ATP production. In TE-1, ECA109 cells, the effect of PRMT1 knockdown on glycolysis was analyzed by assessing the correlation between PRMT1 expression and glycine production and ATP levels. As shown in Fig. 2 E and 2 F, cellular glycine production and ATP levels were reduced by almost 60% after PRMT1 silencing. These data supported the role of PRMT1 as a tumor promoter and manifested a direct relationship between PRMT1 levels and cellular glycolysis. PRMT1 mediates asymmetric methylation of SHMT2 in vitro We conducted RNA sequencing (RNA-seq) to investigate the role of PRMT1 in ESCA cells, and identified 30 differentially expressed genes, including 15 down-regulated and 15 up-regulated genes, in PRMT1-depleted ESCA cells (Fig. 3 A). Besides, KEGG analysis revealed the pathways of these down-regulated genes, the results clearly showed that these pathways included one carbon pool by folate, Glyoxylate and dicarboxylate metabolism and Glycine, serine and threonine metabolism (Fig. 3 B). Then, we found that PRMT1 silencing impeded glucose uptake (Fig. 3 C), weakened ATP levels and glycine production, but increased serine production in both ESCA cells (Fig. 3 D- 4 F). These might be due to the PRMT1 pathway controlling serine and glycine levels through glycolysis and serine/glycine biosynthesis. Considering that protein-protein interactions underlied a broad range of biological processes, to identify potential binding partners of PRMT1, we constructed a protein interaction network of PRMT1 in the STRING database (Fig. 3 G) and identified six important proteins (SHMT2, SHMT1, PSAT1, PHGDH, GLDC, DHFR). Next, we evaluated the interaction between PRMT1 and the above six proteins after immunoprecipitation in ESCA cells. Figure 3 H demonstrated that only SHMT2 formd a complex with PRMT1 in ESCA cells. Secondly, double immunofluorescence staining of SHMT2 and PRMT1 similarly demonstrated that SHMT2 and PRMT1 might form a protein complex and that the complex was localized in the nucleus (Fig. 3 I). In conclusion, the interaction of SHMT2 and PRMT1 might have a potential functional role in ESCA. Cancer cells need to accumulate the building blocks of new cellular components to sustain their own growth and proliferation. Interestingly, amino acid metabolism involving serine and glycine compensates for this requirement. The reversible conversion of serine to glycine is catalyzed by serine hydroxymethyltransferase 2 (SHMT2), which subsequently assumes a crucial role in the one-carbon pathway. Importantly, our data indicated that inhibition of PRMT1 activity appeared to have a negative effect on intracellular glycine production. Therefore, we next asked whether PRMT1 affected SHMT2 function through direct physical binding and subsequently performed immunoprecipitation experiments (Co-IP) using anti-PRMT1 and anti-SHMT2 in TE-1 and ECA109 cells, respectively. As determined by Co-IP, an endogenous interaction between SHMT2 and PRMT1 was reverified in TE-1 and ECA109 cells (Fig. 4 A and 4 B). Previous reports confirmed that PRMT1 catalyzed the appearance of its substrate asymmetric dimethylarginine (ADMA). To explore whether SHMT2 undergoes PRMT1-mediated arginine methylation, we extracted SHMT2 from two ESCA cells and analyzed its methylation using an ADMA antibody. As shown in Fig. 4 A and 4 B, the presence of SHMT2 ADMA signal was detected in both cells, indicating that SHMT2 was methylated by PRMT1. Subsequently, two siPRMT1 (siPRMT1 1# and siPRMT1 2#) were designed to silence the expression of PRMT1 in TE-1 and ECA109 cells. We observed that effective siRNA-mediated silencing of PRMT1 suppressed endogenous SHMT2 ADMA levels. The introduction of the PRMT1 overexpression vector produced the opposite result, which obviously increased the SHMT2 ADMA signal in cell lysates (Fig. 4 C and 4 D). These data supported the asymmetric methylation of endogenous SHMT2 by PRMT1. PRMT1-mediated methylation of SHMT2 protein at arginine 415 is required for its oncogenic functions We searched for four arginine residues (R41, R273, R358, R415) in the SHMT2 sequence that could be methylated by PRMT1, and then we tested whether they were the major arginine sites for PRMT1-mediated methylation (Fig. 5 A). A series of R (arginine)-to-K (lysine) SHMT2 mutants and PRMT1 were co-expressed in HEK293T cells and then analyzed for ADMA changes. No changes in ADMA levels were found in the R41K, R273K, and R358K mutants, but a decrease in ADMA levels was observed in the R415K mutant (Fig. 5 B). In addition, SHMT2 activity was also declined after the R415 mutation. R415 in SHMT2 was highly conserved across species from zebrafish to human (Fig. 5 D), suggesting a potentially critical role for R415 in SHMT2 function. To further analyze the potential contribution of R415 to the asymmetric methylation of SHMT2 protein, plasmids expressing SHMT2-WT and SHMT2-R415K as well as PRMT1 overexpression vector and control vector were transfected into ESCA cells, respectively, to perform Co-IP-SHMT2 experiments and finally obtain immunoprecipitation complexes. Notably, to eliminate the detrimental effect of SHMT2 expression in TE-1 and ECA109 cells, we pre-silenced SHMT2 with a specific shRNA targeting SHMT2. In vitro methylation assays showed that the R415K mutation reduced the interaction of SHMT2 with PRMT1 as well as weakened ADMA signaling (Fig. 5 C), which implied that SHMT2-WT was more susceptible to PRMT1 targeting and asymmetric dimethylation than the SHMT2-R415K mutant. Next, we determined the biological function of PRMT1-mediated SHMT2 in vitro . We found that silencing of PRMT1 induced attenuation of endogenous SHMT2 activity, while enriched PRMT1 expression produced the opposite effect (Fig. 5 E). Furthermore, among TE-1 and ECA109 cells with SHMT2 knockdown, ESCA cells bearing only SHMT2-WT exhibited relatively high SHMT2 activity relative to cells bearing only SHMT2-R415K. Whereas overexpression of PRMT1 caused an increase in endogenous wild-type SHMT2 protein activity, R415K mutation attenuated this effect (Fig. 5 F). The above data suggested that PRMT1-mediated methylation of SHMT2-R415 effectively raised SHMT2 activity in tumor cells. PRMT1-mediated R415 methylation did not have any effect on SHMT2 expression (Fig. 6 A), but we explored its effect on the oncogenic function of SHMT2. The results of MTT, colony formation, and EdU experiments showed that ESCA cells expressing hypomethylated SHMT2 (R415K) had attenuated growth as compared to SHMT2-WT cells, and that PRMT1 overexpression vector treatment effectively alleviated this effect (Fig. 6 B- 6 D). Published reports indicated that SHMT2-enriched cells possessed higher glycolytic capacity under basal conditions [ 21 ]. Based on these results, we hypothesized that the increase of PRMT1-enhanced SHMT2 activity effectively promoted cancer cell glycolysis. As shown in Fig. 6 E and 6 F, similar to the trend of SHMT2 activity, higher glucose uptake capacity and endogenous ATP levels were observed in ESCA cells with only SHMT2-WT than in target cells with only SHMT2-R415K. PRMT1 overexpression significantly induced glucose uptake and endogenous ATP production in ESCA cells with SHMT2-WT, effectively exacerbating the glycolytic capacity. This finding emphasized that the enhanced glycolytic capacity of PRMT1-overexpressed cells was dependent on their dominant arginine methylation in SHMT2. A branch of glycolysis inputs serine, which can be converted to glycine with the help of SHMT2, providing carbon units for one-carbon metabolism. Considering the abnormal alteration of SHMT2 activity, we wanted to further clarify the relationship between SHMT2-R415 methylation and serine or glycine formation. In shSHMT2-treated cells, the PRMT1 + SHMT2-WT group showed a marked defect in serine production and an increase in glycine production relative to the Vector + SHMT2-WT group. Consistently, induction of PRMT1 overexpression resulted in increased serine production and decreased glycine production in SHMT2-R415K cells (Fig. 6 H). Mechanistically, PRMT1 overexpression increased SHMT2-R415 asymmetric methylation and promoted elevated cellular glycolysis, which in turn maintained serine production as well as conversion of serine to glycine. PRMT1 might exert as a vital molecular switch to modulate cell biological behavior changes by detecting SHMT2 arginine methylation. Methylation of SHMT2 at R415 enhances in vivo tumorigenicity of ESCA cells We sought to elucidate whether PRMT1-mediated methylation of SHMT2 at the R415 locus affected esophageal cancer cell growth and proliferation in vivo with the help of a subcutaneous xenograft tumor model. We subsequently injected Vector + SHMT2-WT cells (Vector + WT group), PRMT1 + SHMT2-WT cells (PRMT1 + WT group), Vector + SHMT2-R415K cells (Vector + R415K group) and PRMT1 + SHMT2-R415K cells (PRMT1 + R415K group) subcutaneously into nude mice to drive tumorigenesis. 