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Background: Staphylococcus aureus (S. aureus), a prevalent human pathogen known for its propensity to cause severe infections, has exhibited a growing resistance to antibiotics. Lysine acetylation (Kac) is a dynamic and reversible protein post-translational modification (PTM), played important roles in various physiological functions. Recent studies have shed light on the involvement of Kac modification in bacterial antibiotic resistance. However, the precise relationship between Kac modification and antibiotic resistance in S. aureus remains inadequately comprehended. Methods: We compared the differential expression of acetylated proteins between erythromycin-resistant (Ery-R) and erythromycin-susceptible (Ery-S) strains of S. aureus by 4D label-free quantitative proteomics technology. Additionally, we employed motif analysis, functional annotation and PPI network to investigate the acetylome landscape and heterogeneity of S. aureus. Furthermore, polysome profiling experiments were performed to assess the translational status of ribosome. Results: 6791 Kac sites were identified on 1808 proteins in S. aureus, among which 1907 sites in 483 proteins were quantified. A total of 548 Kac sites on 316 acetylated proteins were differentially expressed by erythromycin pressure. The differentially acetylated proteins were primarily enriched in ribosome assembly, glycolysis and lysine biosynthesis. Bioinformatic analyses implied that Kac modification of ribosomal proteins may play an important role in erythromycin resistance of S. aureus. Western bolt and polysome profiling experiments indicated that the increased Kac levels of ribosomal proteins in the resistant strain may partially offset the inhibitory effect of erythromycin on ribosome function. Conclusions: Our findings confirm that Kac modification is related to erythromycin resistance in S. aureus and emphasize the potential roles of ribosomal proteins. These results expand our current knowledge of antibiotic resistance mechanisms, potentially guiding future research on PTM-mediated antibiotic resistance.
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OBJECTIVES: The rising threat of antibiotic resistance poses a significant challenge to public health. The research on the new direction of resistance mechanisms is crucial for overcoming this hurdle. This study examines metabolic changes by comparing sensitive and experimentally induced ofloxacin-resistant Escherichia coli (E. coli) strains using multi-omics analyses, aiming to provide novel insights into bacterial resistance. METHODS: An ofloxacin-resistant E. coli strain was selected by being exposed to high concentration of ofloxacin. Comparative analyses involving transcriptomics, proteomics, and acetylomics were conducted between the wild-type (WT) and the ofloxacin-resistant (Re-OFL) strains. Enrichment pathways of differentially expressed genes, proteins and acetylated proteins between the two strains were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) method. In addition, the metabolic network of E. coli was mapped using integrated multi-omics analysis strategies. RESULTS: We identified significant differences in 2775 mRNAs, 1062 proteins, and 1015 acetylated proteins between WT and Re-OFL strains. Integrated omics analyses revealed that the common alterations enriched in metabolic processes, particularly the glycolytic pathway. Further analyses demonstrated that 14 metabolic enzymes exhibited upregulated acetylation levels and downregulated transcription and protein levels. Moreover, seven of these metabolic enzymes (fba, tpi, gapA, pykA, sdhA, fumA, and mdh) were components related to the glycolytic pathway. CONCLUSION: The changes of metabolic enzymes induced by antibiotics seem to be a key factor for E. coli to adapt to the pressure of antibiotics, which shed new light on understanding the adaptation mechanism when responding to ofloxacin pressure.
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BACKGROUND: N6-methyladenosine (m6A) mRNA modification and mitochondrial function hold paramount importance in the advancement of metabolic dysfunction-associated steatotic liver disease (MASLD). AIM: The aim of this study was to elucidate the impact of m6A on hepatic mitochondrial dysfunction and provide a novel perspective for a more comprehensive understanding of the pathogenesis of MASLD. METHODS: High-throughput screening methods were used to identify the underlying transcriptome and proteome changes in MASLD model mice. Western blotting, blue native gel electrophoresis (BNGE), dot blot, and Seahorse analyses were conducted to identify and validate the underlying regulatory mechanisms of m6A on mitochondria. RESULTS: In vivo, abnormal m6A modification in MASLD was attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. In vitro, knockdown of Mettl3 inhibited hepatic oxidative phosphorylation (OXPHOS) and the mitochondrial respiratory chain (MRC), while overexpression of Mettl3 promoted these processes. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed mitochondrial OXPHOS. CONCLUSIONS: In MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 axis, particularly by the role of YTHDF1. Modulation of the Mettl3-m6A-YTHDF1 axis has the potential to improve mitochondrial function, alleviate MASLD symptoms, and decrease the likelihood of disease progression.
