General requirements for the production of extracellular vesicles derived from human stem cells.

'General requirements for the production of extracellular vesicles derived from human stem cells' is the first guideline for stem cells derived extracellular vesicles in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the general requirements, process requirements, packaging and labelling requirements and storage requirements for preparing extracellular vesicles derived from human stem cells, which is applicable to the research and production of extracellular vesicles derived from stem cells. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardisation of extracellular vesicles derived from human stem cells.

CCL4 participates in the reprogramming of glucose metabolism induced by ALV-J infection in chicken macrophages.

Interferon and chemokine-mediated immune responses are two general antiviral programs of the innate immune system in response to viral infections and have recently emerged as important players in systemic metabolism. This study found that the chemokine CCL4 is negatively regulated by glucose metabolism and avian leukosis virus subgroup J (ALV-J) infection in chicken macrophages. Low expression levels of CCL4 define this immune response to high glucose treatment or ALV-J infection. Moreover, the ALV-J envelope protein is responsible for CCL4 inhibition. We confirmed that CCL4 could inhibit glucose metabolism and ALV-J replication in chicken macrophages. The present study provides novel insights into the antiviral defense mechanism and metabolic regulation of the chemokine CCL4 in chicken macrophages.

Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

BACKGROUND: Spermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF. METHODS: Semen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis. RESULTS: A total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis. CONCLUSIONS: Proteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Activation of lnc-ALVE1-AS1 inhibited ALV-J replication through triggering the TLR3 pathway in chicken macrophage like cell line.

Endogenous retroviruses (ERVs) are remnants of the historical retroviral infections, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs). We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells. Here, we found that lnc-ALVE1-AS1 was persistently inhibited in chicken macrophages after avian leukosis virus subgroup J (ALV-J) infection. Furthermore, overexpression of lnc-ALVE1-AS1 significantly inhibited the replication of exogenous ALV-J, whereas knockdown of lnc-ALVE1-AS1 promoted the replication of ALV-J in chicken macrophages. This phenomenon is attributed to the induction of antiviral innate immunity by lnc-ALVE1-AS1 in macrophages, whereas knockdown of lnc-ALVE1-AS1 had the opposite effect. Mechanistically, lnc-ALVE1-AS1 can be sensed by the cytosolic pattern recognition receptor TLR3 and trigger the type I interferons response. The present study provides novel insights into the antiviral defense of ERV-derived lncRNAs in macrophages and offers new strategies for future antiviral solutions.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques.

RESEARCH QUESTION: What are the molecular mechanisms leading to human sperm DNA damage? DESIGN: Semen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). RESULTS: A total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. CONCLUSIONS: Differentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

Recent insights into the functions and mechanisms of antisense RNA: emerging applications in cancer therapy and precision medicine.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.

Correlations between elevated basal sperm DNA fragmentation and the clinical outcomes in women undergoing IUI.

Objective: This study aimed to explore the impact of the sperm DNA fragmentation index (DFI) on the clinical outcomes in women undergoing artificial insemination by husband intrauterine insemination (AIH-IUI). Methods: In this retrospective study, the value of sperm DFI was detected by sperm chromatin structure assay (SCSA) in a semen analysis collected before fertility treatment (basal DFI) in 1,500 IUI cycles at the infertility clinic of Northern Jiangsu People's Hospital Reproductive Medicine Center from Jan 2016 to April 2021. Receiver operating characteristic (ROC) curves were used to calculate the cut-off value for the clinical outcomes of IUI, including the biochemical pregnancy rate, clinical pregnancy rate, delivery rate, and live birth rate, and multivariate logistic regression was conducted to analyse the risk factors for clinical outcomes after IUI. Result: In 1,500 IUI cycles, the results showed that there were no statistically significant differences between the normal DFI group and the abnormal DFI group in biochemical pregnancy rate (14.41% vs. 11.3%, P = 0.386), clinical pregnancy rate (12.9% vs. 10.5%, P = 0.433), delivery rate (11.0% vs. 8.9%, P = 0.456), live birth rate (10.9% vs. 8.9%, P = 0.484) or pregnancy loss rate (14.6% vs. 15.4%, P = 1.000). Conclusion: Sperm DFI alone may have limited predictive power for IUI clinical outcomes.

Influence of sperm DNA fragmentation on the clinical outcome of in vitro fertilization-embryo transfer (IVF-ET).