28 days later, it was clearly observed that tumors formed by the PRMT1 + WT group were significantly larger in volume and weight than those from the Vector + WT group or the PRMT1 + R415K group (Fig. 7 A- 7 C). In addition, the size and weight of tumors were smaller in the Vector + R415K group than in the Vector + WT group. At the same endpoints, it was observed under light microscopy that the tumor cancer cells in the PRMT1 + WT group exhibited a high degree of anisotropy and dysregulation of nucleoplasmic ratios, and that the tumor cells in the Vector + R415K group tended to have a normal morphology with tightly packed nuclei (Fig. 7 D). These findings suggested that PRMT1-mediated methylation of SHMT2 at the R415 locus was responsible for accelerated tumor growth in vivo . Cancer cells are highly dependent on serine/one-carbon metabolism for proliferation, so we verified in vivo whether PRMT1 affected one-carbon metabolism. We found that the decreased serine/glycine ratio in the PRMT1 + WT group could be partially alleviated by R415K treatment (Fig. 7 E). Importantly, SHMT2 activity was also highest in the PRMT1 + WT group (Fig. 7 F). These suggested that PRMT1-driven SHMT2 arginine methylation promoted serine/one-carbon metabolism. Discussion In the present study, we investigated the role of PRMT1 in regulating aerobic glycolysis in ESCA cells and preliminarily explored the potential molecular mechanisms of its involvement. We summarized the main findings in this study: (1) PRMT1 was overexpressed in ESCA, enrichment of PRMT1 was more likely to be observed in patients with advanced (III + IV) ESCA, hinting that high PRMT1 levels predicted a poorer prognosis for ESCA patients. (2) The dependence of ESCA on PRMT1 was observed in vitro in cell lines. Specifically, PRMT1 silencing depleted cell growth and survival, and inhibited aerobic glycolysis and one-carbon metabolism in cancer cells. (3) PRMT1 dominated a novel PTM. We demonstrated that PRMT1 could deposit ADMA at SHMT2-R315, and this methylation enhanced SHMT2 protein activity. (4) Excessive accumulation of methylation signals of SHMT2 promoted the levels of glycolysis and indicators related to one-carbon metabolism in ESCA cells. In conclusion, our data revealed an alternative mechanism by which PRMT1 mediated aerobic glycolysis in ESCA, which drove esophageal carcinogenesis by methylating SHMT2. Our results are summarized in a schematic model, as in Fig. 7 G. Overexpression of PRMT1 is frequently observed in human cancers, e.g., lung, breast, and colorectal cancers, and promotes cancer progression by regulating important cellular processes such as transcription and cellular metabolism [ 24 – 26 ]. And its role in these processes may depend on the function of its substrates. A recent study found that enforced PRMT1 was applied to catalyze histone H4R3 methylation in the context of esophageal cancer, mediating the activation of oncogenic transcription through methylated H4R3 [ 14 ]. In addition, PRMT1-dependent methylation also impeded the tumor suppressive function of certain substrates. The methylation of C/EBPα caused by PRMT1 affected its interaction with HDAC3 and also enhanced cell cycle protein D1 expression and subsequent tumor growth [ 27 ]. Thus, PRMT1 could regulate certain biological functions of the disease by methylating certain specific substrates. Here, we first applied RNA-seq transcriptome analysis to probe for differentially expressed genes altered by PRMT1 defects, some of which might be involved in the regulation of intracellular glycolysis. Next, according to KEGG enrichment analysis, knockdown of PRMT1 had an obvious effect on serine/glycine metabolism as well as other important signaling pathways and metabolic processes. Our in vitro functional assays consistently demonstrated that PRMT1 knockdown reduced the levels of glycolysis and one-carbon metabolism-related indicators, including decreased ATP levels and reduced glycine synthesis. Besides, PRMT1-deficient cells were less hungry for glucose. Considering that SHMT2 is a nexus that catalyzes serine metabolism, releases one-carbon units and glycine, and is an important source of folate synthesis, we observed the target binding of PRMT1 to SHMT2 using Co-IP analysis. SHMT2 was found to be asymmetrically methylated at residue R415, and the methylation signal of SHMT2 could be attenuated by PRMT1 silencing. Indeed, the relationship between arginine methylation and metabolism has not been sufficiently studied. For example, another member of the PRMTs family, PRMT6, methylated CRAF in hepatocellular carcinoma (HCC), driving aerobic glycolysis in HCC cells, which in turn accelerated tumor formation [ 28 ]. Consistent with previous studies [ 29 ], our study discovered that SHMT2 was methylated by PRMT1, which enhanced its enzymatic activity and lead to aggravated glycolytic capacity in ESCA cells. We further confirmed the link between these two proteins, as well as arginine methylation and glycolysis. The study of deeper molecular mechanisms will reveal a broader role of arginine methylation in the regulation of glycolysis. SHMT2 is the key enzyme responsible for serine metabolism and reversibly catalyzes the conversion of the substrate serine to glycine [ 30 ], contributing an activated carbon unit to the de novo synthesis of purines (Fig. 7 E). Adequate supply of glycine is a vital reason for the rapid proliferation of cancer cells [ 31 ], and thus SHMT2 often functions as an oncogenic factor in various types of cancers, such as lung [ 32 ], liver [ 33 ], breast [ 34 ], glioblastoma multiforme [ 35 ], bladder [ 36 ] and colorectal [ 37 ] cancers. Some specific mechanisms by which SHMT2 exerted its oncogenic effects have been reported. For example, in human prostate cancer and glioblastoma multiforme, raised SHMT2 level triggered an altered metabolic state and conferred a higher survival advantage to cells in the tumor region [ 38 ]. Overexpression of SHMT2, a vital enzyme mediating serine/one-carbon metabolism, in cancer interfered with the de novo synthesis of glycine and might retard the abnormal biological behaviors of ESCA. In our study, we reported for the first time that PRMT1-mediated asymmetric dimethylation of SHMT2-R415 had a significant effect on SHMT2 protein activity. Furthermore, we found that the R415K mutation largely eliminated the arginine methylation of SHMT2 by PRMT1, indicating that the R415 residue was essential for the oncogenic function of SHMT2 in ESCA. Overall, our study confirmed that glycolysis in ESCA represented a weak point and that diminished SHMT2 activity triggered by PRMT1 silencing had a strong negative impact on tumorigenesis. The above findings raised several new questions that needed to be further explored. Arginine methylation regulated signal transduction cascades, and more efforts were needed to elucidate the detailed molecular mechanisms underlying the effects of PRMT1-dominated methylation on SHMT2 function. Inhibition of PRMT1 and SHMT2 methylation was lethal to cancer cells. Although inhibition of SHMT2 methylation appeared to reduce serine/one-carbon metabolism in ESCA cell lines, resulting in serine accumulation and glycine depletion, this finding revealed a link between serine metabolism and arginine methylation of proteins that needed to be highlighted in future studies. Conclusion In this report, we have characterized the PRMT1-SHMT2 axis in stimulating esophageal cancer development, suggesting that asymmetric dimethylation of SHMT2 at R415 played an essential role in this process. As shown in Fig. 7 E, increased levels of PRMT1 expression in esophageal cancer cells promoted PRMT1-mediated SHMT2-R415 methylation, which enhanced the protein activity of SHMT2. The accumulation of SHMT2 activity promoted the reversible conversion of serine to glycine, contributing an activated carbon unit to the de novo synthesis of purines and ultimately accelerating esophageal cancer tumor growth. These data emphasized the importance of PRMT1-mediated SHMT2 methylation in facilitating esophageal cancer development and growth, and suggested the clinical value of PRMT1 in the diagnosis and treatment of esophageal cancer. Declarations Acknowledgements Not applicable. Author Contributions Zhe Qiao designed the study and draft the manuscript. Yu Li collected the data and processed statistical data. Yao Cheng analyzed and interpreted the data. Shiyuan Liu partly contributed to the experiment. Shaomin Li reviewed, and revised the paper. All authors reviewed the results and approved the final version of the manuscript. Funding Not applicable. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval All participants were provided with written informed consent at the time of recruitment. This study was approved by the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University, and all experiments involving human tissue specimens comply with the Declaration of Helsinki . All animal studies were strictly performed according to protocol approved by the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University. Animal studies were performed in compliance with the ARRIVE guidelines. Consent for publication Not applicable. Conflict of interests The authors declare that they have no conflicts of interest to report regarding the present study. References Cao L, Wang X, Zhu G, Li S, Wang H, Wu J, et al. Traditional Chinese Medicine Therapy for Esophageal Cancer: A Literature Review. 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Yin XK, Wang YL, Wang F, Feng WX, Bai SM, Zhao WW, et al. PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer. Oncogene. 2021;40(7):1375-1389. Malbeteau L, Jacquemetton J, Languilaire C, Corbo L, Le Romancer M, Poulard C. PRMT1, a Key Modulator of Unliganded Progesterone Receptor Signaling in Breast Cancer. Int J Mol Sci. 2022;23(17). Liao HW, Hsu JM, Xia W, Wang HL, Wang YN, Chang WC, et al. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response. J Clin Invest. 2015;125(12):4529-4543. Li M, An W, Xu L, Lin Y, Su L, Liu X. The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLAR(L) in human lung cancer cells. J Exp Clin Cancer Res. 2019;38(1):64. Li HL, Dong LL, Jin MJ, Li QY, Wang X, Jia MQ, et al. A Review of the Regulatory Mechanisms of N-Myc on Cell Cycle. Molecules. 2023;28(3). Chan LH, Zhou L, Ng KY, Wong TL, Lee TK, Sharma R, et al. PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation. Cell Rep. 2018;25(3):690-701.e698. Shen W, Gao C, Cueto R, Liu L, Fu H, Shao Y, et al. Homocysteine-methionine cycle is a metabolic sensor system controlling methylation-regulated pathological signaling. Redox Biol. 2020;28:101322. Wei Z, Song J, Wang G, Cui X, Zheng J, Tang Y, et al. Deacetylation of serine hydroxymethyl-transferase 2 by SIRT3 promotes colorectal carcinogenesis. Nat Commun. 2018;9(1):4468. Guo K, Cao Y, Li Z, Zhou X, Ding R, Chen K, et al. Glycine metabolomic changes induced by anticancer agents in A549 cells. Amino Acids. 2020;52(5):793-809. Kinslow CJ, Chaudhary KR, Ye LF, Upadhyayula PS, Young L, Sun RC, et al. Functional Genomics Screen Identifies SHMT2 as Regulator of Oxidative Stress and Treatment Resistance in Lung Adenocarcinoma. 