Nuclear-embedded mitochondrial DNA segments (NUMT) preserve a record of ongoing mitochondrial-to-nuclear DNA transfer during evolution, with important implications for disease mechanisms and genome organization. Here, we develop a pangenome graph-based NUMT detection approach, achieving a 2.52-fold improvement in sensitivity and generating a high-resolution human NUMT map comprising 774 fixed and 280 polymorphic events, alongside 74 superpopulation-stratified loci. Notably, NUMTs derived from the 3'-end of mtDNA D-loop are less frequently fixed and exhibit cis-regulatory activity, revealing selective pressures shaping their genomic landscape. We also identify seven NUMTs associated with gene expression or splicing, suggesting their potential modulatory functions. Comparative analysis of complete primate genomes reveals lineage-specific NUMT dynamics, with particularly high rates in the Pan lineage. Furthermore, we uncover two NUMT-derived tandem repeats, establishing them as a novel source of complex variants. In summary, the integrated analysis enhances understanding of NUMT genomic architecture, population dynamics, and evolutionary implications, establishing them as dynamic genomic components of biomedical relevance.

East Asian populations, representing over 20% of the global population, remain critically underrepresented in human genomic studies, limiting our understanding of population-stratified genetic variation and its implications for health and disease. Here we present the first phase of the Asian Pan-Genome project (APG), comprising 320 nearly complete, fully phased haploid genome assemblies from 160 East Asian individuals. These assemblies achieve unprecedented quality, with an average contig N50 of 144.3 megabase pairs and an average quality value of 64.5. Leveraging these superior assemblies, we reveal previously uncharacterized diversity in human repeatome, including population-stratified patterns in centromere satellites and rDNA arrays. Compared to existing global human genome assemblies, the newly generated genomes supplement 152 million base pairs of novel sequences, 355 gene gains, 18,300 structural variation loci and 26 large euchromatic inversions missing from current human pangenomes. We perform population stratification analyses of structural variations, and further resolve the structural haplotypes of complex genomic regions such as Major Histocompatibility Complex and Survival Motor Neuron loci across global pangenomes, exemplifying tandem-duplicate and inversion-rich complex locus architectures in the human genome, respectively. This resource provides a critical foundation for human genetic studies, especially for East Asian populations, promoting more accurate variant discovery, reducing bias, and ultimately advancing the equity and efficacy of genomic medicine.

Haplotype phasing, the process of resolving parental allele inheritance patterns in diploid genomes, is critical for precision medicine and population genetics, yet the underlying optimization is NP-hard, posing a scalability challenge. To address this, we introduce QHap, a haplotype phasing tool that leverages quantum-inspired optimization. By reformulating haplotype phasing as a Max-Cut problem and deploying a GPU-accelerated ballistic simulated bifurcation solver, QHap accelerates phasing while maintaining accuracy comparable to established phasing tools. On the highly polymorphic human major histocompatibility complex region, QHap demonstrates 4- to 20-fold acceleration with zero switch error across multiple long read sequencing platforms. The framework implements two strategies: a read-based method for regional phasing, and a single nucleotide polymorphism-based method that, through quality-weighted probabilistic edge construction, efficiently scales to chromosome-scale tasks. Integration of chromatin conformation capture data extends phase block contiguity by up to 15-fold, enabling near-chromosome-spanning haplotype reconstruction. QHap demonstrates that quantum-inspired algorithms operating on classical hardware offer a promising approach to addressing the growing computational demands of sequencing data, establishing a new paradigm for applying physics-inspired optimization to fundamental challenges in computational genomics.