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Adenosina , Metiltransferasas , Proteínas de Unión al ARN , Metiltransferasas/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Masculino , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Fosforilación Oxidativa , Hígado Graso/metabolismo , Humanos , Modelos Animales de EnfermedadRESUMEN
Designing bifunctional catalysts to reduce the oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) reaction barriers while accelerating the reaction kinetics is perceived to be a promising strategy to improve the performance of Zinc-air batteries. Unsymmetric configuration in single-atom catalysts has attracted attention due to its unique advantages in regulating electron orbitals. In this work, a seesaw effect in unsymmetric Fe-Co bimetallic monoatomic configurations is proposed, which can effectively improve the OER/ORR bifunctional activity of the catalyst. Compared with the symmetrical model of Fe-Co, a strong charge polarization between Co and Fe atoms in the unsymmetric model is detected, in whom the spin-down electrons around Co atoms are much higher than those spin-up electrons. The seesaw effect occurred between Co atoms and Fe atoms, resulting in a negative shift of the d-band center, which means that the adsorption of oxygen intermediates is weakened and more conducive to their dissociation. The optimized reaction kinetics of the catalyst leads to excellent performance in ZABs, with a peak power density of 215 mW cm-2 and stable cycling for >1300 h and >4000 cycles. Flexible Zinc-air batteries have also gained excellent performance to demonstrate their potential in the field of flexible wearables.
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Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator.
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Infecciones por Coxsackievirus , Enterovirus , MicroARNs , Miocarditis , ARN Largo no Codificante , Virosis , Animales , Humanos , Ratones , Infecciones por Coxsackievirus/complicaciones , Infecciones por Coxsackievirus/genética , Enterovirus/genética , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Células HeLa/virología , MicroARNs/genética , MicroARNs/metabolismo , Miocarditis/genética , Miocarditis/metabolismo , Miocarditis/virología , ARN Largo no Codificante/genética , Replicación ViralRESUMEN
BACKGROUND: Pancreatic adenocarcinoma (PAAD) is an aggressive solid tumour characterised by few early symptoms, high mortality, and lack of effective treatment. Therefore, it is important to identify new potential therapeutic targets and prognostic biomarkers of PAAD. METHODS: The Cancer Genome Atlas and Genotype-Tissue Expression databases were used to identify the expression and prognostic model of protocadherin 1 (PCDH1). The prognostic performance of risk factors and diagnosis of patients with PAAD were evaluated by regression analysis, nomogram, and receiver operating characteristic curve. Paraffin sections were collected from patients for immunohistochemistry (IHC) analysis. The expression of PCDH1 in cells obtained from primary tumours or metastatic biopsies was identified using single-cell RNA sequencing (scRNA-seq). Real-time quantitative polymerase chain reaction (qPCR) and western blotting were used to verify PCDH1 expression levels and the inhibitory effects of the compounds. RESULTS: The RNA and protein levels of PCDH1 were significantly higher in PAAD cells than in normal pancreatic ductal cells, similar to those observed in tissue sections from patients with PAAD. Aberrant methylation of the CpG site cg19767205 and micro-RNA (miRNA) hsa-miR-124-1 may be important reasons for the high PCDH1 expression in PAAD. Up-regulated PCDH1 promotes pancreatic cancer cell metastasis. The RNA levels of PCDH1 were significantly down-regulated following flutamide treatment. Flutamide reduced the percentage of PCDH1 RNA level in PAAD cells Panc-0813 to < 50%. In addition, the PCDH1 protein was significantly down-regulated after Panc-0813 cells were incubated with 20 µM flutamide and proves to be a potential therapeutic intervention for PAAD. CONCLUSION: PCDH1 is a key prognostic biomarker and promoter of PAAD metastasis. Additionally, flutamide may serve as a novel compound that down-regulates PCDH1 expression as a potential treatment for combating PAAD progression and metastasis.