Purpose: To evaluate the effect of elevated sperm DNA fragmentation index (DFI) on fresh and frozen embryo transfer cycles. Methods: A retrospective study was performed with 549 fresh embryo transfer cycles and 1340 frozen embryo transfer cycles after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) from 2016 to 2021. Results: The statistical results of 549 fresh embryo transfer cycles showed that the delivery rate in the normal sperm DFI group (43.9% vs. 27.1%, P = 0.014) was significantly higher than that in the abnormal sperm DFI group, and there were no significant differences in the biochemical pregnancy rate (59.0% vs. 50.8%, P = 0.232), clinical pregnancy rate (53.1% vs. 40.7%, P = 0.072), or miscarriage rate (17.3% vs. 33.3%, P = 0.098) between the two groups. The results of 1340 frozen embryo transfer cycles showed that the biochemical pregnancy rate (57.9% vs. 45.6%, P = 0.006) and clinical pregnancy rate (50.3% vs. 40.7%, P = 0.027) in the normal sperm DFI group were significantly higher than those in the abnormal sperm DFI group. The delivery rate (40.9% vs. 33.3%, P = 0.074) and miscarriage rate (18.6% vs. 18.0%, P = 0.919) were not significantly different between the two groups. Conclusion: The increase of sperm DFI significantly reduced the delivery rate of fresh embryo transfer cycles and the biochemical pregnancy rate and clinical pregnancy rate of frozen embryo transfer cycles.

Global network mapping research landscape and trends of the endogenous retroviruses: a look through bibliometric analysis.

Endogenous retrovirus (ERV) research amalgamates host-retroviral coevolutionary, phylogenomic, infection, immunity, and cellular studies in various hosts ranging from fish to humans. Henceforth, a bibliometric analysis of these publications may aid in the identification of trends in ERV research. It was the foremost bibliographic study, with the key aim to conduct the bibliometric network analysis (e.g. co-authorship, co-occurrence, citation, bibliographic coupling, and co-citation analysis) to find the most prolific authors, organizations, and countries in ERV research, based on the mapping of bibliographic data. Second, the mapping based on text data comprised to chalk out the research trend over the time. The global literature about endogenous retroviruses published between 1985 and Sep 2021 was searched in the Web of Science (Core Collection) database using the "ENDOGENOUS RETROVIRUS" keyword. The bibliometric analysis of this dataset was carried out using VOSviewer version 1.6.17. According to findings, English was the de facto language of these publications, and 2157 were original articles. Among 2939 published documents, "endogenous retrovirus" was the most frequent keyword. Moreover, it revealed the United States as a core contributor to studies on the ERV. The Journal of Virology published a substantial amount of manuscripts in ERV. Robert Koch Institute and Harvard University were leading organizations for research in this field. The application of ERV research from China could be the research hotspot to follow in the coming years. Current bibliometric analysis provides a comprehensive picture of ERV research progress and has highlighted the contribution of different stakeholders.

Global Maps of Avian Leukosis Viruses: Research Trends and Themes Based on Networking.

Avian leukosis virus (ALV) has a tremendous adverse impact on the poultry industry. Since its discovery, research on different aspects of ALV have been published. Due to the vast academic emphasis and economic importance of the ALV infection in poultry worldwide, this bibliometric analysis explored the scientific output associated with ALV utilizing the Web of Science (Core Collection) database. The relevant data were collected using the search query "AVIAN LEUKOSIS VIRUS", further refined by document types (article, book chapter, and proceedings paper). Finally, 1060 items with full records were imported in Plaintext and tab-delimited formats. The data analysis was carried out using MS Excel, VOS viewer, and R (Biblioshiny) software. Chinese and American research institutions produced the majority of papers during study time period. The Journal of Virology and Avian Diseases appeared as the favorite journal/source for publications. Apart from the avian leukosis virus and the ALV-J, the important keywords mentioned included avian leukosis virus subgroup j, chicken, and retrovirus. The analysis revealed substantial findings on ALV research, with a strong research response from the USA and China.

Fasting and Refeeding Affect the Goose Liver Transcriptome Mainly Through the PPAR Signaling Pathway.