Epigenetic inheritance is fundamental to human development and disease, yet the mechanisms governing the transmission of DNA methylation across generations remain incompletely understood. In this study, we performed haplotype-resolved, whole-genome DNA methylation profiling in a healthy three-generation Chinese family, leveraging high-depth Oxford Nanopore Technologies (ONT) and PacBio HiFi long-read sequencing, anchored to a proband-specific telomere-to-telomere (T2T) genome assembly. We observed globally conserved bimodal methylation landscapes across all individuals and generations. Stratified analyses revealed clear functional compartmentalization of methylation marks, characterized by distinct hypomethylation in centromeres and hypermethylation in retrotransposons and repetitive elements. Chromosome-resolved analysis of ribosomal DNA (rDNA) arrays demonstrated a domain-specific methylation pattern with hypomethylation in the transcriptional core and hypermethylation in the intergenic spacer, with evidence for age-associated epigenetic drift in the transcriptional core domain. Through de novo identification and validation, we mapped 23 high-confidence imprinting control regions (ICRs) showing robust parent-of-origin-specific methylation, all overlapping known imprinted genes and enriched for regulatory element signatures. Haplotype-resolved X chromosome analysis further uncovered sex- and allele-specific methylation patterns linked to X inactivation dynamics. Together, this pedigree-scale, high-resolution study delineates the landscape and principles of intergenerational DNA methylation inheritance, revealing both conserved and dynamic features shaping the human epigenome.

Peptidylprolyl isomerase like 1 (PPIL1) is a component of the Prp19 complex (Prp19C), which plays a crucial role in spliceosome assembly. However, the role of PPIL1 in hepatocellular carcinoma (HCC) progression remains unclear. Analysis of TCGA and LIRI-JP datasets revealed that elevated expression of components from the Prp19 complex (Prp19C) is associated with poor prognosis in HCC. PPIL1, which significantly exacerbated tumor progression in this complex, would be a promising therapy target. To investigate the effect of PPIL1 on HCC cells, CCK-8, colony formation, and sphere-formation assays were performed to evaluate its influence on proliferation. Wound healing and Transwell assays were conducted to assess migration and invasion capabilities. The results demonstrated that knockdown of PPIL1 significantly attenuated the malignant phenotype of HCC cells by reducing their capability for proliferation, as well as by diminishing their potential for migration, invasion, and stemness. Flow cytometry analysis demonstrated that PPIL1 knockdown induces G2/M phase cell cycle arrest in HCC cells. Western blot analysis showed that knockdown of PPIL1 significantly suppressed the mTOR/ERK/NF-κB signaling cascade and the expression of CHK2. Overall, our results demonstrated that PPIL1 is highly expressed in HCC tissues and is associated with poor prognosis. Functionally, PPIL1 drives the malignant phenotype of HCC cells and modulates the mTOR/ERK/NF-κB pathway and the expression of CHK2.

Hepatocellular carcinoma (HCC) is associated with high mortality due to late diagnosis, recurrence, and limited therapeutic options. Thus, there is an urgent need for novel treatment approaches and new therapeutic targets. Cardiotoxicity resulting from anticancer therapies has become increasingly prominent, leading to a higher risk of cardiovascular diseases among cancer survivors. In our study, through a drug library screening, we identified that the cardiovascular therapeutic agent nebivolol exerts antitumor effects against HCC both in vivo and in vitro. Mechanistically, nebivolol suppressed HCC progression by downregulating RHOQ independently of β1-adrenergic receptors. Furthermore, the combination of nebivolol and lenvatinib synergistically inhibited HCC proliferation both in vivo and in vitro. This study provides a rationale for repurposing nebivolol as a combination strategy for HCC therapy.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality, yet mechanisms sustaining malignant progression remain incompletely defined. Integrative analyses across multiple HCC cohorts identified TUBA1B as one of the most consistently upregulated tubulin isotypes whose high expression associates with poor prognosis and metastasis. In hydrodynamic tail-vein injection (HTVi) models, co-expression of Tuba1b with myr-AKT enhanced liver tumor growth, supporting a tumor-promoting role for Tuba1b in vivo. TUBA1B knockdown suppressed HCC cell proliferation, migration, and invasion in vitro and reduced tumor growth and metastasis in orthotopic models. HTVi-mediated delivery of sgTuba1b suppressed hepatocarcinogenesis in NRasV12/myr-AKT- and MYC/sg-p53 HCC models and prolonged survival in the former, whereas sgTuba1b delivery to normal livers caused no detectable histological abnormalities. Mechanistically, TUBA1B bound and stabilized G3BP2 by restraining TRIM25-dependent K48-linked ubiquitination, thereby promoting G3BP2-IκBα association and sustaining basal NF-κB/p65 transcriptional activity; enforced G3BP2 expression rescued the proliferative and metastatic defects caused by TUBA1B loss. Clinically, combined upregulation of TUBA1B and G3BP2 with elevated p65 activity delineated a high-risk HCC subgroup with the poorest outcomes. Collectively, these findings define a TUBA1B-G3BP2 stability axis that supports malignant phenotypes and highlight a potential therapeutic vulnerability in HCC.