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Adenocarcinoma , Neoplasias Pancreáticas , Humanos , Pronóstico , Flutamida , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , ARN , Biomarcadores , Regulación Neoplásica de la Expresión Génica , Protocadherinas , Neoplasias PancreáticasRESUMEN
Osteopontin (OPN), a secreted phosphoglycoprotein, has important roles in tumor growth, invasion and metastasis in numerous types of cancers. Denticleless E3 ubiquitin protein ligase homolog (DTL), one of the CUL4-DDB1-associated factors (DCAFs), has also been associated with the invasion and metastasis of cancer cells. In the present study, OPN was found to induce DTL expression in liver cancer cells, and the results obtained using luciferase activity assays demonstrated that OPN could transcriptionally activate DTL expression in liver cancer cells. Furthermore, the results of the present study demonstrated that OPN could increase the expression of DTL via PI3K/AKT signaling. In conclusion, the present study demonstrated that OPN, as an extracellular matrix protein, is able to promote the growth and invasion of liver cancer cells through stimulation of the expression of DTL via the PI3K/AKT signaling pathway.
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The total number of spikelets (TSPN) and the number of fertile spikelets (FSPN) affect the final number of grains per spikelet in wheat. This study constructed a high-density genetic map using 55K single nucleotide polymorphism (SNP) arrays from a population of 152 recombinant inbred lines (RIL) from crossing the wheat accessions 10-A and B39. Twenty-four quantitative trait loci (QTLs) for TSPN and 18 QTLs for FSPN were localized based on the phenotype in 10 environments in 2019-2021. Two major QTLs, QTSPN/QFSPN.sicau-2D.4 (34.43-47.43 Mb) and QTSPN/QFSPN.sicau-2D.5(32.97-34.43 Mb), explained 13.97%-45.90% of phenotypic variation. Linked kompetitive allele-specific PCR (KASP) markers further validated these two QTLs and revealed that QTSPN.sicau-2D.4 had less effect on TSPN than QTSPN.sicau-2D.5 in 10-A×BE89 (134 RILs) and 10-A×Chuannong 16 (192 RILs) populations, and one population of Sichuan wheat (233 accessions). The alleles combination haplotype 3 with the allele from 10-A of QTSPN/QFSPN.sicau-2D.5 and the allele from B39 of QTSPN.sicau-2D.4 resulted in the highest number of spikelets. In contrast, the allele from B39 for both loci resulted in the lowest number of spikelets. Using bulk-segregant analysis-exon capture sequencing, six SNP hot spots that included 31 candidate genes were identified in the two QTLs. We identified Ppd-D1a from B39 and Ppd-D1d from 10-A and further analyzed Ppd-D1 variation in wheat. These results identified loci and molecular markers with potential utility for wheat breeding and laid a foundation for further fine mapping and cloning of the two loci.
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Prostate cancer (PCa) is one of the most common malignancies in men. Ribosomal protein L22-like1 (RPL22L1), a component of the ribosomal 60 S subunit, is associated with cancer progression, but the role and potential mechanism of RPL22L1 in PCa remain unclear. The aim of this study was to investigate the role of RPL22L1 in PCa progression and the mechanisms involved. Bioinformatics and immunohistochemistry analysis showed that the expression of RPL22L1 was significantly higher in PCa tissues than in normal prostate tissues. The cell function analysis revealed that RPL22L1 significantly promoted the proliferation, migration and invasion of PCa cells. The data of xenograft tumour assay suggested that the low expression of RPL22L1 inhibited the growth and invasion of PCa cells in vivo. Mechanistically, the results of Western blot proved that RPL22L1 activated PI3K/Akt/mTOR pathway in PCa cells. Additionally, LY294002, an inhibitor of PI3K/Akt pathway, was used to block this pathway. The results showed that LY294002 remarkably abrogated the oncogenic effect of RPL22L1 on PCa cell proliferation and invasion. Taken together, our study demonstrated that RPL22L1 is a key gene in PCa progression and promotes PCa cell proliferation and invasion via PI3K/Akt/mTOR pathway, thus potentially providing a new target for PCa therapy.
