The proto-oncogene c-Myc regulates multiple biological processes mainly through selectively activating gene expression. However, the mechanisms underlying c-Myc-mediated gene repression in the context of cancer remain less clear. This study aimed to clarify the role of PRMT5 in the transcriptional repression of c-Myc target genes in gastric cancer.Methods: Immunohistochemistry was used to evaluate the expression of PRMT5, c-Myc and target genes in gastric cancer patients. PRMT5 and c-Myc interaction was assessed by immunofluorescence, co-immunoprecipitation and GST pull-down assays. Bioinformatics analysis, immunoblotting, real-time PCR, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism.Results: We found that c-Myc directly interacts with protein arginine methyltransferase 5 (PRMT5) to transcriptionally repress the expression of a cohort of genes, including PTEN, CDKN2C (p18INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, to promote gastric cancer cell growth. Specifically, we found that PRMT5 was required to promote gastric cancer cell growth in vitro and in vivo, and for transcriptional repression of this cohort of genes, which was dependent on its methyltransferase activity. Consistently, the promoters of this gene cohort were enriched for both PRMT5-mediated symmetric di-methylation of histone H4 on Arg 3 (H4R3me2s) and c-Myc, and c-Myc depletion also upregulated their expression. H4R3me2s also colocalized with the c-Myc-binding E-box motif (CANNTG) on these genes. We show that PRMT5 directly binds to c-Myc, and this binding is required for transcriptional repression of the target genes. Both c-Myc and PRMT5 expression levels were upregulated in primary human gastric cancer tissues, and their expression levels inversely correlated with clinical outcomes.Conclusions: Taken together, our study reveals a novel mechanism by which PRMT5-dependent transcriptional repression of c-Myc target genes is required for gastric cancer progression, and provides a potential new strategy for therapeutic targeting of gastric cancer. Despite advances in early diagnosis, surgical resection, and adjuvant chemotherapy, gastric cancer is still one of the most aggressive malignancies with high morbidity and mortality The proto-oncogene c-Myc is a basic helix-loop- helix/leucine zipper (bHLH-Zip) transcriptional regulator that controls multiple biological processes, including cell growth and differentiation, as well as tumor initiation and progression Arginine methylation of histone tails is a post-translational modifications (PTMs) that has been linked to both transcription activation and repression in vitro and in xenograft models, which has been suggested to occur via epigenetic silencing of the tumor suppressor IRX1 although the role of H4R3me2s was unclear Dysregulation of epigenetic mechanisms is a distinct feature of cancer. Recent reports associate elevated levels of PRMT5 with several human diseases, especially in cancer, including lung cancer, breast cancer, leukemia, lymphoma, gastric cancer, and colorectal cancer INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, to promote gastric cancer cell growth. Our results thus unravel novel mechanisms of c-Myc-mediated transcriptional repression and of PRMT5 recruitment and function within the context of gastric cancer, revealing a new potential strategy for targeted therapy.In the present study, we provide evidence of a direct and functional link between PRMT5-mediated H4R3me2s and c-Myc in gastric cancer. The PRMT5-mediated H4R3me2s mark is enriched on c-Myc-binding CANNTG E-box elements and acts together with c-Myc to selectively repress expression of a cohort of largely cell cycle-related genes, including PTEN, CDKN2C  using short tandem repeat (STR) profiling. All lines were found to be negative for mycoplasma contamination.Human gastric cancer cell lines BGC823 and SGC7901 were obtained from the Cell Bank of the Chinese Academy of Sciences  and cultured in RPMI-1640 medium contained 10% fetal bovine serum  and 1% penicillin- streptomycin (Beyotime Biotechnology) in a humidified atmosphere of 5% COTissue microarrays (TMAs) with clinical pathological data were provided by Shanghai Biochip Co., Ltd. . The arrays contained gastric cancer tissue samples and matched normal tissues adjacent to the tumor. The paraffin-embedded tissues were deparaffinized, rehydrated, and then subjected to antigen retrieval. The tissues slides were incubated with PRMT5, Ki-67, c-Myc, PTEN and p57 antibodies overnight at 4°C. Subsequently, the slides were incubated with horseradish peroxidase (HRP)- conjugated secondary antibody. All gastric cancer tissue sections were reviewed by two experienced pathologists, and staining of target proteins in the tissue was scored independently by two pathologists blinded to the clinical data adopting the semiquantitative immunoreactive score (IRS) system Specific siRNAs targeting PRMT5 and c-Myc were synthesized by GenePharma . Cells were transiently transfected with siRNA duplexes to a final concentration of 100 nM using Lipofectamine 3000 (Invitrogen). siRNA sequences targeting PRMT5 were siRNA-1: 5'-GCACCAGUCUGUUCUGCUA-3'; siRNA-2: 5'-GGCGAUGCAGCAAUUCCAA-3'; siRNA-3: 5'-GGACCUGAGAGAUGAUAUA-3'. siRNA sequences targeting c-Myc were siRNA-1: 5'-CGAUGUUGUUUCUGUGGAA-3'; siRNA-2: 5'-CCAAGGUAGUUAUCCUUAA-3'. ShRNA lentivirus were prepared by the co-transfection of 293T cells with pLKO.1 and packaging plasmids pMD2G and psPAX2. 48 h after transfection, the viral supernatants were collected. The BGC823 or SGC7901 cells were incubated with viral supernatants in the presence of polybrene. The positive cells were selected with 1μg/mL puromycin. The target sequences were PRMT5 shRNA-1: 5'-CCCATCCTCTTCCCTATTAAG-3'; PRMT5 shRNA-2: 5'-GCCCAGTTTGAGATGCCTTAT-3'. PTEN shRNA were 5'-CTAGAACTTATCAAACCCTTT-3'. p18 shRNA were 5'-CTATGGGAGGAATGAGGTTGT-3'. p21 shRNA were 5'-GACAGATTTCTACCACTCCAA-3'. p57 shRNA were 5'-CCACGCACTAGCTCGGTTATT-3'. p63 shRNA were 5'-GCCACATCAAACCTTTGAGTA-3'. Scramble sequence was used as negative controls.3 cells/well. After incubation with CCK-8 reagent  for 1 h at 37°C, the absorbance at 450 nm was determined by a spectrophotometer. EdU incorporation assay was performed using the EdU DNA Cell Proliferation Kit . In brief, cells were incubated with 50 μM EdU for 16 h at 37°C, fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 for 20 min. After washing with PBS, the cells were incubated in Apollo staining solution, and then stained with DAPI. For the colony-formation assay, cells were seeded sparsely in 6-well plates with normal medium at a density of 1000 cells/well. Two weeks later, colonies were fixed with methanol and stained with 0.5% (w/v) crystal violet solution at room temperature. After washing with distilled water, the colonies were counted visually.For the CCK-8 assay, cells were seeded in 96-well plates in RPMI-1640 medium containing 10% FBS at equal density of 2×10E. coli BL21, and cultured with IPTG at 16°C for 12 h until the optical density (OD600) reached 0.5 ~ 0.6. BL21 cells were collected, sonicated in cold PBS and purified with Glutathione S-transferase  beads or Nickel-nitrilotriacetic acid  beads according to the users' manual.pGEX-6p-1 plasmids encoding GST, GST-PRMT5 (wild-type), GST-PRMT5 F1, F2, F3, GST-PRMT5 488-490Δ, GST-PRMT5 R488A and GST-PRMT5 K490A, and pET28a plasmid encoding His-tagged c-Myc were transformed into 3VO4, leupeptin). Equal amounts of protein were separated by 8 ~ 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). The membranes were blocked for 1 ~ 2 h at room temperature in PBST with 5% (w/v) non-fat milk and incubated overnight at 4°C with primary antibodies. After incubation with secondary antibodies, proteins were visualized with the ECL detection system (Thermo Fisher Scientific). The following antibodies were used: PRMT5 , c-Myc , GAPDH , PTEN , p21 , p63 , p18 , p57 , HSP70 , H4R3me2s  and Histone H4 .Cells were trypsinized, washed with phosphate- buffered saline (PBS) and lysed in Cell lysis buffer  according to the manufacturer's instructions. The cDNA was synthesized using a HiScript Q RT SuperMix kit . Quantitative real-time PCR analysis was performed using qPCR SYBR Green Master mix . The qPCR plates were denatured for 5 min at 95°C, and then subjected to 40 cycles of 20 s at 95°C, 15 s at 60°C and 25 s at 72°C in a CFX96 qPCR detection system (Bio-Rad). Relative mRNA expressions were calculated using the 2Cells were cultured on glass coverslips, fixed with 4% paraformaldehyde (Sigma) for 30 min and permeabilized by 0.3%Triton X-100 for 30 min at room temperature. After blocking with 5% goat serum in PBS, cells were incubated with primary antibodies for 60 min and secondary antibodies for 45 min in a dark and humid chamber, respectively. Then, cell were washed with PBS and the nuclei stained with 4, 6, diarnidino-2-phenylindole (DAPI) for 5 ~ 10 min. Immunofluorescence images were captured under a fluorescence microscope .Cells were cultured in RPMI-1640 with 10% FBS, and then cross-linked with 1% formaldehyde at room temperature. 