Abstract Summary Long-read sequencing (LRS) has become essential for genome assembly, structural variations (SVs) detection, haplotype phasing and transcript isoform characterization. However, these applications often require manual inspection of read alignment for validation. Existing visualization tools are either interactive genome browsers that are difficult to scale to large datasets or batch-oriented tools that are not optimized for the unique alignment patterns of long-read data. We developed Bamsnap-LRS, an automated command-line tool for high-throughput LRS alignment visualization. It supports long-read-specific features, phased SNP inspection, and publication-ready batch figure generation within a unified framework for genomic, transcriptomic, and haplotype-aware analyses. Availability and Implementation All codes and examples are freely available at https://github.com/comery/Bamsnap-LRS . Contact Chentao Yang ( yangchentao@genomics.cn ) and Yang Zhou ( zhouyang@genomics.cn ). Supplementary information Supplementary Table 1 and Supplementary Figures 1-8 are available at xxx online.

T cell exhaustion is a critical obstacle for durable treatment response in hepatocellular carcinoma (HCC). Developing drugs that control tumor growth and simultaneously bolster immune function is of great significance. Although high-mobility group box 2 (HMGB2) has been reported to be crucial to HCC prognosis, its role in the tumor microenvironment remains unclear. Here, we found HMGB2 + CD8 + T cells as being associated with immune exhaustion and resistance to anti–PD-1 treatment through single-cell RNA sequencing. Mechanistically, HMGB2 impaired the oxidative phosphorylation in CD8 + T cells and inactivated the interferon-γ response in tumor cells, reducing the antitumor effector function. Tannic acid, a specific inhibitor of HMGB2, synergized with PD-1 antibody to attenuate tumor growth and reverse T cell exhaustion. Our findings highlight the unique role of HMGB2 as an immune exhaustion associated molecule. Targeting HMGB2 on both CD8 + T cells and tumor cells contributed to promising treatment strategies for HCC.

Understanding the driving force of centromere dynamics is crucial for deciphering the complexity of eukaryotic evolution and speciation. Here we assembled 67 rice genomes from the Oryza AA group and analyzed >800 nearly complete centromeres. Through de novo annotation of centromeric satellite CEN155 sequences and employing a progressive compression strategy, we quantified the local homogenization and multilayer structures of rice satellite arrays. Our results indicate that genetic innovations in rice centromeres primarily arise from structural variations and centrophilic retrotransposon insertions. The single-base substitution rate in rice centromeres appears to be lower relative to that in chromosome arms. Comparisons of CEN155 arrays, retrotransposons and functional centromeres highlight their dynamic but correlated interplay. Contrary to the KARMA model for Arabidopsis centromere evolution, we propose a hypothesis that retrotransposon invasion probably contributes to the decline of progenitor centromeric satellite arrays and promotes centromere repositioning, as evidenced by extended CENH3 chromatin immunoprecipitation sequencing enrichment beyond the native satellite arrays.

The crab-eating macaques (Macaca fascicularis) and rhesus macaques (Macacamulatta) are pivotal in biomedical and evolutionary research1, 2-3. However, their genomic complexity and interspecies genetic differences remain unclear4. Here, we present a complete genome assembly of a crab-eating macaque, revealing 46% fewer segmental duplications and 3.83 times longer centromeres than those of humans5,6. We also characterize 93 large-scale genomic differences between macaques and humans at a single-base-pair resolution, highlighting their impact on gene regulation in primate evolution. Using ten long-read macaque genomes, hundreds of short-read macaque genomes and full-length transcriptome data, we identified roughly 2 Mbp of fixed-genetic variants, roughly 240 Mbp of complex loci, 16.76 Mbp genetic differentiation regions and 110 alternative splice events, potentially associated with various phenotypic differences between the two macaque species. In summary, the integrated genetic analysis enhances understanding of lineage-specific phenotypes, adaptation and primate evolution, thereby improving their biomedical applications in human disease research.