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Próstata , Neoplasias de la Próstata , Masculino , Humanos , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular Tumoral , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Próstata/patología , Proliferación Celular/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Movimiento Celular/genéticaRESUMEN
Microcystinleucine arginine (MCLR) is an environmental toxin produced by cyanobacteria and is considered to be a potent carcinogen. However, to the best of our knowledge, the effect of MCLR on colorectal cancer (CRC) cell proliferation has never been studied. The aim of the present study was to investigate the effect of MCLR on CRC cell proliferation and the underlying mechanisms. Firstly, a Cell Counting Kit8 (CCK8) assay was conducted to determine cell viability at different concentrations, and 50 nM MCLR was chosen for further study. Subsequently, a longer CCK8 assay and a cell colony formation assay showed that MCLR promoted SW620 and HT29 cell proliferation. Furthermore, western blotting analysis showed that MCLR significantly upregulated protein expression of PI3K, pAkt (Ser473), pGSK3ß (Ser9), ßcatenin, cmyc and cyclin D1, suggesting that MCLR activated the PI3K/Akt and Wnt/ßcatenin pathways in SW620 and HT29 cells. Finally, the pathway inhibitors LY294002 and ICG001 were used to validate the role of the PI3K/Akt and Wnt/ßcatenin pathways in MCLRaccelerated cell proliferation. The results revealed that MCLR activated Wnt/ßcatenin through the PI3K/Akt pathway to promote cell proliferation. Taken together, these data showed that MCLR promoted CRC cell proliferation by activating the PI3K/Akt/Wnt/ßcatenin pathway. The present study provided a novel insight into the toxicological mechanism of MCLR.
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Neoplasias Colorrectales , beta Catenina , Humanos , Leucina/farmacología , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Microcistinas/toxicidad , Arginina , Proliferación Celular , Proteínas Tirosina Quinasas ReceptorasRESUMEN
The jasmonate-regulated protein Ta-JA1 belongs to the monocot chimeric jacalin (MCJ) family and plays a vital role in stress resistance in wheat. However, the impact of wheat polyploidization on Ta-JA1 remains unclear. In this study, 149 members of the MCJ family were identified among members of Triticeae using a genome-wide approach. The genes were resolved into three clades; MCJ genes in each clade were derived from different donor genes during evolution. Segmental duplication may have been the primary driver, compared with tandem duplication, of expansion in the MCJ family of wheat. Gene loss and acquisition occurred during tetraploidization, and the core expansion of the family occurred after tetraploidization. Sequencing data for 2104 accessions of T. aestivum and 99 accessions of T. dicoccoides showed that Ta-JA1-2A and Ta-JA1 were highly conserved in common wheat, and four alleles (TdJA1-Ax2, TdJA1-Ay2, TdJA1-Ax3, and TdJA1-Ay3) were detected in T. dicoccoides. Using gene-specific markers, one AsJA1-B allele was detected in 11 Ae. speltoides accessions and one TuJA1-Ax1 allele was detected in 70 T. urartu accessions. Six alleles were detected on chromosome 2A: TdJA1-Ax1 (13 accessions), TdJA1-Ay1 (57 accessions), TdJA1-Ax2 (23 accessions), TdJA1-Ay2 (42 accessions), TdJA1-Ax3 (29 accessions), and TdJA1-Ay3 (251 accessions). Only one allele (TdJA1-B) on chromosome 2B was detected in 415 T. dicoccoides accessions. A geographical distribution analysis revealed that Israel hosted higher allelic variation than other regions. Quantitative reverse transcription PCR analysis indicated that divergence in expression has occurred among Ta-JA1 alleles and, notably, TdJA1-Ax1 and TdJA1-Ay1 showed significantly higher expression levels than the other four allelic types in T. dicoccoides. The present results contribute to an improved understanding of the effects of polyploidization on the MCJ gene family and the functions of Ta-JA1, and may be useful to enrich common wheat germplasm resources.
