Differentiation and immunosuppressive function of CD19^(+)CD24^(hi)CD27^(+) regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway

Background: Regulatory B cells(Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs(miRNAs), mi R-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and mi R-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs(m Bregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. Methods: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce mi R-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. Results: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of m Bregs in the circulating blood were significantly impaired. mi R-29a-3p was found to be a regulator of m Bregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5(NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of m Bregs. The inhibition of mi R-29a-3p in CD19~+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into m Bregs. In addition, the observed enhancement of differentiation and immunosuppressive function of m Bregs upon mi R-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. Conclusions: mi R-29a-3p was found to be a crucial regulator for m Bregs differentiation and immunosuppressive function. Silencing mi R-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

Biotin-modified Galactosylated Chitosan-gene Carrier in Hepatoma Cells Targeting Delivery

Our previous studies have successfully grafted biotin and galactose onto chitosan(CS)and synthesized biotin modified galactosylated chitosan(Bio-GC).The optimum N/P ratio of Bio-GC and plasmid DNA was 3:1.At this N/P ratio,the transfection efficiency in the hepatoma cells was the highest with a slow release effect.Bio-GC nanomaterials exhibit the protective effect of preventing the gene from nuclease degradation,and can target the transfection into hepatoma cells by combination with galactose and biotin receptors.The transfection rate was inhibited by the competition of galactose and biotin.Bio-GC nanomaterials were imported into cells’cytoplasm by their receptors,followed by the imported exogenous gene transfected into the cells.Bio-GC nanomaterials can also cause inhibitory activity in the hepatoma cells in the model of orthotopic liver transplantation in mice,by carrying the gene through the blood to the hepatoma tissue.Taken together,bio-GC nanomaterials act as gene vectors with the activity of protecting the gene from DNase degradation,improving the rate of transfection in hepatoma cells,and transporting the gene into the cytoplasm in vitro and in vivo.Therefore,they are efficient hepatoma-targeting gene carriers.

Predicting the prognosis of hepatic arterial infusion chemotherapy in hepatocellular carcinoma

Hepatic artery infusion chemotherapy(HAIC)has good clinical efficacy in the treatment of advanced hepatocellular carcinoma(HCC);however,its efficacy varies.This review summarized the ability of various markers to predict the efficacy of HAIC and provided a reference for clinical applications.As of October 25,2023,51 articles have been retrieved based on keyword predictions and HAIC.Sixteen eligible articles were selected for inclusion in this study.Comprehensive literature analysis found that methods used to predict the efficacy of HAIC include serological testing,gene testing,and imaging testing.The above indicators and their combined forms showed excellent predictive effects in retrospective studies.This review summarized the strategies currently used to predict the efficacy of HAIC in middle and advanced HCC,analyzed each marker's ability to predict HAIC efficacy,and provided a reference for the clinical application of the prediction system.

Diagnosis and therapy of tacrolimus toxicity in a liver transplant recipient during COVID-19 treatment

To the Editor:SARS-CoV-2,the pathogen responsible for the pandemic of coronavirus disease 2019(COVID-19),has had profound impacts on human health,and its antagonist Paxlovid is a commonly used treatment option[1].However,treatment selection for immunosuppressed patients,such as liver recipients,remains uncertain due to potential drug interactions and the risk of immunosuppressant dosage adjustment,which can cause liver injury[2].

Double guidewire technique vs transpancreatic precut sphincterotomy in difficult biliary cannulation

AIM:To compare the outcomes between doubleguidewire technique(DGT) and transpancreatic precut sphincterotomy(TPS) in patients with difficult biliary cannulation.METHODS:This was a prospective,randomized study conducted in single tertiary referral hospital in Korea.Between January 2005 and September 2010.A total of 71 patients,who bile duct cannulation was not possible and selective pancreatic duct cannulation was achieved,were randomized into DGT(n = 34) and TPS(n = 37) groups.DGT or TPS was done for selective biliary cannulation.We measured the technical success rates of biliary cannulation,median cannulation time,and procedure related complications.RESULTS:The distribution of patients after randomization was balanced,and both groups were comparable in baseline characteristics,except the higher percentage of endoscopic nasobiliary drainage in the DGT group(55.9% vs 13.5%,P < 0.001).Successful cannulation rate and mean cannulation times in DGT and TPS groups were 91.2% vs 91.9% and 14.1 ± 13.2 min vs 15.4 ± 17.9 min,P = 0.732,respectively.There was no significant difference between the two groups.The overall incidence of post-endoscopic retrograde cholangiopancreatography(ERCP) pancreatitis was 38.2% vs 10.8%,P < 0.011 in the DGT group and the TPS group;post-procedure pancreatitis was significantly higher in the DGT group.But the overall incidence of post-ERCP hyperamylasemia was no significant difference between the two groups;DGT group vs TPS group:14.7% vs 16.2%,P < 1.0.CONCLUSION:When free bile duct cannulation was difficult and selective pancreatic duct cannulation was achieved,DGT and TPS facilitated biliary cannulation and showed similar success rates.However,post-procedure pancreatitis was significantly higher in the DGT group.