Serine arginine-rich splicing factor 1 (SRSF1) is a key oncogenic splicing factor in various cancers, promoting abnormal gene expression through post-translational regulation. Although the protumoral function of SRSF1 is well-established, the effects of inhibiting tumor-intrinsic SRSF1 on the tumor microenvironment and its impact on CD8+ T cell-mediated antitumor immunity remain unclear. Our findings indicate that depleting SRSF1 in CD8+ T cells improve antitumor immune function, glycolytic metabolism, and the efficacy of adoptive T cell therapy. The inactivation of SRSF1 in tumor cells reduces transcription factors, including c-Jun, c-myc, and JunB, facilitating glycolytic metabolism reprogramming, which restores CD8+ T cell function and inhibits tumor growth. The small-molecule inhibitor TN2008 targets SRSF1, boosting antitumor immune responses and improving immunotherapy effectiveness in mouse models. We therefore introduce a paradigm targeting SRSF1 that simultaneously disrupts tumor cell metabolism and enhances the antitumor immunity of CD8+ T cells.

ABSTRACTThe crab-eating macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) are widely studied nonhuman primates in biomedical and evolutionary research. Despite their significance, the current understanding of the complex genomic structure in macaques and the differences between species requires substantial improvement. Here, we present a complete genome assembly of a crab-eating macaque and 20 haplotype-resolved macaque assemblies to investigate the complex regions and major genomic differences between species. Segmental duplication in macaques is ∼42% lower, while centromeres are ∼3.7 times longer than those in humans. The characterization of ∼2 Mbp fixed genetic variants and ∼240 Mbp complex loci highlights potential associations with metabolic differences between the two macaque species (e.g.,CYP2C76andEHBP1L1). Additionally, hundreds of alternative splicing differences show post-transcriptional regulation divergence between these two species (e.g.,PNPO). We also characterize 91 large-scale genomic differences between macaques and humans at a single-base-pair resolution and highlight their impact on gene regulation in primate evolution (e.g.,FOLH1andPIEZO2). Finally, population genetics recapitulates macaque speciation and selective sweeps, highlighting potential genetic basis of reproduction and tail phenotype differences (e.g.,STAB1,SEMA3F, andHOXD13). In summary, the integrated analysis of genetic variation and population genetics in macaques greatly enhances our comprehension of lineage-specific phenotypes, adaptation, and primate evolution, thereby improving their biomedical applications in human diseases.

Cancer-associated fibroblasts (CAFs) are the main components in the tumor microenvironment. Tumors activate fibroblasts from quiescent state into activated state by secreting cytokines, and activated CAFs may in turn promote tumor progression and metastasis. Therefore, studies targeting CAFs could enrich the therapeutic options for tumor treatment. In this study, we demonstrate that the content of lipid droplets and the expression of autophagosomes were higher in CAFs than in peri-tumor fibroblasts (PTFs), which was inhibited by 5-(tetradecyloxy)-2-furoic acid(TOFA). The expression of CD36 in CAFs was higher than that in PTFs at both mRNA and protein levels. Inhibition of CD36 activity using either the CD36 inhibitor SSO or siRNA had a significant negative impact on the proliferation and migration abilities of CAFs, which was associated with reduced levels of relevant activated genes (α-SMA, FAP, Vimentin) and cytokines (IL-6, TGF-β and VEGF-α). SSO also inhibited HCC growth and tumorigenesis in nude mice orthotopically implanted with CAFs and HCC cells. Our data further show that CD36+CAFs affected the expression of PD-1 in CTLs leading to CTL exhaustion, and that patients with high CD36 expression in CAFs were correlated with shorter overall survival (OS). Together, our data demonstrate that CAFs were active in lipid metabolism with increased lipid content and lipophagy activity. CD36 may play a key role in the regulation of the biological behaviors of CAFs, which may influence the proliferation and migration of tumor cells by reprograming the lipid metabolism in tumor cells. Thus, CD36 could be an effective therapeutic target for the treatment of HCC.