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Oxilipinas , Triticum , Alelos , Ciclopentanos , Oxilipinas/metabolismo , Lectinas de Plantas , Triticum/genética , Triticum/metabolismoRESUMEN
Microcystin-LR (MC-LR) is an environmental toxin that is synthesized by cyanobacteria and considered a potential human carcinogen. However, the role of MC-LR in prostate cancer progression has not been elucidated. The purpose of this study was to investigate the effect of MC-LR on prostate cancer cell invasion and its underlying mechanisms. Transwell assay was performed, and the result showed that MC-LR increased DU145 cell invasion in a concentration-dependent manner. The result of Western blot showed that MC-LR promoted ERK phosphorylation, while enhancing VASP and ezrin phosphorylation. Moreover, PD0325901 was used to verify the role of the ERK/VASP/ezrin axis in MC-LR-promoted cell invasion. The results revealed that MC-LR promoted microfilament rearrangement and cell invasion by activating the ERK/VASP/ezrin pathway in DU145 cells. Finally, in vivo assay was performed, and the result suggested that MC-LR promoted p-ERK, p-VASP and p-ezrin expression and local invasion in nude mice model. Taken together, our data proved that MC-LR induced microfilament rearrangement and cell invasion by activating the ERK/VASP/ezrin pathway in DU145 cells.
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Citoesqueleto de Actina , Microcistinas , Animales , Proteínas del Citoesqueleto , Masculino , Toxinas Marinas , Ratones , Ratones Desnudos , Microcistinas/toxicidadRESUMEN
Koolen-de Vries syndrome (KdVS) is a rare disorder caused by haploinsufficiency of KAT8 regulatory NSL complex subunit 1 (KANSL1), which is characterized by intellectual disability, heart failure, hypotonia, and congenital malformations. To date, no effective treatment has been found for KdVS, largely due to its unknown pathogenesis. Using siRNA screening, we identified KANSL1 as an essential gene for autophagy. Mechanistic study shows that KANSL1 modulates autophagosome-lysosome fusion for cargo degradation via transcriptional regulation of autophagosomal gene, STX17. Kansl1+/- mice exhibit impairment in the autophagic clearance of damaged mitochondria and accumulation of reactive oxygen species, thereby resulting in defective neuronal and cardiac functions. Moreover, we discovered that the FDA-approved drug 13-cis retinoic acid can reverse these mitophagic defects and neurobehavioral abnormalities in Kansl1+/- mice by promoting autophagosome-lysosome fusion. Hence, these findings demonstrate a critical role for KANSL1 in autophagy and indicate a potentially viable therapeutic strategy for KdVS.