10 ~ 15 min later, the reaction was quenched with Glycine for 10 min at a final concentration of 125 mM. Subsequently, the chromatin was sonicated to produce DNA fragments (200 bp ~ 500 bp). 100 μg chromatin was incubated overnight with rotation at 4°C with primary antibodies or IgG (2 μg) followed by 1 h incubation with protein A sepharose beads. After washing with low-salt buffer and high-salt buffer, the beads were incubated with elution buffer and proteinase K at 65°C overnight to immunoprecipitate DNA. Total DNA fragments were isolated by phenol/chloroform extraction and ethanol precipitation. After isolation, the DNA was diluted in water and subjected to real-time PCR analysis. The primers used for the analysis are listed in To overexpress PRMT5 or mutant in PRMT5-depleted BGC823 cells, PRMT5 (wild-type), PRMT5 Δ or PRMT5 R368A , and PRMT5 K490A cDNAs were cloned into the eukaryotic expression pcDNA3.1 plasmid. The shRNA-resistant PRMT5 or mutant cDNAs were generated by introducing point mutations into the target site of PRMT5 shRNA-1. The target sequence 5'-CCCATCCTCTTCCCTATTAAG-3' was mutated to 5'-CCGATTTTGTTTCCCATAAAA-3'-. The amino acid sequence of protein did not change.2. 20 days later, all nude mice were sacrificed and tumors were excised, photographed and weighed.All animal care and handling procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Review Board of Nanjing University . BALB/c nude mice  were purchased from the Model Animal Research Center of Nanjing University . Scr, sh_PRMT5- treated and sh_PRMT5 + sh_p57-treated BGC823 cells were suspended in PBS containing 10% Matrigel (BD Biosciences) and injected subcutaneously into the flanks of nude mice (1×107 cells per mouse). The sizes of tumors (Length and Width) were measured every 4 days and tumor volumes were calculated using the formula: Volume (cm3) = 0.5 × (Length) × (Width)Publicly available H4R3me2s ChIP-seq data sets from mouse embryonic stem cells were obtained from the GEO database GSE37604). To identify DNA sequence motifs enriched in H4R3me2s regions, we used Hypergeometric Optimization of Motif EnRichment (HOMER) for the motif analysis. Reducing redundancy by clustering and merging motifs was performed as in a previous report 604. To it test for comparing two groups using the GraphPad Prism software. Data are shown as mean ± SD. Differences in the mean values were considered to be significant at P < 0.05.Statistical analysis was performed by Student's To investigate the role of PRMT5 in gastric cancer, we first examined PRMT5 expression by immunohistochemistry using tissue arrays containing 90 pairs of gastric cancer and their matched non-tumorous tissues . The sumin vitro.Based on this PRMT5 expression analysis in gastric cancer samples, we reasoned that depleting PRMT5 would attenuate the malignancy of gastric cancer cells. Therefore, we designed three specific siRNAs targeting PRMT5, and their efficacy was verified by Western blot analysis in gastric cancer cells BGC823 and SGC7901 , CDKN1B (p27KIP1), CDKN1C (p57KIP2), CDKN2A (p16INK4), CDKN2B (p15INK4b), CDKN2C (p18INK4C), KNTC1, MKI67, RAD9A, RB1, SKP2, TFDP1, TFDP2, GADD45A, E2F4, DDX11, CKS1B, CDK1, CDK2, CDK4, CDK5R1, CDK6, CDK7, CDK8, CDC16, CDC20, CCNT2, CCNC, CCND, CCND2, CCNE1, CCNF, CCNH, CCNT1, CCNB2, CCNB1, BRCA2, BCL2, BCCIP, ATR, ANAPC2, ANAPC4, ABL1, ATM, DIRAS3, PTEN, p53 and p63. Our findings revealed that five of these genes, specifically PTEN, CDKN2C (p18INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, were significantly upregulated in PRMT5-depleted BGC823 and SGC7901 cells , CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63 expression were consistently increased in PRMT5-knockdown cells, validating the quantitative RT-PCR results  by site-directed mutagenesis In order to test whether PRMT5 directly regulates PTEN, p18, p21, p57 and p63 expression, we analyzed the enrichment of PRMT5-mediated H4R3me2s on the promoters of these candidate genes in BGC823 and SGC7901 cells using chromatin immunoprecipitation (ChIP) assays. Herein, we designed 4 to 6 pairs of walking primers across the core promoter regions of these candidate genes. We found that, in comparison with IgG controls, H4R3me2s was indeed enriched and distributed at the proximal promoter regions of PTEN, p18, p21, p57 and p63 genes in both BGC823 and SGC7901 cells . A total of 16 highly enriched motifs were identified in increased H4R3me2s regions , 355-453 (F2) and 454-637 (F3), according to its subdomain structure. Purified His-tagged c-Myc protein pre-adsorbed to nitrilotriacetic acid-nickel beads was incubated with purified GST or GST fusion PRMT5 proteins containing fragments 1-354 (F1), 355-453 (F2) or 454-637 (F3) respectively. We found that only fragment 3 (F3) of PRMT5 was able to interact with c-Myc . Interestingly, GST pull-down assays showed that the direct interaction between this PRMT5 deletion mutant and c-Myc was largely lost  or p57  exhibited a significant inverse correlation with PRMT5 levels in these gastric cancer samples calculated by Pearson correlation  on a tissue array containing 90 pairs of gastric cancer samples and their matched non-tumorous tissues. We found that PTEN and p57 protein were significantly downregulated in tumor tissues compared with matched adjacent normal tissues Figure A-B. Notan Figure C. Moreovn Figure D. These INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, to promote gastric cancer cell growth  and CDKN2B (p15INK4B) In this study, we found that c-Myc could interact directly with PRMT5 to transcriptionally repress the expression of a cohort of genes, including PTEN, CDKN2C p18INK4C, CDet al. provided evidence proposing a perspective for c-Myc function where the transcription machinery rather than DNA sequence elements plays a major role in recruiting the c-Myc-Max heterodimer to genomic sites  in vitroThrough the analysis of publicly available H4R3me2s ChIP-Seq datasets, we found that a DNA motif, CAGCTG, was indeed observed in the H4R3me2s-enriched regions and exactly corresponds to an E-box variant element that c-Myc binds, suggesting it may also play a functional role in PRMT5-mediated transcriptional repression. Subsequent ChIP assays showed that c-Myc was co-enriched with H4R3me2s at the PRMT5-targeted genes and repressed their expression, suggesting that PRMT5-mediated H4R3me2s may have a preferential neighboring E-box or its variants. Of note, previously Guo et al. found that PRMT5-mediated IRX1 inactivation plays an important role in promoting tumorigenicity and metastasis of gastric cancer cells CIP1/WAF1) as well as the tumor repressor PTEN in other malignancies As an epigenetic regulator, PRMT5 could transcriptionally regulate the expression of a wide spectrum of cellular events, including cell growth/ proliferation et al. showed that PRMT1 associates with Myc/PRMT5 in both HEK293T cells and glioblastoma stem cells and that Myc is both symmetrically and asymmetrically dimethylated by PRMT5 and PRMT1, respectively et al. demonstrated that PRMT1 is necessary for c-Myc-dependent transcription through altering promoter recruitment of acetyltransferase p300 et al. suggested that PRMT5 physically associated with c-Myc and posttranslationally modulated the stability of c-Myc Interestingly, previous reports suggested PRMT5 and N-Myc proteins physically interact and N-Myc protein stability is regulated by PRMT5 in neuroblastoma cells et al. observed a diffused cellular localization of PRMT5 