2020;108. Wang M, Yuan F, Bai H, Zhang J, Wu H, Zheng K, et al. SHMT2 Promotes Liver Regeneration Through Glycine-activated Akt/mTOR Pathway. Transplantation. 2019;103(7):e188-e197. Xie SY, Shi DB, Ouyang Y, Lin F, Chen XY, Jiang TC, et al. SHMT2 promotes tumor growth through VEGF and MAPK signaling pathway in breast cancer. Am J Cancer Res. 2022;12(7):3405-3421. Tanaka K, Sasayama T, Nagashima H, Irino Y, Takahashi M, Izumi Y, et al. Glioma cells require one-carbon metabolism to survive glutamine starvation. Acta Neuropathol Commun. 2021;9(1):16. Zhang P, Yang Q. Overexpression of SHMT2 Predicts a Poor Prognosis and Promotes Tumor Cell Growth in Bladder Cancer. Front Genet. 2021;12:682856. Liu C, Wang L, Liu X, Tan Y, Tao L, Xiao Y, et al. Cytoplasmic SHMT2 drives the progression and metastasis of colorectal cancer by inhibiting β-catenin degradation. Theranostics. 2021;11(6):2966-2986. Marrocco I, Altieri F, Rubini E, Paglia G, Chichiarelli S, Giamogante F, et al. Shmt2: A Stat3 Signaling New Player in Prostate Cancer Energy Metabolism. Cells. 2019;8(9). Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3291514","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":388287490,"identity":"3625baef-aad4-4397-aae6-d250fbe2dc92","order_by":0,"name":"Zhengshui Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhengshui","middleName":"","lastName":"Xu","suffix":""},{"id":388287491,"identity":"d6396653-e23a-4dbb-b1a0-01e758ca1d49","order_by":1,"name":"Changchun Ye","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Changchun","middleName":"","lastName":"Ye","suffix":""},{"id":388287492,"identity":"33232682-2510-41e8-82bc-16485948a92f","order_by":2,"name":"Yao Cheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Cheng","suffix":""},{"id":388287493,"identity":"6b7f693f-8a06-41e6-a8dc-75aea565b644","order_by":3,"name":"Feng Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Zhao","suffix":""},{"id":388287494,"identity":"22edd348-4eef-4a2a-b725-5371a6c044af","order_by":4,"name":"Jianzhong Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jianzhong","middleName":"","lastName":"Li","suffix":""},{"id":388287495,"identity":"ccbb5081-62d9-4d09-b7a7-c2b747407348","order_by":5,"name":"Jiantao Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiantao","middleName":"","lastName":"Jiang","suffix":""},{"id":388287496,"identity":"fba033cc-dff7-4b59-b6ac-ebf0e16b1343","order_by":6,"name":"Shiyuan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACPmYGBmYGAxCT+QCDBFgsAb8WNoQWtgQGiQRitICMhzB5DKCqCWlh5zH+XFBwOLF/ds83Ccsfhxn42XMMGH7uwOcwHjPpGQaHE2fcObvZQCLhMINkzxsDxt4z+LUw8wC1NNzI3fgApMXgRo4BM2MbXi3Gn0Fa5t/IeXAApMWeCC0G0iAtG27kMEJskSCoha0MqCXdeOONNGMDibR0HokzzwoO9uLRws9/ePNnnj/WsvNuJD+TlrCxluNvT9744CceLVDQDCaZgbHPA2IcIKiBgaEOTDJ+IELpKBgFo2AUjDwAABcUSIEL32Y+AAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Shiyuan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2023-08-24 07:19:00","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3291514/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-3291514/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72841044,"identity":"84c1e880-ca53-43f5-8569-50c6e9c851d8","added_by":"auto","created_at":"2025-01-02 18:17:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1938332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT1 is present in a high expression pattern in a variety of malignancies. \u003c/strong\u003e(A) Box plots were applied to represent the differential expression of PRMT1 in the 24 cancer types. The blue and red box lines indicated normal and tumor tissues, respectively. (B) Heat map from the GEO database (GSE189830) showed 50 up-regulated and 50 down-regulated genes differentially expressed between ESCA samples and normal subjects. (C) Volcano map from the GEO database (GSE189830) demonstrated genes up- and down-regulated in ESCA. (D) TCGA database presented high expression of PRMT1 in ESCA. (E) Expression of PRMT1 in 30 collected clinical ESCA tumor tissues (Tumor) and paired normal tissues (Normal) was examined by RT-qPCR assay. (F) Higher PRMT1 levels were seen in stage III-IV ESCA patients relative to stage I-II ESCA patients. (G) Kaplan-Meier analysis revealed the correlation between PRMT1 levels and overall survival in ESCA patients. (H and I) Western blotting and IHC were used to verify PRMAT1 levels in ESCA tissues and paired tissues, respectively. (J) Differences in PRMT1 expression levels in human normal esophageal epithelial cells HEEC and representative ESCA cells (KYSE150, TE-10, TE-1, ECA109). **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/12b652bf183db79e237926ad.jpg"},{"id":72841887,"identity":"29238cd9-0fde-4bb6-87a2-eb08378b3fda","added_by":"auto","created_at":"2025-01-02 18:25:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1748061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eESCA cell proliferation and glycolysis are limited by decreased levels of PRMT1. \u003c/strong\u003e(A) RT-qPCR exhibited the levels of PRMT1 in ECA109 and TE-1 cells after siNC or siPRMT1 transfection. (B) CCK-8 assay was employed to obtain data on the change in viability of ECA109 (right) and TE-1 (left) cells with PRMT1 knockdown. (C) Colony formation assay was conducted to obtain changes in the proliferative capacity of PRMT1 knockdown cells. The number of ECA109 and TE-1 cell clones was assessed by the captured microscopic images. (D) EdU experiments illustrated the inhibition of PRMT1-silenced ECA109 and TE-1 cell growth. EdU-positive ratios of ECA109 and TE-1 cells were calculated from the obtained images. (E) Glycine production was significantly decreased in PRMT1 knockdown ECA109 and TE-1 cells. (F) ATP levels were measured in PRMT1 knockdown ECA109 and TE-1 cells. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/121ad1225a16e7a188c1c449.jpg"},{"id":72841042,"identity":"7a63711a-3b95-4e1b-aac5-1180f1e5722a","added_by":"auto","created_at":"2025-01-02 18:17:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4557317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of PRMT1 affects one-carbon metabolism (glycine production) in cancer cells.\u003c/strong\u003e (A) Heatmap revealed the top 15 up-regulated and top 15 down-regulated genes in ECA109 cells expressing shNC (n=3) or shPRMT1 (n=3). (B) KEGG analysis of the enrichment pathway in ESCA cells with PRMT1 knockdown. (C-F) Changes in glucose uptake, ATP levels, serine production, and glycine production in cells expressing siNC or siPRMT1. (G) Interaction network of PRMT1 constructed by STRING. (H) Co-IP was performed to verify the interaction of PRMT1 with SHMT2. (I) Confocal pictures of ESCA cells co-stained for PRMT1 and SHMT2. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/65775b8ccd8737e24c0f58eb.jpg"},{"id":72842346,"identity":"03899748-7941-45b9-a26c-75d01ceeb010","added_by":"auto","created_at":"2025-01-02 18:33:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2628665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT1 interacts and methylates with SHMT2.\u003c/strong\u003e (A-D) Immunoprecipitation of PRMT1 and SHMT2 proteins from TE-1 and ECA109 cells, and western blot analysis was carried out to detect the interaction between the two, as well as ADMA signaling. (E, F) Immunoprecipitation of SHMT2 protein from TE-1 and ECA109 cells expressing siNC, siPRMT1 1#, siPRMT1 2#, oeNC and oePRMT1, and westernblot analysis was employed to detect their interaction with PRMT1, as well as ADMA signaling.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/1043cd525c1a8963848a54e6.jpg"},{"id":72841889,"identity":"63e028a8-4681-46bc-946a-b49028eb8a56","added_by":"auto","created_at":"2025-01-02 18:25:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1749886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT1 mediates ADMA modification of SHMT2 at R415.\u003c/strong\u003e (A) Search for arginine residues in the SHMT2 sequence that might be methylated by PRMT1. (B) The R415K mutation eliminated PRMT1-mediated ADMA modification of SHMT2 and attenuated SHMT2 protein activity. (C) WT and R415K mutant SHMT2 proteins were immunoprecipitated with HA beads from PRMT1 overexpressing and SHMT2 knockdown TE-1 cells or control cells, followed by western blotting analysis to examine ADMA levels. (D) Evolutionary conservation of SHMT2 R415 residues among different species. (E) The SHMT2 activity was measured in TE-1 and ECA109 cells as shown. (F) Changes of SHMT2 activity in SHMT2-depleted cells when WT-SHMT2 and R415K-SHMT2 mutations were transfected into PRMT1 overexpressing and control cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/43e3028cd5dfc3f002723350.jpg"},{"id":72842857,"identity":"7460be3b-9e28-44cd-a7b6-daf4b0e9bc9e","added_by":"auto","created_at":"2025-01-02 18:41:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3274125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHMT2 methylation is important for PRMT1-mediated glycolysis.\u003c/strong\u003e (A) R415K mutation and PRMT1 overexpression did not significantly affect SHMT2 expression. (B-D) Changes in cell viability, proliferative capacity of ESCA cells transfected with WT-SHMT2 or R415K-SHMT2 with or without PRMT1 overexpression. (E-H) Changes of glucose uptake (E), ATP levels (F), serine production (G), and glycine production (H) in TE-1 and ECA109 cells shown. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/6602d2ca385a2feee8e70962.jpg"},{"id":72841048,"identity":"bea3b1ce-a2d9-469f-8c77-b2a8825dc61a","added_by":"auto","created_at":"2025-01-02 18:17:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3939819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMethylation of SHMT2 at R415 enhances in vivo tumorigenicity of esophageal cancer cells. \u003c/strong\u003e(A) Representative images clearly presented xenograft tumors in nude mice injected subcutaneously with ECA109 cells. (B) Tumor volumes in xenograft mice were monitored regularly (four days/time). (C) The R415K mutation significantly reduced the weight of xenografts. (D) Representative H\u0026amp;E stained images exhibited the cellular morphological changes of xenografts in the indicated groups. (E, F) Serine/glycine ratio (E) and SHMT2 protein activity (F) monitoring in xenografts. (G) Schematic diagram demonstrated the specific mechanism by which PRMT1-enhanced SHMT2 arginine methylation modifications exacerbated glycolysis ultimately driving ESCA adverse progression. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/b7b9b6b20465c34ebc201e7b.jpg"},{"id":72842927,"identity":"3d614238-62eb-4040-b7e6-bd4894db475e","added_by":"auto","created_at":"2025-01-02 18:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20558332,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3291514/v2/f33bcad9-2d6e-42e4-9935-ace3f5ff6c70.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"SHMT2 arginine methylation by PRMT1 facilitates esophageal cancer progression by enhancing glycolysis and one-carbon metabolism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer (ESCA) has the seventh and sixth incidence and mortality worldwide, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence of ESCA differs by region and gender, with 70% of ESCA occurring in men and the highest prevalence in East Asia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. ESCA represents a biologically aggressive disease with an unfavorable prognosis. The frequently reported short survival is mainly attributed to the limited benefit of systemic radiotherapy and the increased incidence of margin involvement in surgical specimens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, surgery combined with chemotherapy remains the preferred treatment for this specific population of patients with advanced ESCA, but more efforts should be devoted to elucidating the specific mechanisms of disease progression and establishing mechanism-based therapeutic strategies for patients with potential clinical consequences (recurrence or metastasis) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the study of tumor biomarkers is a promising approach to control disease progression.\u003c/p\u003e \u003cp\u003eDysregulation of arginine methylation is a driver of adverse cancer progression. Only the protein arginine methyltransferases (PRMTs) family catalyzes the methylation of protein arginine residues, and this post-transcriptional modification (PTM) functions a critical role in the subsequent biological consequences of their substrates [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, an obvious future goal is to elucidate downstream pathways by identifying novel substrates of PRMTs, which will help us understand the mechanisms of various diseases (including cancer) and establish therapeutic strategies. Importantly, over 90% of the activity of PRMTs in mammalian cells is accounted for by PRMT1, in other words, 85% of asymmetric dimethylarginine (ADMA) in mammals occurs in substrates of PRMT1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Evidence of a direct link between PRMT1 and disease progression has emerged. Several cancer types have shown overexpression or aberrant splicing of PRMT1, so the oncogenic involvement of PRMT1 in specific cancer types is not in question. This \u0026ldquo;oncogenic\u0026rdquo; effect of PRMT1 is essentially dependent on the methylation of EZH2 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Twist1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], FOXO1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], EGFR [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], C/EBPα [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], H4 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and other proteins. It interrupted E3 ligase TRAF6-mediated ubiquitination of EZH2 by methylating histone methyltransferase EZH2 to form meR342-EZH2, which led to enhanced levels of meR342-EZH2, and high levels of EMT were usually predictive of poor clinical outcomes in breast cancer patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The regulation of PRMTs is a complex process because in some cases the enzymatic activity of PRMTs can be altered by other PTMs (e.g., phosphorylation) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, the mechanism of arginine methylation mediated by PRMT1 is a concept that must be explored. However, the role of PRMT1 in ESCA is very limitedly understood.\u003c/p\u003e \u003cp\u003eReprogramming and dysregulation of energy metabolism is a prevalent phenomenon in cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Unlike normal cells, rapidly dividing cancer cells rely on aerobic glycolysis to take up and consume more glucose and convert it to lactate in the presence of sufficient oxygen. This process produces a large number of intermediate metabolites that support the rapid growth of cancer cells, and the accumulation of lactic acid contributes to the poor prognosis of human cancers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, the glycolytic switch in cancer cells greatly limits the development of anti-tumor therapeutic strategies, and useful ways to disrupt glycolysis in tumors need to be found. Several studies have identified the interaction between glycolysis and arginine methylation in cancer cells. For example, arginine methyltransferase 1 (CARM1)-mediated PKM2 methylation in breast cancer became a contributor to aerobic glycolysis in cancer cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, whether PRMT1-mediated protein methylation regulates energy metabolism in ESCA cells remains unknown. Importantly, a branch source intermediate of glycolysis is imported into serine, which is subsequently converted to glycine to provide carbon units for one-carbon metabolism [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Serine hydroxymethyltransferases (SHMTs) are critical proteins that modulate one-carbon metabolism and include cytoplasmic isozymes (SHMT1) and mitochondrial isozymes (SHMT2) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Available reports indicated that SHMT2 overexpressing cancer cells had strengthened glycolytic capacity under basal conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Qi et al. also pointed that silencing SHMT2 was a key target to suppress the malignant phenotype and glycolysis of tumor cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, few studies have pointed out the aberrant metabolic mechanisms by which SHMT2 modulated cancer glycolysis.\u003c/p\u003e \u003cp\u003eHere, we demonstrated that PRMT1 was overexpressed in ESCA tissues, cells and provided direct evidence of its correlation with tumor stage and poor patient prognosis. Furthermore, silencing of PRMT1 restricted glucose uptake, ATP production and one-carbon metabolism (serine to glycine conversion) in cancer cells. Additionally, we found that PRMT1-SHMT2 interaction was a major contributor to the metabolic reprogramming of cancer. PRMT1 deposited ADMA at SHMT2-R415, and methylated SHMT2 accelerated aerobic glycolysis and one-carbon metabolism in tumor cells. Thus, we aimed to elucidate how PRMT1 regulated ESCA progression in a SHMT2-dependent mechanism, demonstrating the possibility of identifying novel targets for cancer therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical tissue samples and cell cultures\u003c/h2\u003e \u003cp\u003ePatients with ESCA who were diagnosed and treated at our institution were recruited for this study. Fresh tumor tissue and paracancerous non-cancerous tissue specimens were surgically removed from 30 patients who had signed a written informed consent. Thirty patients with ESCA were identified as having clinical stage I-II (n\u0026thinsp;=\u0026thinsp;15) or stage III-IV (n\u0026thinsp;=\u0026thinsp;15). Patients receiving any preoperative anticancer therapy or other therapeutic measures affecting the level of gene expression \u003cem\u003ein vivo\u003c/em\u003e were excluded. Complete clinical information was available for all cases. The collection and use of human tissues for the study was licensed and authorized by our ethics committee, in accordance with the \u003cem\u003eDeclaration of Helsinki\u003c/em\u003e. For immunohistochemical (IHC) staining, tissue sections were stained with anti-PRMT1 (Abcam, ab190892, 1:500).\u003c/p\u003e \u003cp\u003eFour cell lines from esophageal cancer (KYSE150, hypofractionated squamous cell carcinoma; TE-10, hypofractionated squamous cell carcinoma; TE-1, highly differentiated squamous cell carcinoma; ECA109, highly differentiated squamous cell carcinoma) and human normal esophageal epithelial cells HEEC were purchased from the Shanghai Cell Bank (Shanghai, China) and maintained in a humid incubator at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. All five cell lines were grown in commercially available RPMI-1640 medium (Gibco\u0026trade;, 31870074) supplemented with 10% FBS (Gibco-BRL) and 100 U/ml penicillin/100 U/ml streptomycin (Sigma, USA). Cells were tested for mycoplasma contamination by PCR method at regular intervals (twice/month). Unless otherwise stated, all experiments were performed in medium containing serine and glycine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003eFor IF analysis, cultured ESCA cells were spread on slides for growth, and the next day the cells were fixed with 4% paraformaldehyde solution for 10 min at room temperature (Beyotime, P0099). Subsequently, cells were permeabilized in permeabilization solution containing 0.2% Triton X-100 for 10 min at room temperature and then closed in 5% BSA for 1 h. Cells were detected with the following primary antibodies: anti-PRMT1 (Abcam, ab190892, 1:80), anti-SHMT2 (Abcam, ab180786, 1:100). After rinsing three times with PBS, Alexa Fluor-coupled secondary antibodies (Thermo Fisher Scientific) diluted in the blocking solution were then added to the slides and incubated for 1 h at room temperature. Nuclei were restained with DAPI (Sigma) at a concentration of 500 nM. Finally, images were observed using a confocal microscope (Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eDifferences