Nanopore sequencing, a third-generation sequencing technology, has revolutionized the gene sequencing industry with its advantages of long reads, fast speed, real-time sequencing and analysis, and potential in detecting base modifications. This technology allows researchers to sequence longer DNA fragments in a single read, providing more comprehensive genomic information compared to previous methods. Nanopore sequencing operates on electrical signals generated by a nanopore embedded in a membrane separating two electrolyte-filled chambers. When single-stranded DNA (ssDNA) passes through the nanopore, it creates variations in the current that correspond to different DNA bases. By analyzing these current fluctuations with machine learning algorithms, the DNA sequence can be determined. In this study, we introduced several improvements to nanopore sequencing, including nanopore local chemistry sequencing, novel motor and pore proteins, chip design, and basecalling algorithms. Our new nanopore sequencing platform, CycloneSEQ, demonstrated long-duration sequencing (107 hours) on a single chip with high yield (>50 Gb). In human genomic DNA sequencing, CycloneSEQ was able to produce long reads with N50 33.6 kb and modal identity 97.0%. Preliminary findings on human whole-genome de novo assembly, variant calling, metagenomics sequencing, and single-cell RNA sequencing have further highlighted CycloneSEQ's potential across different areas of genomics. ### Competing Interest Statement All authors are employees of the BGI Group. The authors have submitted patent applications related to the methods or results presented in this manuscript.

MOTIVATION:Recent advances in long-read sequencing technologies have significantly facilitated the production of high-quality genome assembly. The telomere-to-telomere (T2T) gapless assembly has become the new golden standard of genome assembly efforts. Several recent efforts have claimed to produce T2T-level reference genomes. However, a universal standard is still missing to qualify a genome assembly to be at T2T standard. Traditional genome assembly assessment metrics (N50 and its derivatives) have no capacity in differentiating between nearly T2T assembly and the truly T2T assembly in continuity either globally or locally. Additionally, these metrics are independent of raw reads, making them inflated easily by artificial operations. Therefore, a gaplessness evaluation tool at single-nucleotide resolution to reflect true completeness is urgently needed in the era of complete genomes. RESULTS:Here, we present a tool called Genome Continuity Inspector (GCI), designed to assess genome assembly continuity at single-base resolution, and evaluate how close an assembly is to the T2T level. GCI utilizes multiple aligners to map long reads from various sequencing platforms back to the assembly. By incorporating curated mapping coverage of high-confidence read alignments, GCI identifies potential assembly issues. Meanwhile, it provides GCI scores that quantify overall assembly continuity on the whole genome or chromosome scales. AVAILABILITY AND IMPLEMENTATION:The open-source GCI code is freely available on Github (https://github.com/yeeus/GCI) under the MIT license.

Background The agamid dragon lizard Pogona vitticeps is one of the most popular domesticated reptiles to be kept as pets worldwide. The capacity of breeding in captivity also makes it emerging as a model species for a range of scientific research, especially for the studies of sex chromosome origin and sex determination mechanisms. Results By leveraging the CycloneSEQ and DNBSEQ sequencing technologies, we conducted whole genome and long-range sequencing for a captive-bred ZZ male to construct a chromosome-scale reference genome for P. vitticeps . The new reference genome is ∼1.8 Gb in length, with a contig N50 of 202.5 Mb and all contigs anchored onto 16 chromosomes. Genome annotation assisted by long-read RNA sequencing greatly expanded the P. vitticeps lncRNA catalog. With the chromosome-scale genome, we were able to characterize the whole Z sex chromosome for the first time. We found that over 80% of the Z chromosome remains as pseudo-autosomal region (PAR) where recombination is not suppressed. The sexually differentiated region (SDR) is small and occupied mostly by transposons, yet it aggregates genes involved in male development, such as AMH , AMHR2 and BMPR1A . Finally, by tracking the evolutionary origin and developmental expression of the SDR genes, we proposed a model for the origin of P. vitticeps sex chromosomes which considered the Z-linked AMH as the master sex-determining gene. Conclusions Our study provides novel insights into the sex chromosome origin and sex determination of this model lizard. The near-complete P. vitticeps reference genome will also benefit future study of amniote evolution and may facilitate genome-assisted breeding. ### Competing Interest Statement The authors have declared no competing interest. CycloneSEQ long-read WGS data, DNBSEQ short-read WGS data, CycloneSEQ long-read RNA-seq data, DNBSEQ short-read RNA-seq data, CycloneSEQ based Pore-C data, and Hi-C data in this study are deposited in NCBI Sequence Read Archive (SRA) under BioProject accession no. PRJNAxxxxxx and in the CNGB Nucleotide Sequence Archive (CNSA) of China National Gene Bank DataBase (CNGBdb) under accession no. CNP0005509. Genome assembly, annotation, other supporting data, and material are available in the GigaScience GigaDB database. * LncRNA : Long non-coding RNA PAR : pseudo-autosomal region SDR : sexually differentiated region EBP : Earth BioGenome Project VGP : Vertebrate Genomes Project WGS : whole genome sequencing PCR : polymerase chain reaction HMW : high molecular weight TRUs : telomeric repeat units T2T : telomere-to-telomere BUSCO : Benchmarking Universal Single-Copy Orthologs QV : consensus quality value RNA-seq : RNA-sequencing TPM : Transcripts Per Million NCBI : The National Center for Biotechnology information UTR : Untranslated Region 5’-UTRs : five prime untranslated regions 3’-UTR : three prime untranslated regions A3 : alternative 3’ splice site A5 : alternative 5’ splice site AF : alternative first exon AL : alternative last exon MX : mutually exclusive exons RI : retained intron SE : skipped exon GSD : genetic sex determination chr2qter : the terminal region of the long arm of chromosome 2 TSD : Temperature-dependent sex determination TSO : template switch oligo sequence RTP : reverse transcription primer NOR : nucleolus organizer region tRNAs : transfer RNAs rRNAs : ribosomal RNAs.