both in the cytoplasm and nucleus of glioblastoma cells Cellular localization of PRMT5 helps dictate its role in cells. It is interesting that we observed a dominant nuclear localization of PRMT5 in both gastric cancer tissues and cells, although a small fraction was distributed in the cytoplasm as well. PRMT5 has been shown to predominantly localize in the cytoplasm in lung Our study demonstrated that PRMT5-dependent epigenetic repression of c-Myc target genes regulates gastric cancer progression. However, we still do not understand how PRMT5-mediated H4R3me2s is fully recognized and thereby affects other epigenetic modifications to repress downstream gene expression and promote tumor progression. Large-scale cohort studies and detailed ChIP-seq analysis of PRMT5- mediated H4R3me2s and other histone marks would facilitate understanding their roles in transcriptional repression and gastric cancer progression. Nevertheless, our findings reveal important insights that link PRMT5-dependent transcription repression of c-Myc target genes and gastric cancer progression. Collectively, we unraveled a novel mechanism by which PRMT5-dependent transcriptional repression of c-Myc target genes is required for gastric cancer progression. Our results will help contribute to the development of new therapeutic strategies for gastric cancer in the future.Supplementary figures and tables.Click here for additional data file.


Age-related macular degeneration (AMD) is considered as the main worldwide cause of blindness in elderly adults. Exudative AMD type represents 10 to 15% of macular degeneration cases, but is the main cause of vision loss and blindness. Circadian rhythm changes are associated with aging and could further accelerate it. However, the link between circadian rhythms and exudative AMD is not fully understood. Some evidence suggests that dysregulation of circadian functions could be manifestations of diseases or could be risk factors for the development of disease in elderly adults. Biological rhythms are complex systems interacting with the environment and control several physiological pathways. Recent findings have shown that the dysregulation of circadian rhythms is correlated with exudative AMD. One of the main pathways involved in exudative AMD is the canonical WNT/β-catenin pathway. Circadian clocks have a main role in some tissues by driving the circadian expression of genes involved in physiological and metabolic functions. In exudative AMD, the increase of the canonical WNT/β-catenin pathway is enhanced by the dysregulation of circadian rhythms. Exudative AMD progression is associated with major metabolic reprogramming, initiated by aberrant WNT/β-catenin pathway, of aerobic glycolysis. This review focuses on the interest of circadian rhythm dysregulation in exudative AMD through the aberrant upregulation of the canonical WNT/β-catenin pathway. Age-related macular degeneration (AMD) is considered as the main worldwide cause of blindness in elderly adults . AMD proCircadian rhythm (CR) changes are associated with aging and could further accelerate it . HoweverThe endogenous characteristic of CRs is an innate oscillation associated with a period of over one day. All of the studied organisms show this oscillatory process. Numerous cell functions present temporal variations driven by these oscillatory and circadian ways including gene expression, metabolic reprogramming, and molecular and cellular pathways. Different integration levels allow for the study of CRs as endocrinal, physiological, and neuronal cell behaviors. Although the coordination and the modulation of CRs are organized by specific pacemaker structures, the primary circadian oscillations are controlled at the cell level. These oscillations are determined by numerous clock genes . The conEndogenous oscillations generate a freewheeling period, which is close to 24 h, at constant ambient conditions to maintain the organism. These oscillators, at the molecular level, are based on the products of clock regulator genes organized in a transcriptional feedback loop. Circadian oscillations are the product of post-transcriptional modifications of proteins . A complMoreover, the output pathways are predicted to be rhythmic and then controlled by the clock gene transcription factors. These compounds, in turn, regulate downstream the circadian clock genes in a time-of-day-specific manner . This syPhoto-entrainment of the suprachiasmatic nucleus processes through pathways from a subpopulation of retinal ganglion cells (RGCs), which are melanospin-expressing and intrinsically photosensitive (ipRGCs) . These rClock), brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 , Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), and Cryptochrome (Cry 1 and Cry 2) ..Erg. ErgLRP5/LRP6 [LRP5 has a main role while LRP6 has a minor role in retinal vascularization [AXIN, and this prevents the phosphorylation of β-catenin by glycogen synthase kinase-3β (GSK-3β). Then, β-catenin accumulation in the cytosol is observed and translocates to the nucleus and binds the T-cell factor/lymphoid enhancer factor (TCF/LEF) co-transcription factors. This nuclear binding allows the transcription of WNT-responsive genes such as cyclin D1, c-Myc, PDK1, and MCT-1 [Stimulation of FZD4/β-catenin signaling needs the presence of the complex RP5/LRP6 . LRP5 harization ,119. Disnd MCT-1 ,121. GSK-3β. In the absence of WNT ligands, cytosolic β-catenin is phosphorylated by AXIN, GSK-3β, and β-catenin. Then, phosphorylated β-catenin is destroyed into the proteasome. WNT inhibitors including DKKs and SFRPs control the WNT/β-catenin pathway by preventing its ligand–receptor interactions [A destruction complex is composed of tumor suppressor adenomatous polyposis coli (APC), ractions .GSK-β, an intracellular serin-threonin kinase, is a regulator of the WNT pathway [GSK-3β regulates numerous pathophysiological pathways  [GSK-3β downregulates β-catenin cytosolic accumulation and then its nuclear translocation [GSK-3β diminishes β-catenin, the mTOR pathway, HIF-1α, and VEGF expression [ pathway . GSK-3β mmation) ,125,126.location . GSK-3β pression . SERPIN) family, can lead to anti-angiogenic and anti-inflammatory properties [LRP6 to inhibit the WNT/β-catenin pathway [Aberrant activation of the WNT/β-catenin pathway is associated with focal retinal degeneration and exudative AMD lesions . Kallistoperties ,131,132. pathway . TF), a transmembrane cell-surface receptor for plasma coagulation factor VII, is considered as the major effector of the extrinsic coagulation pathway [TF activates VEGF expression and participates in vascular formation by activating the WNT/β-catenin pathway [LRP6, and its use shows the main role played by the WNT/β-catenin pathway in exudative AMD. Tissue factor . They allow for the control of peripheral clocks ,39. Per1ll cycle ,161. HIF-1α [PDK) to phosphorylate PDH and inactivates it, leading to cytosolic pyruvate being shunted into lactate by LDH-A [HIF-1α is transcriptionally activated by the PI3K/Akt/mTOR pathway through 4E-BP1 and STAT3 [Some reports have highlighted that the WNT/β-catenin pathway is a main effector of aerobic glycolysis  10,15,2,66,104. HIF-1α . HIF-1α by LDH-A . HIF-1α nd STAT3 ,169,170.c-Myc directly stimulates HIF-1α [PDK, and lactate transporter (MCT-1) expression [HK), pyruvate kinase (PK), PDK1, and LDH-A [Numerous studies have observed that the WNT/β-catenin pathway can downregulate pyruvate oxidation in the TCA cycle ,171. Thes HIF-1α , PDK, annd LDH-A ,177,178.Changes in energy metabolism are associated with metabolic and thermodynamic alterations and abnormal CRs in exudative AMD. In exudative AMD, the canonical WNT/β-catenin pathway is increased. Energy behaviors of metabolic enzymes in exudative AMD are modified by this upregulation of the WNT/β-catenin pathway, leading to the enhancement of aerobic glycolysis and thus the production of lactate. This explains the glucose hyper-metabolism observed in exudative AMD cells. The WNT pathway is driven by the CRs and operates under a circadian regime evolving to changes in energy metabolism. Regulation of the WNT/β-catenin pathway is directly controlled by CRs and the impairment of these rhythms is involved in reprogramming energy metabolism, enabling exudative AMD.


Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Lenvatinib oral chemotherapy is approved as a first-line treatment of patients with unresectable HCC. The efficacy and therapeutic duration of lenvatinib are limited by drug resistance, and the mechanism is unclear. IRF2 is a constitutive transcription factor associated with the development of various cancers by regulating cancer cell growth, apoptosis, and drug resistance. However, the potential role of IRF2 in lenvatinib resistance in HCC has not been explored. In this study, we found that IRF2 promoted proliferation, inhibited apoptosis, and increased lenvatinib resistance of HCC cells by regulating β-catenin expression. Silencing IRF2 downregulated the expression of β-catenin, while overexpressing IRF2 upregulated β-catenin. Moreover, the expression of β-catenin and IRF2 was positively correlated in HCC tissues. Inhibiting β-catenin with XAV-939 effectively abrogated β-catenin expression caused by lenvatinib treatment. These findings identify an important function of IRF2 in HCC and demonstrate a mechanism of lenvatinib resistance of HCC cells. Targeting IRF2 may be a potential strategy to improve the therapeutic effect of lenvatinib on HCC. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, with a high incidence of HCC in the African and Asian-Pacific regions Lenvatinib, an oral multi-kinase inhibitor, is recommended as the first-line treatment of patients with unresectable HCC in the USA, EU, Japan, and China since 2019. Lenvatinib shows non-inferiority for overall survival (OS), but significantly improves the objective response rate (ORR) and progression-free survival (PFS) Interferon regulatory factors (IRFs) are crucial nuclear transcription factors, consisting of 9 members (IRF1–9) in mammals. The IRF family is best known for regulation of gene expression that underlies IFN responses. IRF proteins have a conserved amino-terminal DNA-binding domain (DBD), and recognize a consensus DNA sequence element termed ISRE β-catenin, a subunit of the cell surface cadherin protein complex, is a critical component of Wnt signaling, which is involved in several physiologic and pathophysiologic processes during embryonic development and carcinogenesis. Activation of Wnt signaling induces nuclear accumulation of β-catenin to form a transcription complex and activate the transcription of downstream target genes In this study, we found that IRF2 was positively correlated with the malignancy of HCC. IRF2 promoted proliferation, inhibited apoptosis, and increased lenvatinib resistance of HCC cells through regulating β-catenin expression. The inhibitor of β-catenin XAV-939 effectively inhibited β-catenin expression caused by lenvatinib treatment. Therefore, our findings suggest that targeting IRF2 may be a potential strategy to improve the therapeutic effect of lenvatinib for HCC.Human HCC cells HepG2 and Huh7 cells were obtained from the American Type Culture Collection and cultured as recommended. The cells were tested negative for mycoplasma contamination and were authenticated by short tandem repeat (STR) fingerprinting. The HCC tissue samples and paired adjacent tissue samples were obtained from University of Pittsburgh Medical Center (UPMC). Written informed consent was obtained from all the patients and participants. This study was approved by the Ethics Committee of University of Pittsburgh Institutional Review Board (IRB).Ⓡ AQueous Non-Radioactive Cell Proliferation Assay  was used to detect the viability of HCC cells. 5000 HepG2 and Huh7 cells infected with control or IRF2 overexpression adenoviruses. Control or IRF2-targeting siRNA were plated in 96 well plates. 20μl of the combined MTS/PMS solution was pipetted into each well of the 96 well assay plate containing 100μl of cells in the culture medium. Plates were incubated for 1–4 h at 37 °C in a humidified, 5% CO2 atmosphere. Absorbance was recorded at 490 nm using an ELISA plate reader.CellTiter 96Ⓡ TUNEL Alexa FluorⓇ Imaging Assay  was used to detect the apoptotic HCC cells. Briefly, HepG2 and Huh7 cells infected with control or IRF2 overexpressing adenoviruses, or control and IRF2-targeting siRNA were fixed, and cellular permeabilization was conducted. TdT reaction cocktail was added to each sample and incubated for 60 min at 37 °C. Click-iT reaction cocktail was added to each sample and incubated on coverslips for 30 min at room temperature. For DNA staining, the DAPI mounting reagent was used.Click-iT25–50 μg of each protein sample was loaded and separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked by 5% milk for 0.5–1 h, and incubated with antibodies against Bcl-2 , survivin , PARP , cleaved Caspase-3 , c-Myc , Cyclin-D1 , IRF2 , β-catenin , VEGFR2 , p65 , Phospho-p65 (Ser536)  and β-actin  overnight at 4 °C and incubated with secondary antibodies for 2 h at room temperature.H + L). For nuclear staining, DAPI mounting reagent was used.HepG2 and Huh7 cells treated with 0 or 7.5 µM of lenvatinib for 48 h were fixed, blocked by 5% bovine serum albumin (BSA) for 30 min, and incubated with the antibodies against IRF2 or β-catenin at 4 °C overnight. Then the cells were incubated with anti-rabbit or anti-mouse IgG . Real-time PCR was conducted by using GoTaq qPCR Master Mix . Data were analyzed and normalized to the β-Actin data.P-value < 0.05 was considered to indicate statistical significance. Kaplan–Meier method and the log-rank test were used to analyze the correlation between IRF2 expression and prognosis. Pearson correlation coefficients were used to analyze the expression correlations between IRF2 and β-catenin.The data are shown as the mean ± standard deviation and were analyzed by Student's t-test, a To address the correlation between the expression of IRF2 and the malignancy of HCC. We analyzed the expression of IRF2 in 14 HCC tumors and paired adjacent tissues. IRF2 was dramatically increased in HCC compared to adjacent non-cancer liver tissues A. To verSince β-catenin mutation occurs in many HCC tumors, we examined the relationship between β-catenin expression and IRF2. Interestingly, we found a positive correlation between IRF2 and β-catenin expression C. To exaSince IRF2 and β-catenin were co-induced in many HCC tumors, this raised the possibility that β-catenin may be a target gene of IRF2, and that IRF2 affects the growth of HCC cells through β-catenin.116kDaPARP, which inhibit cellular apoptosis, and promoted the expression of β-catenin in HCC cells  decreased the expression of endogenous IRF2, and likewise decreased the expression of β-catenin in HCC cells A. IntereNext, to examine the functional effect on apoptosis of the HCC cells, TUNEL staining was performed. Knockdown of IRF2 increased the percentage of apoptotic cells in HepG2 and Huh7 cells B. Furthein situ  cell apoptosis and impaired proliferation through promoting the expression of TP53 se model ,31. FurtG12D-mediated repression of IRF2 subsequently enhanced the expression of CXCL3 that binds to CXCR2 on myeloid-derived suppressor cells (MDSCs) and increases the migration of MDSCs to the CRC microenvironment, which results in higher IRF2 expression and more sensitive response to anti-PD-1 therapy in vivoOthers have shown that KRASin vitro and in vivo [RET induced by miR-449 inactivated β-catenin-mediated transcription, cell proliferation, and transformation of PTC Lenvatinib, used as the first-line treatment for HCC tumors, represses tumor cells by targeting FGF/FGFR, RET protooncogene, and cKIT. The functions of these targets depend on the β-catenin pathway. FGF10 and FGFR-mediated signaling play a crucial role in a β-catenin signaling-dependent manner in liver stem cells, hepatic tumor-initiating stem cells ,21 and C in vivo ,24; The We also analyzed the downstream effects of β-catenin in IRF2-overexpressed/deficient HCC. c-Myc is known as an oncogene and a target of β-catenin We identified IRF2 overexpression as an important mechanism underlying the drug resistance of Lenvatinib in human HCC. The expression of β-catenin and IRF2 was positively correlated in HCC tumors. Silencing endogenous IRF2 downregulated the expression of β-catenin, while overexpressing IRF2 upregulated β-catenin. These findings identify an important function of IRF2 in mediating Lenvatinib resistance of HCC cells. Targeting IRF2 may be a potential strategy to improve the therapeutic effect of Lenvatinib on HCC.This work was sponsored by grants from the NIH NIDDH HHSN276201200017C and P30DK120531 (DAG).Design: Yarong Guo, David. A. Geller; Methodology: Yarong Guo, Qiang Du, Yihe Yan; Clinical and animal data: Yarong Guo, Qiang Du; Data analysis: Yarong Guo, David. A. Geller, Jun Xu; Writing and reviewing manuscript: Yarong Guo, David. A. Geller; Technique support: Yarong Guo, Qiang Du, Yihe Yan; Study supervision: David. A. Geller.The authors declare no conflicts of interest.