Nutrition and energy are essential for poultry growth and production performance. Fasting and refeeding have been widely used to study the effects of nutrition, energy, and related mechanisms in chicken. Previous studies have shown that geese have a strong capacity for fat synthesis and storage; thus, changes in the goose liver transcriptome may be different from those in chicken assessed with a model of fasting and refeeding. However, the responses of the goose liver transcriptome to fasting and refeeding have not yet been addressed. In this study, 36 70-day-old Si Ji geese with similar body weight were randomly assigned to three groups: control (ad libitum feeding), fasting (fasted for 24 h), and refeeding (fast for 24 h followed by 2-h feeding) groups. After treatment, eight geese per group were sacrificed for sample collection. Liver samples from four geese in each group were subjected to transcriptome analysis, followed by validation of differentially expressed genes (DEGs) using quantitative polymerase chain reaction with the remaining samples. As a result, 155 DEGs (73 up-regulated) were identified between the control and fasting groups, and 651 DEGs (321 up-regulated) were identified between the fasting and refeeding groups. The enrichment analyses of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed that fasting mainly influenced material metabolism in the liver, especially lipid metabolism; in contrast, refeeding affected not only lipid metabolism but also glucose and amino acid metabolism. In addition, the peroxisome proliferator-activated receptor (PPAR) signaling pathway may play an important role in lipid metabolism. In conclusion, fasting and refeeding have a strong effect on lipid metabolism in the goose liver; specifically, fasting promotes fatty acid oxidation and inhibits fatty acid synthesis, and refeeding has the opposite effect. The model of fasting and refeeding is suitable for goose nutrition studies.

Screening of MicroRNAs with Potential Systemic Effects Released from Goose Fatty Liver.

Communication between tissues and organs plays an important role in the maintenance of normal physiological functions as well as the occurrence and development of diseases. Communication molecules act as a bridge for interactions between tissues and organs, playing not only a local role in the tissues and organs where they are secreted but also in exerting systemic effects on the whole body via circulation. In this study, blood microRNA-omics analysis of overfed vs. normally fed (control) Landes geese revealed that the content of each of the 21 microRNAs (miRNAs) in the blood of overfed geese was significantly higher than that in the blood of control geese. These miRNAs may have systematic effects in the development of goose fatty liver as well as being candidate markers for the diagnosis of goose fatty liver. We determined the expression of miR-143, miR-455-5p, miR-222a-5p, miR-184, miR-1662, and miR-129-5p using quantitative PCR in goose fatty liver vs. that in normal liver. The expression of these miRNAs, except miR-129-5p, in goose fatty liver was also significantly higher than that in normal liver (P<0.05), suggesting that these blood miRNAs are released from goose fatty liver. In addition, we found that expression of IGFBP5, the predicted target gene of miR-143, was significantly decreased in goose fatty liver vs. the normal liver (P<0.05), indicating that miR-143 may exert both local and systematic effects by inhibiting the expression of IGFBP5, thus promoting the development of goose fatty liver. In conclusion, we identified several miRNAs, including those we validated (i.e., miR-143, miR-455-5p, miR-222a-5p, miR-184, miR-1662, and miR-129-5p) that may serve as candidate markers in the diagnosis of goose fatty liver as well as local and global regulators contributing to the development of goose fatty liver.

An Endogenous Retroviral LTR-Derived Long Noncoding RNA lnc-LTR5B Interacts With BiP to Modulate ALV-J Replication in Chicken Cells.

Infection with the avian leukosis virus subgroup J (ALV-J) impairs host genes and facilitates the establishment of chronic infection and the viral life cycle. However, the involvement of long noncoding RNAs (lncRNAs) in ALV-J infection remains largely unknown. In this study, we identified a novel chicken lncRNA derived from LTR5B of the ERV-L family (namely lnc-LTR5B), which is significantly downregulated in ALV-J infected cells. lnc-LTR5B was localized in the cytoplasm and was relatively high expressed in the chicken lung and liver. Notably, the replication of ALV-J was inhibited by the overexpression of lnc-LTR5B but enhanced when lnc-LTR5B expression was knocked down. We further confirmed that lnc-LTR5B could bind to the binding immunoglobulin protein (BiP), a master regulator of endoplasmic reticulum (ER) function. Mechanistically, lnc-LTR5B serves as a competing endogenous RNA for BiP, restricting its physical availability. Upon ALV-J infection, the reduction of lnc-LTR5B released BiP, which facilitated its translocation to the cell surface. This is crucial for ALV-J entry as well as pro-survival signaling. In conclusion, we identified an endogenous retroviral LTR-activated lnc-LTR5B that is involved in regulating the cell surface translocation of BiP, and such regulatory machinery can be exploited by ALV-J to complete its life cycle and propagate.