in PRMT1 gene expression between 24 pan-cancer types and normal tissues in the TCGA dataset were analyzed by entering \u0026ldquo;PRMT1\u0026rdquo; in the \u0026ldquo;Gene_DE\u0026rdquo; module of the Tumor Immune Evaluation Resource (TIMER2.0) database. Download PRMT1 expression data from the TCGA database for 184 ESCA patients and 11 normal samples. The GSE189830 dataset was download from the GEO database, which included mRNA expression profiles of four pairs of ESCA tumor tissues and paired normal tissues. The identification of differentially expressed mRNAs was analyzed in the R software environment through the Limma package. Based on the KEGG database, pathway analysis was applied to screen important pathways of differentially expressed genes, and the enriched pathways were demonstrated using the R packages \u0026ldquo;tidyr\u0026rdquo; and \u0026ldquo;ggplot2\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid and cell transfection\u003c/h2\u003e \u003cp\u003eIndependent lentiviral vectors encoding shPRMT1 and shSHMT2 were designed and constructed by Genechem (Shanghai, China), respectively. Serum-containing medium was supplemented with polyclathrin (5 \u0026micro;g/mL) and ESCA cells were subsequently infected with purchased lentiviral vectors and co-incubated with puromycin (5 \u0026micro;g/mL) to screen and obtain shPRMT1 or shSHMT2 stably expressed ECA109 and TE-1 cells. Specific small interfering RNAs (siPRMT1 1#, siPRMT1 2#) targeting specific regions of PRMT1 and a negative control siNC were transiently transfected into ESCA cells via Lipofectamine 3000 transfection reagent (Invitrogen, USA). For PRMT1 overexpression, human PRMT1 cDNA was cloned into the pcDNA3.1 vector provided by Genechem (Shanghai, China), labeled as oePRMT1 or PRMT1. HA-tagged SHMT2 was also cloned into the pcDNA3.1 vector, labeled as SHMT2-WT, and the plasmid (wild type) was constructed to generate Mutants of HA-tagged SHMT2, including the following point mutations: only Arg-415 was mutated to Lys (R415K). Flag-SHMT2 was cloned into pCDH-CMV, labeled as Flag-SHMT2-WT, and Flag-SHMT2 R-to-K mutants (R41K, R273K, R358K, R415K) were next developed using a targeted mutagenesis kit (Agilent, 200515). Transfection of specific plasmids was achieved in ECA109 and TE-1 cells with the aid of Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT, EdU and colony formation assay\u003c/h2\u003e \u003cp\u003eThe viability of ECA109 and TE-1 cells was monitored using MTT assay. 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e ESCA cells with different treatments were grown in 96-well plates (100 \u0026micro;L) maintained for different times (0h, 24h, 48h, 72h). Each group of cells was set with 5 replicate wells. Next, MTT working solution (Beyotime, 10 \u0026micro;L) was added to the wells at the set time points (0h, 24h, 48h, 72h) and incubation was continued for 4 h at room temperature. Finally, the optical density (OD) values of each well were quantified at 490 nm using an enzyme marker (BioTek Instruments).\u003c/p\u003e \u003cp\u003eECA109 and TE-1 cell proliferation capacity analysis was done by colony formation assay and EdU assay. For colony formation analysis, equal amounts of ESCA cells (ECA109 and TE-1, 600 cells/well) with different treatments were spread in six-well plates. Colonies were clearly visualized by maintaining them in an incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) for 14 days, followed by fixing and staining of target cell colonies with paraformaldehyde fixative (4%, Beyotime, P0099) and crystalline violet staining solution (Beyotime, C0121). The staining solution was discarded, the cells were rinsed with PBS, and representative pictures were captured under a microscope (Olympus Inc.) EdU analysis was completed with the aid of the BeyoClick\u0026trade; EdU-488 Cell Proliferation Assay Kit (Beyotime, C0071S). Briefly, cells were incubated in 10 \u0026micro;M EdU working solution (obtained by dilution of complete medium) for 2 h, fixed with immunostaining fixative (Beyotime, P0098,1 mL) for 15 min at room temperature, and permeabilized with 1 mL of permeabilization solution (0.3% Triton X-100) for 15 min. EdU was then detected by Click reaction mixture at room temperature. To quantify the proportion of cell proliferation, EdU-positive cells were visualized using HOECHST 33342 staining of cell nuclei and the ratio of EDU-positive cells was subsequently calculated (Image-Pro Plus 6.0, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of glycolytic activity\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, glucose uptake, lactate production, and ATP levels were monitored by the Glucose Uptake Assay Kit (ab136955, Abcam, USA), the Lactate Assay Analysis Kit (K607-100, Biovision, USA), and the Luminescent ATP Assay Kit (ab113849, Abcam, USA), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting and Co-IP Analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed according to standard western blotting protocols. Briefly, cultured ESCA cell or tumor tissue protein lysates were obtained from RIPA lysis buffer (P0013E, Beyotime, China). After quantification of the proteins, aliquots of protein samples were loaded and separated on SDS-PAGE and then transferred to PVDF membranes. After incubation with the corresponding antibodies, blots were visualized using the ECL chemiluminescence kit (Millipore). Immunoprecipitation assays were performed according to the previously described steps [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, collected ECA109 and TE-1 cells were lysed (on ice, 30 min) in RIPA lysis buffer (P0013E, Beyotime, China) as indicated, requiring the addition of a protease inhibitor (PMSF, Beyotime) to this lysate. After removal of cell debris by centrifugation (13,000g, 15min, 4\u0026deg;C), protein concentration in cell lysates was determined by the BCA method. For IP of HA-tagged/Flag-tagged proteins, lysates were incubated with Anti-HA magnetic beads (P2185S, Beyotime) or Flag microspheres (MilliporeSigma, A2220) overnight at 4\u0026deg;C. For IP of endogenous PRMT1 and SHMT2 proteins, clarified lysates were co-incubated (4\u0026deg;C) with primary antibodies to the indicated proteins (anti-PRMT1, Abcam, ab190892; anti-SHMT2, Abcam, ab240606) or isotype-matched IgG. Protein A/G agarose beads (Abcam, ab286842) were then added and incubated overnight (4\u0026deg;C). Beads were collected to release immunoprecipitated protein samples and used for subsequent western blotting analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid and enzyme activity analysis\u003c/h2\u003e \u003cp\u003eThe concentration of serine in ESCA cell lysates (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) was measured via the DL-Serine Assay Kit (ab241027, Abcam). Cell supernatant samples were pretreated and deproteinated with the sample cleanup mixture provided in the kit according to the manufacturer's protocol, and then assayed. Glycine in ESCA cell lysates was quantified using a fluorometric assay (Glycine Assay Kit, Biovision). SHMT2 activity analysis was done by Serine Hydroxymethyltransferase ELISA Assay Kit (JianglaiBio, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, qPCR and RNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA from ESCA tissues, paired normal tissue samples and ESCA cell lines was isolated by TRIzol (Invitrogen, USA). The purity of the total RNA obtained was determined by a NanoDrop 2000C ultra-micro spectrophotometer (Thermo Scientific). RNA was synthesized into cDNA using a reverse transcription kit (PrimeScript RT Master Mix, Takara). SYBR Premix Ex Taq II kit (Takara) and an ABI Prism\u0026reg; 7500 fluorescent quantitative PCR system (Applied Biosystems, USA) were performed to quantify the PCR amplification of PRMT1, SHMT2 and GAPDH. The relative transcript levels of the target RNAs were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, and their expression levels were normalized to the expression of GAPDH. The primers used in this study were as follows.\u003c/p\u003e \u003cp\u003ePRMT1, F, 5'-CTTTGACTCCTACGCACACTT-3' and R, 5'-GTGCCGGTTATGAAACATGGA-3'. SHMT2, F, 5'-TGATTCCCTCGCCTTTCAAGC-3' and R, 5'-TTTCCGGTAGAAGATGAGCCC-3'. '-TGATTCCCTCGCCTTTCAAGC-3' and R, 5'-TTTCCGGTAGAAGATGAGCCC-3'.\u003c/p\u003e \u003cp\u003eRNA-seq was completed in three biological replicates. We compared the differential gene expression between shNC and shPRMT1 cohorts. A cDNA library for each RNA sample was constructed using the RNA Library Preparation Kit (Illumina, USA). RNA sequencing was then performed with the Illumina NovaSeq 6000 sequencing platform and analysis was executed using the OmicStudio tool. The criteria for differential genes were set at a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eresearch\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwenty 6\u0026ndash;8 week female BALB/c nude mice (weight 18\u0026ndash;20 g, SLAC Laboratory Animals Ltd., Shanghai, China) were employed for the \u003cem\u003ein vivo\u003c/em\u003e xenograft tumor assay. The use of animals and experimental procedures were in strict accordance with institutional guidelines and were approved by the Second Affiliated Hospital of Xi'an Jiaotong University Ethics Committee. For \u003cem\u003ein vivo\u003c/em\u003e tumor formation, ECA109 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) transfected with Vector\u0026thinsp;+\u0026thinsp;SHMT2-WT (Vector\u0026thinsp;+\u0026thinsp;WT group), PRMT1\u0026thinsp;+\u0026thinsp;SHMT2-WT (PRMT1\u0026thinsp;+\u0026thinsp;WT group), Vector\u0026thinsp;+\u0026thinsp;SHMT2-R415K (Vector\u0026thinsp;+\u0026thinsp;R415K group) and PRMT1\u0026thinsp;+\u0026thinsp;SHMT2-R415K (PRMT1\u0026thinsp;+\u0026thinsp;R415K group) were injected subcutaneously into the right side of nude mice (n\u0026thinsp;=\u0026thinsp;5). All mice were monitored, and the tumor volume and size were estimated every four days starting from the fourth day after inoculation, and were calculated as 0.5 \u0026times; (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e). After four weeks, all nude mice were executed and tumor tissues were immediately excised for photographing and weighing. Additional, acquired tumor tissues were also executed with hematoxylin-eosin (H\u0026amp;E) staining. Briefly, 5 \u0026micro;m paraffin sections of tissue were prepared and sections were processed using the H\u0026amp;E staining kit (C0105S, Beyotime). Sections were captured under the microscope after blocker sealing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8.0 software was employed in analysis of the data in this study. All experiments were performed at least three times independently, and the data obtained were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical differences between two consecutive data sets were compared using Student's \u003cem\u003et\u003c/em\u003e-test. Kaplan-Meier curves were plotted to demonstrate the correlation between PRMT1 expression and patient survival. \u003cem\u003eP\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePRMT1 overexpression is associated with poor clinical outcomes in patients with esophageal cancer\u003c/h2\u003e \u003cp\u003eAbnormally high expression of the PRMT1 gene has been seen in multiple cancer types. Existing studies on PRMT1 expression in cancer have applied inconsistent study protocols, and most have focused on only a single cancer type. In the current study, we comprehensively analyzed the differential expression of PRMT1 in 24 cancers (BLCA、BRCA、CESC、COAD、ESCA、GBM、HNSC-HPV+、HNSC、KICH、KIRC、KIRP、LIHC、LUAD、LUSC、PAAD、PCPG、PRAD、READ、SKCM、STAD、THCA、UCEC) using the TIMER2.0 database. We could observe from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA that PRMT1 was significantly differentially expressed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all 17 cancers including ESCA, among which it was significantly elevated in 16 cancer types (BLCA, BRCA, CHOL, COAD, ESCA, GBM, HNSC-HPV+, HNSC, KIRC, LIHC, LUAD, LUSC READ, STAD, THCA, UCEC) and significantly low expressed in only one cancer type (KICH). To perform further validation of differentially expressed genes (DEGs) in human ESCA, we performed analysis on the obtained publicly available GSE189830 dataset to identify the important drivers involved in ESCA. The volcano map revealed multiple DEGs between ESCA (n\u0026thinsp;=\u0026thinsp;4) and normal samples (n\u0026thinsp;=\u0026thinsp;4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Subsequently, 100 differentially expressed genes, including 50 up-regulated and 50 down-regulated, were visualized in the heat map (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Further analysis of PRMT1 expression in the ESCA cohort based on sample types in the TCGA database showed that PRMT1 levels were increased in tissues from patients with esophageal cancer (n\u0026thinsp;=\u0026thinsp;184) compared to normal samples (n\u0026thinsp;=\u0026thinsp;11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThereafter, PRMT1 expression was examined in 30 pairs of collected clinical specimens, and the results demonstrated that the level of PRMT1 mRNA was higher in all ESCA tissues than in paired non-tumor tissue samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). We also analyzed the correlation between PRMT1 expression and tumor stage in 30 ESCA tissue specimens, and the results presented that higher PRMT1 expression was more likely to be found in advanced ESCA samples (clinical stage III-IV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, patients with lower PMRT1 levels had significantly higher survival rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In addition, western blotting (containing 5 pairs of well-annotated patients) and IHC analyses demonstrated consistently elevated PRMT1 expression in ESCA tissue samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). We also determined PMRT1 expression in acquired ESCA cells (KYSE150, TE-10, TE-1, ECA109). Enhanced PMRT1 expression was measured in all ESCA cells relative to HEEC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), and the cells with the most upregulated expression (TE-1, ECA109) were chosen for subsequent experiments. The above data supported the correlation between high PRMT1 expression and poor prognosis of esophageal cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSilencing of PRMT1 hinders ESCA cell growth and glycolysis\u003c/h2\u003e \u003cp\u003eNext, we planned to determine the PRMT1-mediated oncogenic properties in esophageal cancer. The two cell lines with the highest PRMT1 expression (TE-1, ECA109) were selected to generate PRMT1 knockdown cells to carry out the next step of this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Then, we found that the presence of siPRMT1 effectively attenuated the viability of TE-1 and ECA109 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similarly, unlike siNC treatment, silencing of PRMT1 reduced the number of colony formation in ESCA cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). EdU assay showed that EdU incorporation was reduced in TE-1 and ECA109 cells after transfection with siPRMT1, and the proportion of EdU-positive cells decreased dramatically (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The enhanced glycolysis of tumor cells conferred a proliferative advantage to achieve tumor cell overgrowth. Furthermore, the activation of glycolytic pathway in tumor cells was characterized by more glycine production and ATP production. In TE-1, ECA109 cells, the effect of PRMT1 knockdown on glycolysis was analyzed by assessing the correlation between PRMT1 expression and glycine production and ATP levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, cellular glycine production and ATP levels were reduced by almost 60% after PRMT1 silencing. These data supported the role of PRMT1 as a tumor promoter and manifested a direct relationship between PRMT1 levels and cellular glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePRMT1 mediates asymmetric methylation of SHMT2\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe conducted RNA sequencing (RNA-seq) to investigate the role of PRMT1 in ESCA cells, and identified 30 differentially expressed genes, including 15 down-regulated and 15 up-regulated genes, in PRMT1-depleted ESCA cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Besides, KEGG analysis revealed the pathways of these down-regulated genes, the results clearly showed that these pathways included one carbon pool by folate, Glyoxylate and dicarboxylate metabolism and Glycine, serine and threonine metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Then, we found that PRMT1 silencing impeded glucose uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), weakened ATP levels and glycine production, but increased serine production in both ESCA cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These might be due to the PRMT1 pathway controlling serine and glycine levels through glycolysis and serine/glycine biosynthesis. Considering that protein-protein interactions underlied a broad range of biological processes, to identify potential binding partners of PRMT1, we constructed a protein interaction network of PRMT1 in the STRING database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) and identified six important proteins (SHMT2, SHMT1, PSAT1, PHGDH, GLDC, DHFR). Next, we evaluated the interaction between PRMT1 and the above six proteins after immunoprecipitation in ESCA cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH demonstrated that only SHMT2 formd a complex with PRMT1 in ESCA cells. Secondly, double immunofluorescence staining of SHMT2 and PRMT1 similarly demonstrated that SHMT2 and PRMT1 might form a protein complex and that the complex was localized in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). In conclusion, the interaction of SHMT2 and PRMT1 might have a potential functional role in ESCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCancer cells need to accumulate the building blocks of new cellular components to sustain their own growth and proliferation. Interestingly, amino acid metabolism involving serine and glycine compensates for this requirement. The reversible conversion of serine to glycine is catalyzed by serine hydroxymethyltransferase 2 (SHMT2), which subsequently assumes a crucial role in the one-carbon pathway. Importantly, our data indicated that inhibition of PRMT1 activity appeared to have a negative effect on intracellular glycine production. Therefore, we next asked whether PRMT1 affected SHMT2 function through direct physical binding and subsequently performed immunoprecipitation experiments (Co-IP) using anti-PRMT1 and anti-SHMT2 in TE-1 and ECA109 cells, respectively. As determined by Co-IP, an endogenous interaction between SHMT2 and PRMT1 was reverified in TE-1 and ECA109 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Previous reports confirmed that PRMT1 catalyzed the appearance of its substrate asymmetric dimethylarginine (ADMA). To explore whether SHMT2 undergoes PRMT1-mediated arginine methylation, we extracted SHMT2 from two ESCA cells and analyzed its methylation using an ADMA antibody. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the presence of SHMT2 ADMA signal was detected in both cells, indicating that SHMT2 was methylated by PRMT1. Subsequently, two siPRMT1 (siPRMT1 1# and siPRMT1 2#) were designed to silence the expression of PRMT1 in TE-1 and ECA109 cells. We observed that effective siRNA-mediated silencing of PRMT1 suppressed endogenous SHMT2 ADMA levels. The introduction of the PRMT1 overexpression vector produced the opposite result, which obviously increased the SHMT2 ADMA signal in cell lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data supported the asymmetric methylation of endogenous SHMT2 by PRMT1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePRMT1-mediated methylation of SHMT2 protein at arginine 415 is required for its oncogenic functions\u003c/h2\u003e \u003cp\u003eWe searched for four arginine residues (R41, R273, R358, R415) in the SHMT2 sequence that could be methylated by PRMT1, and then we tested whether they were the major arginine sites for PRMT1-mediated methylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A series of R (arginine)-to-K (lysine) SHMT2 mutants and PRMT1 were co-expressed in HEK293T cells and then analyzed for ADMA changes. No changes in ADMA levels were found in the R41K, R273K, and R358K mutants, but a decrease in ADMA levels was observed in the R415K mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, SHMT2 activity was also declined after the R415 mutation. R415 in SHMT2 was highly conserved across species from zebrafish to human (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting a potentially critical role for R415 in SHMT2 function. To further analyze the potential contribution of R415 to the asymmetric methylation of SHMT2 protein, plasmids expressing SHMT2-WT and SHMT2-R415K as well as PRMT1 overexpression vector and control vector were transfected into ESCA cells, respectively, to perform Co-IP-SHMT2 experiments and finally obtain immunoprecipitation complexes. Notably, to eliminate the detrimental effect of SHMT2 expression in TE-1 and ECA109 cells, we pre-silenced SHMT2 with a specific shRNA targeting SHMT2. In vitro methylation assays showed that the R415K mutation reduced the interaction of SHMT2 with PRMT1 as well as weakened ADMA signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), which implied that SHMT2-WT was more susceptible to PRMT1 targeting and asymmetric dimethylation than the SHMT2-R415K mutant. Next, we determined the biological function of PRMT1-mediated SHMT2 \u003cem\u003ein vitro\u003c/em\u003e. We found that silencing of PRMT1 induced attenuation of endogenous SHMT2 activity, while enriched PRMT1 expression produced the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, among TE-1 and ECA109 cells with SHMT2 knockdown, ESCA cells bearing only SHMT2-WT exhibited relatively high SHMT2 activity relative to cells bearing only SHMT2-R415K. Whereas overexpression of PRMT1 caused an increase in endogenous wild-type SHMT2 protein activity, R415K mutation attenuated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The above data suggested that PRMT1-mediated methylation of SHMT2-R415 effectively raised SHMT2 activity in tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePRMT1-mediated R415 methylation did not have any effect on SHMT2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), but we explored its effect on the oncogenic function of SHMT2. The results of MTT, colony formation, and EdU experiments showed that ESCA cells expressing hypomethylated SHMT2 (R415K) had attenuated growth as compared to SHMT2-WT cells, and that PRMT1 overexpression vector treatment effectively alleviated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePublished reports indicated that SHMT2-enriched cells possessed higher glycolytic capacity under basal conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Based on these results, we hypothesized that the increase of PRMT1-enhanced SHMT2 activity effectively promoted cancer cell glycolysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, similar to the trend of SHMT2 activity, higher glucose uptake capacity and endogenous ATP levels were observed in ESCA cells with only SHMT2-WT than in target cells with only SHMT2-R415K. PRMT1 overexpression significantly induced glucose uptake and endogenous ATP production in ESCA cells with SHMT2-WT, effectively exacerbating the glycolytic capacity. This finding emphasized that the enhanced glycolytic capacity of PRMT1-overexpressed cells was dependent on their dominant arginine methylation in SHMT2. A branch of glycolysis inputs serine, which can be converted to glycine with the help of SHMT2, providing carbon units for one-carbon metabolism. Considering the abnormal alteration of SHMT2 activity, we wanted to further clarify the relationship between SHMT2-R415 methylation and serine or glycine formation. In shSHMT2-treated cells, the PRMT1\u0026thinsp;+\u0026thinsp;SHMT2-WT group showed a marked defect in serine production and an increase in glycine production relative to the Vector\u0026thinsp;+\u0026thinsp;SHMT2-WT group. Consistently, induction of PRMT1 overexpression resulted in increased serine production and decreased glycine production in SHMT2-R415K cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Mechanistically, PRMT1 overexpression increased SHMT2-R415 asymmetric methylation and promoted elevated cellular glycolysis, which in turn maintained serine production as well as conversion of serine to glycine. PRMT1 might exert as a vital molecular switch to modulate cell biological behavior changes by detecting SHMT2 arginine methylation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethylation of SHMT2 at R415 enhances\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003etumorigenicity of ESCA cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe sought to elucidate whether PRMT1-mediated methylation of SHMT2 at the R415 locus affected esophageal cancer cell growth and proliferation \u003cem\u003ein vivo\u003c/em\u003e with the help of a subcutaneous xenograft tumor model. We subsequently injected Vector\u0026thinsp;+\u0026thinsp;SHMT2-WT cells (Vector\u0026thinsp;+\u0026thinsp;WT group), PRMT1\u0026thinsp;+\u0026thinsp;SHMT2-WT cells (PRMT1\u0026thinsp;+\u0026thinsp;WT group), Vector\u0026thinsp;+\u0026thinsp;SHMT2-R415K cells (Vector\u0026thinsp;+\u0026thinsp;R415K group) and PRMT1\u0026thinsp;+\u0026thinsp;SHMT2-R415K cells (PRMT1\u0026thinsp;+\u0026thinsp;R415K group) subcutaneously into nude mice to drive tumorigenesis. 28 days later, it was clearly observed that tumors formed by the PRMT1\u0026thinsp;+\u0026thinsp;WT group were significantly larger in volume and weight than those from the Vector\u0026thinsp;+\u0026thinsp;WT group or the PRMT1\u0026thinsp;+\u0026thinsp;R415K group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In addition, the size and weight of tumors were smaller in the Vector\u0026thinsp;+\u0026thinsp;R415K group than in the Vector\u0026thinsp;+\u0026thinsp;WT group. At the same endpoints, it was observed under light microscopy that the tumor cancer cells in the PRMT1\u0026thinsp;+\u0026thinsp;WT group exhibited a high degree of anisotropy and dysregulation of nucleoplasmic ratios, and that the tumor cells in the Vector\u0026thinsp;+\u0026thinsp;R415K group tended to have a normal morphology with tightly packed nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These findings suggested that PRMT1-mediated methylation of SHMT2 at the R415 locus was responsible for accelerated tumor growth \u003cem\u003ein vivo\u003c/em\u003e. Cancer cells are highly dependent on serine/one-carbon metabolism for proliferation, so we verified \u003cem\u003ein vivo\u003c/em\u003e whether PRMT1 affected one-carbon metabolism. We found that the decreased serine/glycine ratio in the PRMT1\u0026thinsp;+\u0026thinsp;WT group could be partially alleviated by R415K treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Importantly, SHMT2 activity was also highest in the PRMT1\u0026thinsp;+\u0026thinsp;WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These suggested that PRMT1-driven SHMT2 arginine methylation promoted serine/one-carbon metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we investigated the role of PRMT1 in regulating aerobic glycolysis in ESCA cells and preliminarily explored the potential molecular mechanisms of its involvement. We summarized the main findings in this study: (1) PRMT1 was overexpressed in ESCA, enrichment of PRMT1 was more likely to be observed in patients with advanced (III\u0026thinsp;+\u0026thinsp;IV) ESCA, hinting that high PRMT1 levels predicted a poorer prognosis for ESCA patients. (2) The dependence of ESCA on PRMT1 was observed \u003cem\u003ein vitro\u003c/em\u003e in cell lines. Specifically, PRMT1 silencing depleted cell growth and survival, and inhibited aerobic glycolysis and one-carbon metabolism in cancer cells. (3) PRMT1 dominated a novel PTM. We demonstrated that PRMT1 could deposit ADMA at SHMT2-R315, and this methylation enhanced SHMT2 protein activity. (4) Excessive accumulation of methylation signals of SHMT2 promoted the levels of glycolysis and indicators related to one-carbon metabolism in ESCA cells. In conclusion, our data revealed an alternative mechanism by which PRMT1 mediated aerobic glycolysis in ESCA, which drove esophageal carcinogenesis by methylating SHMT2. Our results are summarized in a schematic model, as in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG.\u003c/p\u003e \u003cp\u003eOverexpression of PRMT1 is frequently observed in human cancers, e.g., lung, breast, and colorectal cancers, and promotes cancer progression by regulating important cellular processes such as transcription and cellular metabolism [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. And its role in these processes may depend on the function of its substrates. A recent study found that enforced PRMT1 was applied to catalyze histone H4R3 methylation in the context of esophageal cancer, mediating the activation of oncogenic transcription through methylated H4R3 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, PRMT1-dependent methylation also impeded the tumor suppressive function of certain substrates. The methylation of C/EBPα caused by PRMT1 affected its interaction with HDAC3 and also enhanced cell cycle protein D1 expression and subsequent tumor growth [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thus, PRMT1 could regulate certain biological functions of the disease by methylating certain specific substrates. Here, we first applied RNA-seq transcriptome analysis to probe for differentially expressed genes altered by PRMT1 defects, some of which might be involved in the regulation of intracellular glycolysis. Next, according to KEGG enrichment analysis, knockdown of PRMT1 had an obvious effect on serine/glycine metabolism as well as other important signaling pathways and metabolic processes. Our \u003cem\u003ein vitro\u003c/em\u003e functional assays consistently demonstrated that PRMT1 knockdown reduced the levels of glycolysis and one-carbon metabolism-related indicators, including decreased ATP levels and reduced glycine synthesis. Besides, PRMT1-deficient cells were less hungry for glucose. Considering that SHMT2 is a nexus that catalyzes serine metabolism, releases one-carbon units and glycine, and is an important source of folate synthesis, we observed the target binding of PRMT1 to SHMT2 using Co-IP analysis. SHMT2 was found to be asymmetrically methylated at residue R415, and the methylation signal of SHMT2 could be attenuated by PRMT1 silencing. Indeed, the relationship between arginine methylation and metabolism has not been sufficiently studied. For example, another member of the PRMTs family, PRMT6, methylated CRAF in hepatocellular carcinoma (HCC), driving aerobic glycolysis in HCC cells, which in turn accelerated tumor formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consistent with previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], our study discovered that SHMT2 was methylated by PRMT1, which enhanced its enzymatic activity and lead to aggravated glycolytic capacity in ESCA cells. We further confirmed the link between these two proteins, as well as arginine methylation and glycolysis. The study of deeper molecular mechanisms will reveal a broader role of arginine methylation in the regulation of glycolysis.\u003c/p\u003e \u003cp\u003eSHMT2 is the key enzyme responsible for serine metabolism and reversibly catalyzes the conversion of the substrate serine to glycine [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], contributing an activated carbon unit to the de novo synthesis of purines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Adequate supply of glycine is a vital reason for the rapid proliferation of cancer cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and thus SHMT2 often functions as an oncogenic factor in various types of cancers, such as lung [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], liver [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], breast [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], glioblastoma multiforme [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], bladder [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and colorectal [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] cancers. Some specific mechanisms by which SHMT2 exerted its oncogenic effects have been reported. For example, in human prostate cancer and glioblastoma multiforme, raised SHMT2 level triggered an altered metabolic state and conferred a higher survival advantage to cells in the tumor region [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Overexpression of SHMT2, a vital enzyme mediating serine/one-carbon metabolism, in cancer interfered with the de novo synthesis of glycine and might retard the abnormal biological behaviors of ESCA. In our study, we reported for the first time that PRMT1-mediated asymmetric dimethylation of SHMT2-R415 had a significant effect on SHMT2 protein activity. Furthermore, we found that the R415K mutation largely eliminated the arginine methylation of SHMT2 by PRMT1, indicating that the R415 residue was essential for the oncogenic function of SHMT2 in ESCA. Overall, our study confirmed that glycolysis in ESCA represented a weak point and that diminished SHMT2 activity triggered by PRMT1 silencing had a strong negative impact on tumorigenesis.\u003c/p\u003e \u003cp\u003eThe above findings raised several new questions that needed to be further explored. Arginine methylation regulated signal transduction cascades, and more efforts were needed to elucidate the detailed molecular mechanisms underlying the effects of PRMT1-dominated methylation on SHMT2 function. Inhibition of PRMT1 and SHMT2 methylation was lethal to cancer cells. Although inhibition of SHMT2 methylation appeared to reduce serine/one-carbon metabolism in ESCA cell lines, resulting in serine accumulation and glycine depletion, this finding revealed a link between serine metabolism and arginine methylation of proteins that needed to be highlighted in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this report, we have characterized the PRMT1-SHMT2 axis in stimulating esophageal cancer development, suggesting that asymmetric dimethylation of SHMT2 at R415 played an essential role in this process. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, increased levels of PRMT1 expression in esophageal cancer cells promoted PRMT1-mediated SHMT2-R415 methylation, which enhanced the protein activity of SHMT2. The accumulation of SHMT2 activity promoted the reversible conversion of serine to glycine, contributing an activated carbon unit to the de novo synthesis of purines and ultimately accelerating esophageal cancer tumor growth. These data emphasized the importance of PRMT1-mediated SHMT2 methylation in facilitating esophageal cancer development and growth, and suggested the clinical value of PRMT1 in the diagnosis and treatment of esophageal cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhe Qiao\u0026nbsp;designed the study and draft the manuscript.\u0026nbsp;Yu Li\u0026nbsp;collected the data and processed statistical data. Yao Cheng\u0026nbsp;analyzed and interpreted the data.\u0026nbsp;Shiyuan Liu\u0026nbsp;partly contributed to the experiment.\u0026nbsp;Shaomin Li\u0026nbsp;reviewed, and\u0026nbsp;revised\u0026nbsp;the paper. All authors reviewed the results and approved the final version of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\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\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants were provided with written informed consent at the time of recruitment. This study was approved by the Ethics Committee of The Second Affiliated Hospital of Xi\u0026apos;an Jiaotong University, and all experiments involving human tissue specimens comply with the \u003cem\u003eDeclaration of Helsinki\u003c/em\u003e. All animal studies were strictly performed according to protocol approved by the Ethics Committee of The Second Affiliated Hospital of Xi\u0026apos;an Jiaotong University. Animal studies were performed in compliance with the \u003cem\u003eARRIVE\u003c/em\u003e guidelines.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest to report regarding the present study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCao L, Wang X, Zhu G, Li S, Wang H, Wu J, et al. Traditional Chinese Medicine Therapy for Esophageal Cancer: A Literature Review. Integr Cancer Ther.\u003cem\u003e \u003c/em\u003e2021;20:15347354211061720.\u003c/li\u003e\n\u003cli\u003eHamdi Y, Abdeljaoued-Tej I, Zatchi AA, Abdelhak S, Boubaker S, Brown JS, et al. Cancer in Africa: The Untold Story. 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Cells.\u003cem\u003e \u003c/em\u003e2019;8(9).\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":"Esophageal cancer, PRMT1, SHMT2, glycolysis, one-carbon metabolism","lastPublishedDoi":"10.21203/rs.3.rs-3291514/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3291514/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eProtein arginine methyltransferase 1 (PRMT1) is the main enzyme that directly responsible for the production of asymmetric dimethylarginine (ADMA), and upregulation of PRMT1 is observed in a variety of malignancies, including esophageal cancer (ESCA). Dysregulation of arginine methylation caused by PRMT1 overexpression is a driver of poor cancer progression, and the detailed mechanism of modulation is currently unknown.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe present study confirmed a novel oncogenic mechanism of PRMT1 in ESCA. PRMT1 levels were significantly upregulated in ESCA, and its high expression correlated with TNM stage and poor patient prognosis. We continued to find the mechanisms by which PRMT1 expression was more relevant to ESCA progression. RNA-seq and KEGG enrichment analyses revealed that differentially expressed genes after PRMT1 silencing in ESCA might modulate serine/one-carbon metabolism. Knockdown of PRMT1 \u003cem\u003ein vitro\u003c/em\u003e resulted in a significant reduction in ESCA cell growth, and indicators related to serine/one-carbon metabolism and glycolysis, whereas its overexpression showed opposite results. The catalytic activity of PRMT1 was crucial in mediating these biological processes. We found that PRMT1 mediated the ADMA modification of serine hydroxymethyltransferase 2 (SHMT2) at arginine 415 (R415), which activated SHMT2 activity and enhanced serine/one-carbon metabolism and glycolysis. The R415K mutation largely eliminated the arginine methylation of SHMT2 by PRMT1, and weakened PRMT1-induced glycolysis and serine/one-carbon metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study further confirmed the link between the two proteins, PRMT1 and SHMT2, as well as arginine methylation and glycolysis. The study of deeper molecular mechanisms will reveal a broader role of arginine methylation in the regulation of glycolysis.\u003c/p\u003e","manuscriptTitle":"SHMT2 arginine methylation by PRMT1 facilitates esophageal cancer progression by enhancing glycolysis and one-carbon metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-01-02 18:17:24","doi":"10.21203/rs.3.rs-3291514/v2","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}},{"code":1,"date":"2023-09-06 13:56:59","doi":"10.21203/rs.3.rs-3291514/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":"7238265b-2825-4ec4-8934-1be02b9a2a95","owner":[],"postedDate":"January 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-23T00:10:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-02 18:17:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-3291514","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3291514","identity":"rs-3291514","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}