Mice were fed with a methionine-choline-deficient (MCD) diet for 8 weeks to induce steatohepatitis-related liver fibrosis and were treated with HO-1 inducer Hemin and inhibitor ZnPP. Mouse sera were collected for the biochemical analysis, and livers were obtained for further histological observation and gene expression analysis. HSC-T6 cells were cultured for the β-catenin and NFAT5 were inhibited by Hemin, but GSK-3β was upregulated compared to the MCD group, which were consistent with the in vitro study. Hemin markedly inhibited the TGF-β1/Smad signaling pathway in both in vivo and in vitro studies.  MCD-fed mice developed marked macrovesicular steatosis, focal necrosis, and inflammatory infiltration and pericellular fibrosis in liver sections. Administration of Hemin could significantly ameliorate the severity of steatosis, inflammation, and fibrosis and also could decrease the serum ALT and AST. We demonstrated that HO-1 induction was able to downregulate the key regulator of the canonical Wnt pathway Wnt1 and the noncanonical Wnt pathway Wnt5a. The downstream factors of the Wnt pathway  Our study demonstrated that HO-1 inhibited the activation of canonical and noncanonical Wnt signaling pathways in NASH-related liver fibrosis. Thus, these results may suggest a new therapeutic strategy for NASH-related liver fibrosis. Nonalcoholic fatty liver disease (NAFLD) has been recognized as the most important cause of chronic liver disease in developed countries and has been of considerable interest in recent years . NAFLD eHeme oxygenase-1 (HO-1), known as heat shock protein 32, is one of the key antioxidant defenses that inhibit ROS by the degradation of heme to carbon monoxide (CO), biliverdin (BV), and iron . Increasβ-catenin signaling pathway [The Wnt signaling pathway, which could be stimulated by hepatic stress, plays a pivotal role in the pathology and physiology of the liver . It is r pathway . HoweverFor this study, we have hypothesized that HO-1 could regulate the Wnt signaling pathway in dietary steatohepatitis-related liver fibrosis. To investigate this hypothesis, we established NASH animal models with a methionine-choline-deficient (MCD) diet and treated mice with HO-1 inducer Hemin and inhibitor ZnPP, simultaneously. The expression of Wnt and downstream genes was detected. We believe that understanding the interactions between the HO-1 and the Wnt signaling pathway in NASH-related fibrosis will lead to the development of therapeutic strategies to fight hepatic dysfunction.ad libitum as previously described [μmol/kg) by intraperitoneal (i.p.) injections three times per week; and (4) MCD+ZnPP group: mice were fed a MCD diet and treated with HO-1 inhibitor ZnPP-IX (20 μmol/kg) by i.p. injections three times per week. At the end of the experiment for 8 weeks, all animals were fasted overnight before being euthanized. The livers were fixed in 10% formalin for histological analysis or snap-frozen in liquid nitrogen followed by storage at -80°C freezer for RNA and protein. All the protocols and procedures were carried out in accordance with the guidelines of the Hebei Committee for Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee of the Hebei Medical University.Male C57BL/6J mice were maintained in a 12 h light/dark cycle with free access to water and food escribed . Mice weμm thick) to observe hepatic steatosis, inflammation, and fibrosis, respectively. The quantification of liver fibrosis was determined by METAVIR Scoring System. Serum ALT and AST levels were measured by the enzymatic kinetic method using an automatic biochemical analyzer  according to the manufacturer's instructions. Hepatic ROS levels were assessed by flow cytometry by dihydrodichlorofluorescein diacetate , the most sensitive probe that becomes fluorescent upon oxidation by ROS.Hematoxylin and eosin staining and Masson's Trichrome staining were performed on paraffin-embedded liver sections  using a Lipofectamine 2000 transfection reagent  according to the manufacturer's instructions. Four hours after treatment, the serum-free medium was replaced with complete medium for 24 h, and the cells were harvested.The HSC-T6 cells were maintained in Dulbecco's modified Eagle's high glucose medium (DMEM), supplemented with 100 U/ml penicillin, 100 g/ml streptomycin , and 10% fetal bovine serum . The cells were incubated at 37°C in a humidified atmosphere with 5% COΔΔCt- method, and gene relative expression was normalized to GAPDH. The specific primers were designed using Primer 5.0 and are shown in Total RNA was isolated from frozen liver tissues and cell palate using a TRIzol Reagent  according to the manufacturer's instructions. The first-strand cDNA was synthesized using a PrimeScript RT Master Mix . qRT-PCR were performed using the ABI 7500 sequence detection system  with a TB Green Premix Ex Taq II kit . The relative amount of each gene was measured using the 2μg/well proteins were loaded onto 10% SDS-PAGE for each sample, and proteins were transferred onto equilibrated polyvinylidene difluoride membranes  by electroblotting. Membranes were blocked with TBST containing 5% milk and incubated overnight at 4°C with primary antibodies against HO-1 , Wnt1 , Wnt5a , β-catenin, GSK-3β, phospho-GSK-3β (Ser9) , and NFAT5 . After incubation with the secondary antibody (ProteinTech), proteins expression was corrected by the amount of β-actin (ProteinTech) in the same sample. Lamin A (ProteinTech) and tubulin (ProteinTech) were used as the marker for nuclear and cytosolic proteins, respectively. The bands were quantified by scanning densitometry using Quantity One 4.6.3 software (Bio Rad).Total protein was extracted by RIPA , and the concentration was measured by bicinchoninic acid (Solarbio). The nuclear and cytoplasmic fractions were isolated by a Nuclear Extraction Kit  according to the manufacturer‘s instruction. 80 3 cells per well for 1, 2, 3, 4, and 5 d. The cells were assessed for proliferation using the Cell Counting Kit-8 , according to the manufacturer's instructions. The experiments were conducted three times, independently.Apoptosis of HSC-T6 cells were assessed using an Annexin V-FITC/PI apoptosis detection kit (BD Bioscience) following the manufacturer's instruction by flow cytometry. To investigate the effect of HO-1 on the proliferation of HSC-T6 cells, five hours after treatment, HSC-T6 cells were reseeded on 96-well plates, at a density of 5 × 10P value of less than 0.05 was considered statistically significant.All data are expressed as mean standard deviation (SD). Statistical analysis was carried out by one-way analysis of variance (ANOVA) and the Student-Newman-Keuls test for evaluating differences between groups using SPSS 19.0. Mice fed with a MCD diet for 8 weeks exhibited macrovesicular steatosis, ballooning degeneration, disordered lobule structure in Zone 3, focal necrosis, and inflammatory infiltration and pericellular fibrosis . AdminisTo further investigate the effect of HO-1 modulation on ROS in the pathogenesis of NASH, we determined hepatic ROS by flow cytometry in each group of mice. The results showed that MCD could significantly induce the increase of hepatic ROS. With the induction of HO-1 by Hemin, the ROS level was significantly decreased. And the hepatic ROS level of mice with the administration of ZnPP was further increased as compared with that of the MCD group mice .