Metformin Triggers Apoptosis and Induction of the G0/G1 Switch 2 Gene in Macrophages.

Metformin is a widely used antidiabetic drug for the treatment of type 2 diabetes and has been recently demonstrated to possess anti-inflammatory properties via AMPK-mediated modulation of M2 macrophage activation. However, the anti-inflammatory mechanisms of metformin on inflammatory macrophages are still not fully elucidated. In this study, we found that metformin induced apoptosis in macrophages. In particular, metformin induced apoptosis of M1 macrophages, based on M1 marker genes in apoptotic macrophages. Next, we comprehensively screened metformin-responsive genes in macrophages by RNA-seq and focused on the extrinsic apoptotic signaling pathway. The G0/G1 switch 2 gene (G0S2) was robustly up-regulated by metformin in macrophages. Overexpression of G0S2 significantly induced apoptosis of macrophages in a dose-dependent manner and blunted the function of the crucial anti-apoptotic gene Bcl-2, which was significantly reduced by metformin. These findings show that metformin promoted apoptosis of macrophages, especially M1 macrophages, via G0S2 induction and provides a novel anti-inflammatory mechanism of metformin through induction of macrophage apoptosis.

Fasting and overfeeding affect the expression of the immunity- or inflammation-related genes in the liver of poultry via endogenous retrovirus.

It is known that nutrition and immunity are connected, but the mechanism is not very clear. Endogenous retroviruses (ERV) account for 8 to 10% of the human and mouse genomes and play an important role in some biological processes of animals. Recent studies indicate that the activation of ERV can affect the expression of the immunity- or inflammation-related genes, and the activities of ERV are subjected to regulation of many factors including nutritional factors. Therefore, we hypothesize that nutritional status can affect the expression of the immunity- or inflammation-related genes via ERV. To verify this hypothesis, the nutritional status of animals was altered by fasting or overfeeding, and the expression of intact ERV (ERVK18P, ERVK25P) and immunity- or inflammation-related genes (DDX41, IFIH1, IFNG, IRF7, STAT3) in the liver was determined by quantitative PCR, followed by overexpressing ERVK25P in goose primary hepatocytes and determining the expression of the immunity- or inflammation-related genes. The data showed that compared with the control group (no fasting), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the fasted chickens but decreased in the liver of the fasted geese. Moreover, compared with the control group (routinely fed), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the overfed geese. In addition, overexpression of ERVK25P in goose primary hepatocytes can induce the expression of the immunity- or inflammation-related genes. In conclusion, these findings suggest that ERV mediate the effects of fasting and overfeeding on the expression of the immunity- or inflammation-related genes, the mediation varied with poultry species, and ERV and the immunity- or inflammation-related genes may be involved in the development of goose fatty liver. This study provides a potential mechanism for the connection between nutrition and immunity.

Narrow H3K4me2 is required for chicken PGC formation.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.

Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is known to cause immunosuppression and various types of cancer in chickens. Recent reports have shown that epigenetic changes in DNA and chromatin are widely implicated in the life cycle of diverse viruses, and reversal of these changes in host cells can lead to alterations in the propagation of viruses. In the present study, we found that disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine79 (H3K79) methyltransferase, was upregulated during ALV-J infection in chicken macrophage HD11 cells. Subsequently, we show that targeting DOT1L with a specific inhibitor can significantly decrease the ALV-J replication and viral production. By generating of DOT1L-knockout (KO) HD11 cells using the CRISPR/Cas9 system, we show that deletion of the DOT1L led to an increase in the induction of IFNß and interferon-stimulated genes (ISGs) in HD11 cells in response to ALV-J infection. Importantly, we confirmed that ALV-J infection impaired the activation of the melanoma differentiation-associated protein 5 (MDA5)-mediated-IFN pathway by suppressing the MDA5 expression, and knockout DOT1L rescued the expression of MDA5 and signal transducer and activator of transcription 1 (STAT1), both of which tightly control the antiviral innate immunity. Collectively, it can be deduced from the current data that blocking DOT1L activity or deletion of DOT1L can lead to ALV-J replication inhibition and restoration of the virally suppressed host innate immunity. Thus, we suggest that DOT1L might be a potential drug target for modulating host innate immune responses to combat ALV-J infection.

Aberrant NSUN2-mediated m5C modification of H19 lncRNA is associated with poor differentiation of hepatocellular carcinoma.

RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m6A modification of mRNA, but very little is known about the m5C modification. NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.