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Anomalías Múltiples/genética , Discapacidad Intelectual/genética , Mitofagia/genética , Proteínas Nucleares/genética , Anomalías Múltiples/tratamiento farmacológico , Anomalías Múltiples/inmunología , Anomalías Múltiples/patología , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/patología , Corteza Cerebral/citología , Corteza Cerebral/patología , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 17/inmunología , Modelos Animales de Enfermedad , Femenino , Haploinsuficiencia/inmunología , Células HeLa , Humanos , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/inmunología , Discapacidad Intelectual/patología , Isotretinoína/farmacología , Isotretinoína/uso terapéutico , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Ratones Transgénicos , Mitofagia/efectos de los fármacos , Mitofagia/inmunología , Neuronas , Proteínas Nucleares/metabolismo , Cultivo Primario de CélulasRESUMEN
BACKGROUND: Emerging evidence reveals that the initiation and development of human cancers, including colorectal cancer (CRC), are associated with the deregulation of circular RNAs (circRNAs). Our study intended to disclose the role of circ_0026416 in the malignant behaviors of CRC. METHODS: The detection for circ_0026416 expression, miR-545-3p expression, and myosin VI (MYO6) mRNA expression was performed using quantitative real-time PCR (qPCR). CCK-8 assay, colony formation assay, transwell assay, and flow cytometry assay were applied for functional analysis to monitor cell proliferation, migration, invasion, and apoptosis. The protein levels of MYO6 and epithelial mesenchymal-transition (EMT) markers were detected by western blot. Mouse models were used to determine the role of circ_0026416 in vivo. The potential relationship between miR-545-3p and circ_0026416 or MYO6 was verified by dual-luciferase reporter assay and RIP assay. RESULTS: The expression of circ_0026416 was increased in CRC tumor tissues and cell lines. Circ_0026416 downregulation inhibited CRC cell proliferation, colony formation, migration, invasion, and EMT but induced cell apoptosis in vitro, and circ_0026416 knockdown also blocked tumor growth in vivo. MiR-545-3p was a target of circ_0026416, and rescue experiments indicated that circ_0026416 knockdown blocked CRC development by enriching miR-545-3p. In addition, miR-545-3p targeted MYO6 and inhibited MYO6 expression. MiR-545-3p enrichment suppressed CRC cell malignant behaviors by sequestering MYO6. Importantly, circ_0026416 knockdown depleted MYO6 expression by enriching miR-545-3p. CONCLUSION: Circ_0026416 downregulation blocked the development of CRC through depleting MYO6 expression by enriching miR-545-3p. HIGHLIGHTS: 1. Circ_0026416 downregulation inhibits CRC development in vitro and in vivo. 2. Circ_0026416 regulates the expression of MYO6 by targeting miR-545-3p. 3. Circ_0026416 governs the miR-545-3p/MYO6 axis to regulate CRC progression.
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Neoplasias Colorrectales , MicroARNs , Cadenas Pesadas de Miosina/genética , Animales , Neoplasias Colorrectales/genética , Regulación hacia Abajo , Ratones , MicroARNs/genética , Pronóstico , ARN CircularRESUMEN
Raltitrexed has shown efficacy and safety in many tumor types; however, the clinical data on the treatment of hepatocellular carcinoma is rare. In this report, we aim to assess the efficacy and safety of raltitrexed plus oxaliplatin (OXA)-based transarterial chemoembolization (TACE) in patients with unresectable hepatocellular carcinoma (uHCC). Patients with uHCC were recruited from multi-centers in China and assigned randomly to raltitrexed+OXA-based (n=76), fluorouracil+OXA-based (n=76), and doxorubicin+OXA-based (n=75) TACE treatment. The primary end point was overall survival (OS). Tumor response was assessed using response evaluation criteria in solid tumors (RECIST), modified response evaluation criteria in solid tumors (mRECIST), and European Association for the Study of the Liver criteria (EASL). Safety and toxicity were evaluated using the National Cancer Institute Common Toxicity Criteria. The raltitrexed group showed a better disease control rate evaluated using RECIST (raltitrexed vs. fluorouracil vs. doxorubicin: 96.1 vs. 84.2 vs. 86.7%, P=0.05) and a better overall response rate on the basis of mRECIST (67.1 vs. 47.4 vs. 50.7%, P=0.03) and EASL (67.1 vs. 47.4 vs. 49.3%, P=0.02). The median OS and median progression-free survival (PFS) were higher in the raltitrexed group (median OS: 13.4 vs. 9.6 vs. 8.5 months; median PFS: 6.7 vs 4.9 vs 4.6 months). The most common toxicities included elevated aspartate aminotransferase (78.9 vs. 86.8 vs. 81.3%) and abdominal nonspecific pain (68.4 vs. 81.6 vs. 78.7%). No significant differences were found in the overall number of patients who experienced any toxicity. Raltitrexed plus OXA-based TACE suggested a safe and efficacious regimen in uHCC patients. The results warrant further clinical investigation.