β-catenin and NFAT5 were inhibited by the HO-1 inducer Hemin compared to those in the MCD group, but p-GSK-3β and GSK-3β expression was upregulated. However, the expression of Wnt1, Wnt5a, and downstream factors β-catenin and NFAT5 was further increased in mice treated with ZnPP, and the expression of p-GSK-3β and GSK-3β was decreased compared to that of the MCD group (β (Ser9) to total GSK-3β in To address the role of HO-1 on Wnt signaling pathway, we investigated the hepatic expression of canonical and noncanonical Wnt pathways in MCD diet-induced steatohepatitis-related liver fibrosis. We demonstrated that HO-1 induction by Hemin was able to downregulate the key regulator of the canonical Wnt pathway Wnt1 and the noncanonical Wnt pathway Wnt5a Figures . As showCD group . Densitoβ1 (TGF-β1), alpha-smooth muscle actin (α-SMA), collagen I (Col-1), Smad4, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3) were significantly increased in MCD diet-fed mice, which were blunted by Hemin administration. Conversely, hepatic expressions of TGF-β1, α-SMA, Col-1, and Smad4 were further increased by the treatment with ZnPP as compared with those of MCD-induced steatohepatitis-related liver fibrosis (Figures To investigate the mechanisms of HO-1 on steatohepatitis-related liver fibrosis, we assessed the hepatic expression levels of fibrogenic factors. Hepatic mRNA  and prot Figures .μM Hemin for 12 hours. We found that with the increased concentration of Hemin, the HO-1 level was not further increased in HSC-T6 at the concentration of 40 μM (μM Hemin for 12 hours in the following study. After Hemin treatment, the protein expressions of Wnt1 and the downstream factor β-catenin were significantly downregulated. To examine the effects of HO-1 on β-catenin accumulation and GSK-3β activation, we quantified the cytosolic and nuclear β-catenin and total and p-GSK-3β at Ser9 by Western blot. The results showed that nuclear and cytosolic β-catenin were reduced after Hemin administration. More importantly, there was a more significant decrease in nuclear β-catenin (β and GSK-3β expressions were upregulated in Hemin-treated HSC-T6 compared to those of the control (β-catenin. As shown in To determine the effects of HO-1 induction on the Wnt pathway in HSC-T6, we treated HSC-T6 with the HO-1 inducer Hemin and harvested cells at 0, 6, 12, and 24 hours after cell treatment. HO-1 mRNA and protein levels began to increase by 6 hours, and a more striking increase in HO-1 was evident after 12 hours of Hemin treatment, which was not further increased after 24 hours of treatment . We thenof 40 μM . Thus, H-catenin . However control . These dβ1, α-SMA, Col-1, Smad4, p-Smad2, and p-Smad3 in HSC-T6 (β/Smad signaling pathway by blocking canonical and noncanonical Wnt signaling pathways.As shown in n HSC-T6 . These dβ-catenin and NFAT5 were significantly upregulated, but p-GSK-3β and GSK-3β expressions were downregulated in HSC-T6 after si-HO-1 transfection compared to those of the control (β-catenin expression and found that the knockdown of HO-1 could significantly increase β-catenin translocation to the nucleus (β1, α-SMA, Col-1, Smad4, p-Smad2, and p-Smad3 were increased in si-HO-1-transfected HSC-T6 (We next investigated the effect of HO-1 knockdown on the Wnt signaling pathway in HSC-T6. We found that by transfecting HSCs with si-HO-1, the protein level of HO-1 was significantly decreased compared to that of the control . After t control . We furt nucleus . Further nucleus  and inhi nucleus . We also nucleus . Additiod HSC-T6 . These rTo clarify the pathophysiological mechanisms of NASH, an ideal NASH animal model should replicate the pathophysiology of human patients, including insulin resistance, steatohepatitis, and liver fibrosis. The MCD diet is one of the widely employed dietary models for NAFLD . The MCDIn the progression of NAFLD, continuous oxidative stress leads to the imbalance of ROS and the antioxidant capacity of hepatocytes and then causes lipid peroxidation, intracellular organelle damage, and activation of HSCs . Our preβ-catenin pathway and the noncanonical Wnt pathway [β-catenin, GSK-3β, and NFAT5, which was evident by the β-catenin, NFAT5 and inhibition of the phosphorylation of GSK-3β in MCD diet-fed mice. In the canonical Wnt signaling pathway, activated Wnt1 binds to its receptor frizzled and then is stimulated by GSK-3β phosphorylation, preventing the major downstream effector β-catenin phosphorylation, which subsequently translocates to the nucleus from the cytoplasm [β-catenin in hepatocytes developed more severe steatohepatitis and fibrosis with MCD diet compared with wild-type mice [β expression, which is a key factor in activating quiescent HSCs. TGF-β has been implicated to be activated by the Wnt signaling pathway [β, α-SMA, Col-1, Smad4, p-Smad2, and p-Smad3 was detected in in the MCD diet-induced steatofibrosis. Thus, as the causal link between Wnt signaling and liver fibrosis, inhibition of the Wnt signaling pathway may be potential therapeutic targets for preventing steatohepatitis-related liver fibrosis.Wnts belong to a family of secreted proteins involved in cell proliferation, differentiation, and activity . Recentl pathway . However pathway , 30, 31. pathway . Howeverytoplasm . Accumulytoplasm . Accumulype mice . Early rype mice . Researcype mice . Consistype mice . We moni pathway . Parallein vivo and in vitro. Impaired Wnt1 and Wnt5a then regulate their downstream targets β-catenin, GSK-3β, and NFAT5 expression. These results were supported by a recent study that demonstrated that overexpression of HO-1 suppressed the activation of canonical Wnt/β-catenin signaling in renal fibrosis [β, α-SMA, Col-1, Smad4, p-Smad2, and p-Smad3, were significantly inhibited by the HO-1 induction in HSC-T6 with the treatment of Hemin, and transfection of si-HO-1 could further upregulate the expression of these fibrogenic genes, which were consistent with the in vivo study. These results demonstrated that HO-1 may play an antifibrotic role partially via suppressing the Wnt signaling pathway in steatohepatitis-related liver fibrosis.As mentioned above, diet-induced steatohepatitis could upregulate HO-1 and the Wnt signaling pathway. However, the effects of HO-1 modulation on the Wnt signaling pathway have seldom been referred to in NAFLD. In the present study, the association between HO-1 and the Wnt signaling pathway in steatohepatitis-related liver fibrosis is a novel finding for further exploring the pathogenesis of NASH. Our investigations demonstrated that targeted induction of HO-1 by Hemin could suppress the Wnt signaling pathway, including the canonical Wnt1 pathway and the noncanonical Wnt5a pathway both fibrosis . Other rfibrosis . StrikinIn conclusion, our study demonstrated that the Wnt signaling pathway plays a pivotal role in the pathogenesis of diet-induced steatohepatitis-related liver fibrosis. Moreover, it is evident for the first time that HO-1 inhibited the activation of the canonical and noncanonical Wnt signaling pathways in NASH-related liver fibrosis. Thus, these findings may suggest a new therapeutic strategy for NASH-related liver fibrosis.