PURPOSE:GPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown. METHODS:Immunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry. RESULTS:The GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8. CONCLUSION:The findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Nuclear receptor subfamily 4 group A member 3 (NR4A3) is a member of the orphan nuclear receptor superfamily, and exhibits transcription factor activity by binding to sequence-specific DNA. Considering that the specific mechanism by which NR4A3 regulates gene transcription in HCC (hepatocellular carcinoma) has not yet been elucidated, our study aimed to explore the transcriptional role of NR4A3 in regulating the target gene CDKN2AIP (CDKN2A interacting protein), which will suppress the development of HCC. Our data show that NR4A3 is downregulated in human HCC tissues, and that low expression of NR4A3 is correlated with poor prognosis, indicating that NR4A3 could act as a tumor suppressor gene in HCC. NR4A3 overexpression suppresses cell proliferation, clone formation, cell cycle arrest at G0/G1 phase and tumor growth in vitro and in vivo and promote DNA damage. NR4A3 could directly regulate the expression of CDKN2AIP at the transcriptional level, suggesting that NR4A3 may play a role as a transcription factor in HCC and may serve as a potential biomarker for predicting prognosis for HCC patients.

PURPOSE:Terminal nucleotidyltransferase 5A (TENT5A), recently predicted as a non-canonical poly(A) polymerase, is critically involved in several human disorders including retinitis pigmentosa, cancer and obesity. However, the exact biological role of TENT5A in hepatocellular carcinoma (HCC) has not been elucidated. METHODS:The transcription level of TENT5A and clinical correlation were analyzed using the LIRI-JP cohort, the TCGA-LIHC cohort, and clinical tissue samples of HCC patients in our laboratory. Proliferation, migration, and invasion were detected with stably TENT5A overexpressing and knockdown HCC cells in vitro and in vivo. Chromatin immunoprecipitation and dual-luciferase reporter assay were performed to verify the binding of the target protein to DNA. Co-immunoprecipitation and GST pull-down assay combined with mass spectrometry (MS) were used to identify protein interactions. RESULTS:Our study presented here shows that TENT5A is downregulated in HCC tissues, suggesting a shorter overall survival for patients. Gain- and loss-of-function experiments reveal that TENT5A suppresses the proliferation and metastasis, and the residue Gly122 is of great importance to the role of TENT5A in HCC. More importantly, EGR1 (Early growth response 1) directly binds to the TENT5A promoter and promotes TENT5A expression. By interacting with RPL35, TENT5A is involved in ribosome biogenesis and exerts a negative regulatory effect on the mTOR pathway. CONCLUSIONS:Our findings illustrate the role of the oncosuppressive function of TENT5A in HCC and suggest that the EGR1/TENT5A/RPL35 regulatory axis may be a promising target for therapeutic strategies in HCC.