Attending assessment is a critical part of endoscopic education for gastroenterology fellows. The aim of this study was to develop and validate a concise, web-based assessment tool to evaluate real-time fellow performance in upper endoscopy.We developed the Skill Assessment in Fellow Endoscopy Training (SAFE-T) upper endoscopy tool to capture both summative and formative feedback in a concise, five-part questionnaire. The tool mirrors the previously validated SAFE-T colonoscopy tool and is administered electronically via a web-based application. We evaluated the tool in a prospective study of 15 gastroenterology fellows (5 fellows each from Years 1–3 of training) over the 2018–2019 academic year. An independent reviewer evaluated a subset of these procedures and completed both the SAFE-T and Assessment of Competency in Endoscopy (ACE) upper endoscopy forms for reliability testing.P < 0.001) and second-year cases having lower performance than third-year cases . The mean SAFE-T overall score decreased with increasing case-complexity score, with straightforward compared with average cases  and average compared with challenging cases . In dual-observed procedures, the SAFE-T tool showed excellent inter-rater reliability with a Kappa agreement statistic of 0.815 (P = 0.001). The SAFE-T overall score also highly correlated with the ACE upper endoscopy overall hands-on score .Twenty faculty completed 413 SAFE-T evaluations of the 15 fellows in the study. The mean SAFE-T overall score differentiated each sequential fellow year of training, with first-year cases having lower performance than second-year cases (3.31 vs 4.25, We developed and validated the SAFE-T upper endoscopy tool—a concise and web-based means of assessing real-time gastroenterology fellow performance in upper endoscopy. Learning how to perform endoscopy is a key objective of gastroenterology (GI) fellowship programs . AlthougA handful of assessment tools for upper endoscopy have been developed to help faculty to assess fellow endoscopic skills and guide the delivery of feedback . Of thesTo improve the continuous assessment of fellow endoscopic performance, we recently developed and provided validity evidence for the Skill Assessment in Fellow Training (SAFE-T) tool for colonoscopy . This evOur aim was to develop and validate a concise and analogous SAFE-T upper endoscopy tool that included both summative and formative feedback and utilized a web-based application to facilitate its use as a point-of-care evaluation tool for GI fellow endoscopic performance.We developed a five-part questionnaire specific to upper endoscopy training based on the framework of the SAFE-T colonoscopy tool . The firAll ACGME-accredited general GI fellows in a single GI fellowship program participated in the study. Five fellows each from Years 1–3 of training were included, for a total of 15 fellows. All attending gastroenterologists who supervise fellows performing upper endoscopy were eligible to participate in the study as evaluators. The study was conducted over a 12-month period from July 2018 (start of the academic year) through June 2019. Evaluations were completed at three different clinical sites affiliated with the fellowship program. The study was reviewed by the Institutional Review Board at Partners Healthcare and was given exempt status.We adapted the SAFE-T upper endoscopy tool into a web-based application that was optimized for use on smartphones as well as computers. Participating faculty downloaded the SAFE-T application onto their smartphones and added the associated icon to their smartphone home screen. Selection of the web-based application icon directly navigated the faculty member to a website that contained the five-part SAFE-T upper endoscopy evaluation form. Each faculty member had a unique login and password to access the application. Completed SAFE-T evaluations were electronically logged and transmitted to a password-protected central repository. Prior to each endoscopy session with a fellow, the web-based application sent an automated email reminder to faculty members to complete SAFE-T evaluations for each observed upper endoscopy.SAFE-T upper endoscopy evaluations were solicited and completed for each upper endoscopy performed by a fellow from 15 July 2018 to 30 June 2019. Nested within this prospective data collection, an independent reviewer observed an additional 10 upper endoscopies performed by a fellow  under the guidance of a supervising gastroenterologist in the endoscopy procedure room. The independent reviewer completed the SAFE-T upper endoscopy tool as well as the ACE upper endoscopy form for each of these 10 observed upper endoscopies. The attending gastroenterologist completed the SAFE-T evaluation tool per the normal protocol for these same procedures. The independent reviewer was blinded to the SAFE-T data completed by the attending gastroenterologist for these procedures.n = 19).Surveys on the user experience of the SAFE-T tool were sent to participating faculty who completed at least three prior SAFE-T upper endoscopy evaluations by the conclusion of the study period  and 4  to improve the accuracy of responses. To minimize error from data entry, the web-based application automatically uploaded the completed evaluation forms to a central repository. Prior to the start of the study, we conducted a 2-week trial period  using the web-based application to address any issues with the electronic logging of data.To assess how the SAFE-T upper endoscopy tool correlated with other relevant variables, we compared the mean SAFE-T overall performance scores across (i) trainee year, (ii) successful vs failed intubation of the second part of the duodenum (D2), and (iii) case complexity. We hypothesized that fellows with more training experience would perform better, cases with successful intubation of D2 would be associated with higher performance scores, and more complex cases would be associated with lower performance scores. Given that we expected a steep learning curve for early fellows, we also compared the mean SAFE-T overall performance score for the first half of the academic year vs the second half for the first-year fellows. We hypothesized that the first-year fellows would perform significantly better with more experience during their first year of training.For the subset of dual-observed procedures, we calculated the Kappa agreement statistic to compare the SAFE-T overall performance score between the supervising faculty member and the independent reviewer.r) to assess the correlation between the SAFE-T overall performance score and the overall hands-on score of the ACE upper endoscopy tool.From the data generated by the independent reviewer in these dual-observed procedures, we calculated the Pearson correlation coefficient .We calculated means and 95% confidence intervals (CIs) for continuous variables and counts and percentages for categorical variables. We used independent During the study period, 20 faculty used the SAFE-T tool to complete 413 evaluations of upper endoscopies performed by the 15 fellows in the study . First-yP < 0.001; Year 2 vs Year 3, P < 0.001). The SAFE-T overall performance score also increased over the length of the study period. First-year fellows had significantly higher SAFE-T overall performance scores in the second half of the academic year compared with the first half . Overall, first-year fellows received the lowest percentage of superior SAFE-T overall performance scores at 13.4% , second-year fellows was 4.25 (95% CI 4.12–4.37), and third-year fellows was 4.56 (95% CI 4.44–4.68). The difference between each subsequent year of training was significant .In addition to differentiating fellows based on experience, the mean SAFE-T overall performance score was also significantly higher according to D2 intubation rates . FellowsP < 0.001) and average vs challenging cases (P = 0.042).With increasing case complexity, fellows required additional support from their supervising faculty . The meaP = 0.001). The SAFE-T overall performance score also correlated highly with the previously validated ACE upper endoscopy overall hands-on score .For the 10 dual-observed procedures, the inter-rater reliability for the SAFE-T overall performance score assigned by the supervising faculty member and the independent reviewer was excellent, with a Kappa agreement statistic of 0.815 (The survey response rate for participating faculty was 74% (14/19). All respondents reported that completing a SAFE-T evaluation using the web-based application took <2 minutes. All respondents also agreed or strongly agreed with the statement “it was easy to complete a SAFE-T evaluation form.” The majority of participating faculty (78.6%) also agreed or strongly agreed that the SAFE-T tool encouraged them to deliver feedback to fellows after supervised upper endoscopies.Faculty members emphasized the ease of use and role as a reminder to provide feedback to fellows as particular strengths of the SAFE-T tool in the optional comment section of the survey. One faculty member noted that the tool was “easy to use” and a “helpful reminder to give verbal feedback about each procedure to the fellow.” Another faculty member similarly remarked: “The SAFE-T tool is extremely user-friendly and most importantly, it is an excellent reminder to give feedback.”In this prospective study of ACGME-accredited GI fellows, we developed and provided validity evidence for the SAFE-T upper endoscopy tool as a concise and web-based means of assessing GI fellow performance in upper endoscopy. The SAFE-T overall score differentiated performance based on year of training, D2 intubation, and case complexity. In addition, the SAFE-T overall score showed excellent inter-rater reliability in dual-observed procedures and high correlation with a previously validated tool. Administered electronically via a web application, the SAFE-T tool functions as a point-of-care assessment tool that can readily be used after each supervised upper endoscopy. The vast majority of users reported that the tool was easy to use, took <2 minutes to complete, and encouraged faculty to provide feedback to fellows. By capturing both summative and formative feedback, the SAFE-T upper endoscopy tool may be used by GI fellowship programs to trend fellow performance over time, advise fellows on specific areas to improve, and help faculty tailor teaching to a specific fellow.We provided validity evidence for the SAFE-T upper endoscopy tool as outlined by Downing, including content validity, response process, and relationship to other variables . We desi minutes to complete a SAFE-T upper endoscopy form. Faculty members also all agreed or strongly agreed that it was easy to complete a SAFE-T upper endoscopy form using the web-based application. As such, we believe the SAFE-T upper endoscopy tool can function as a point-of-care assessment tool for real-time assessment in actual clinical practice.Given the limited time that attendees have in their busy schedules to complete trainee evaluations, we placed a priority on designing the SAFE-T upper endoscopy tool as a concise means of assessment that was easy to use. We thus limited the evaluation form to five questions and adapted the tool into a web-based application that was optimized for smartphone use and automatically updated all data to a central repository. Our end-of-study survey provided evidence that we met our design goals, as all faculty respondents noted that it took <2The previously validated ACE and DOPS forms for upper endoscopy are both more comprehensive in their skill assessment than the SAFE-T upper endoscopy form. These two scores require evaluators to rate trainee performance along several cognitive and motor parameters beyond an overall performance score. Of note, the DOPS form does so with 34 distinct ratings of endoscopic skills, whereas the ACE form narrows the skill evaluation to 6 different entities. As a result, these two scoring tools provide more granular data of individual fellow performance in upper endoscopy. However, these data come at the cost of additional questions, which places additional stress on the evaluator and limits their usage for continuous assessment after each procedure. Indeed, the ACE form was completed only after each set of 50 procedures in the validation study . FurtherWe envision the SAFE-T upper endoscopy tool working in cohort with the ACE upper endoscopy form to provide a continuous and comprehensive assessment of a trainee’s endoscopic skills over the entirety of GI fellowship. Given the ease of use and concise nature of the form, the SAFE-T upper endoscopy tool can be completed after each supervised procedure without significant burden on the attending physician. This continuous assessment produces a wealth of data on the individual fellow as well as the entire fellowship. The resultant learning curves allow for quick identification of fellows in need of additional training and the areas to improve help faculty to tailor their teaching to each individual fellow. As the SAFE-T tool lacks a rating system for individual motor and cognitive skills, the ACE upper endoscopy form can then be used at regular intervals  for more comprehensive assessment. By taking advantage of the strengths of each tool, this combined approach produces the most complete assessment of a fellow’s endoscopic performance.The SAFE-T upper endoscopy tool joins the previously validated SAFE-T colonoscopy tool as a more complete means of continuously assessing GI fellow performance in the standard endoscopic procedures outlined by the Gastroenterology Core Curriculum . Both toThis study does have some important limitations. First, this was a single-center study and thus the results may not be generalizable to other fellowship programs. As such, we intend to conduct a multicenter study of the SAFE-T upper endoscopy tool to provide further validity evidence. Second, we were unable to blind faculty to the fellow they were supervising and thus it is possible that faculty ratings were biased by this knowledge (e.g. fellow’s year of training). However, one can imagine that this would be exceedingly difficult and resource-intensive, and thus was similarly not done in prior studies , 10. ThiIn conclusion, the SAFE-T upper endoscopy tool is a valid and concise means of assessing fellow performance in upper endoscopy. Administered via a web-based application, the tool allows for point-of care assessment and joins the previously validated SAFE-T colonoscopy tool to provide an efficient means of evaluating fellow performance in standard endoscopy. When used after each supervised procedure, the SAFE-T tool produces an immense amount of data that allow fellowship programs to track fellow performance over time and identify those who may need additional endoscopic training. Particularly from the formative assessment piece, the tool allows faculty to tailor their teaching to a specific fellow, thereby enriching the educational experience. The SAFE-T tool may also be used to measure the impact of educational interventions on fellow endoscopic performance. As future steps, we aim to make the SAFE-T tool available to any interested fellowship program site and utilize large-scale data to establish performance benchmarks for each fellowship year of training.N.L.K. conceived the idea, developed the study design, and wrote the manuscript. G.K. developed the web-based application and oversaw data collection and statistical analysis with N.L.K. K.E.H., M.L.P., and K.J. assisted with study design/implementation and edited the manuscript. J.R.S. provided guidance on the study idea, design, analysis, and manuscript preparation. All authors approved the final manuscript.This work was supported by the Clinical Education Research Scholars Program of the Department of Medicine at Brigham and Women’s Hospital (award recipient—N.L.K.) and the NIH T32 training grant .