Silencing SMYD3 in hepatoma demethylates RIZI promoter induces apoptosis and inhibits cell proliferation and migration

AIM： To investigate the role of SMYD3 in hepatocellular carcinoma （HCC） development and progression and to verify whether its regulation activity was through RIZ1 inactivation. METHODS： Expression of SMYD3 in HCC cell lines and tissues were measured; silencing of SMYD3 by RNA interference （RNAi） was effectuated, hepatoma cell proliferation, migration and apoptosis were tested, with RIZl CpG promoter methylation, and corresponding mRNA expression were investigated. RESULTS： SMYD3 over-expression in HCC was associated with RIZl hypermethylation and mRNA down-expression. Suppression of SMYD3 expression de- methylated RIZl CpG promoter （P 〈 0.01） and increased RIZl mRNA expression （P 〈 0.01）. Consequently, SMYD3 down-expression with RIZl de-methylation strongly inhibited hepatoma cell growth （MTT inhibitory rates： Pgenesil-1-s1 60.95%± 7.97%, Pgenesil-1-s2 72.14% ± 9.68% vs Pgenesil-1-hk 6.89% ± 4.12%, P 〈 0.01） and migration （Pgenesil-1-s1 4.24% ± 1.58%, Pgenesil- 1-s1 4.87% ± 0.73% vs Pgenesil-1 19.03% ± 4.63%, Pgenesil-1-hk 19.95% ±5.21%, P 〈 0.01） and induced apoptosis （FCM subG1 phase Pgenesil-1-s1 19.07% ＋ 1.78%, Pgenesil-1-s2 17.68% ± 2.36% vs Pgenesil-1 0.47% ± 0.12%, Pgenesil-1-hk 1.46% ± 0.28%, P 〈 0.01. TUNEL-positive cells： Pgenesil-1-s1 40.24%± 5.18%, Pgenesil-1-s2 38.48% ± 4.65% vs Pgenesil-1 2.1B% - 1.34%, Pgenesil-1-hk 2.84%± 1.22%, P 〈 0.01） in HepG2 cells. CONCLUSION： These results demonstrate that SMYD3plays a critical role in the carcinogenesis and progression of HCC, The proliferation, migration induction and apoptosis inhibition activities of SMYD3 may be mediated through RIZl CpG promoter hypermethylation.

Antiviral effects of hepatitis B virus S gene-specific anti-gene locked nucleic acid in transgenic mice

AIM To assess the antiviral effects of hepatitis B virus(HBV) S gene-specific anti-gene locked nucleic acid(LNA) in transgenic mice.METHODS Thirty HBV transgenic mice were acclimatized to laboratory conditions and positive for serum HBV surface antigen(HBs Ag) and HBV DNA, were randomly divided into 5 groups(n = 7), including negative control(blank control, unrelated sequence control), positive control(lamivudine, anti-sense-LNA), and anti-gene-LNA experimental group. LNA was injected into transgenic mice by tail vein while lamivudine was administeredby gavage. Serum HBV DNA and HBs Ag levels were determined by fluorescence-based PCR and enzymelinked immune sorbent assay, respectively. HBV S gene expression amounts were assessed by reverse transcription polymerase chain reaction. Positive rates of HBsA g in liver cells were evaluated immunohistochemistry.RESULTS Average rate reductions of HBs Ag after treatment on the 3 rd, 5 th, and 7 th days were 32.34%, 45.96%, and 59.15%, respectively. The inhibitory effect of antigene-LNA on serum HBs Ag peaked on day 7, with statistically significant differences compared with pretreatment(0.96 ± 0.18 vs 2.35 ± 0.33, P < 0.05) and control values(P < 0.05 for all). Average reduction rates of HBV DNA on the 3 rd, 5 th, and 7 th days were 38.55%, 50.95%, and 62.26%, respectively. This inhibitory effect peaked on the 7 th day after treatment with anti-gene-LNA, with statistically significant differences compared with pre-treatment(4.17 ± 1.29 vs 11.05 ± 1.25, P < 0.05) and control values(P < 0.05 for all). The mR NA levels of the HBV S gene(P < 0.05 for all) and rates of HBsA g positive liver cells(P < 0.05 for all) were significantly reduced compared with the control groups. Liver and kidney function, and histology showed no abnormalities. CONCLUSION Anti-gene-LNA targeting the S gene of HBV displays strong inhibitory effects on HBV in transgenic mice, providing theoretical and experimental bases for gene therapy in HBV.

Diverse Roles of Immune Cells in Transplant Rejection and Immune Tolerance

Organ transplant rejection(OTR)is a complex immune reaction involving multiple cells,and it determines graft survival and patient prognosis.At present,most transplant recipients are administered a combination of immunosuppressive and biological agents to protect them from OTR.However,immunosuppressive agents negatively impact the immune system of the patients,causing them to suffer from serious complications,such as chronic infection and malignant tumors.Therefore,a thorough understanding of the mechanisms involved in immune tolerance and immune rejection with regard to organ transplant(OT)is essential for developing better treatment options and improving patient outcomes.This article reviews the role of immune cells in OTR and organ transplant tolerance(OTT),including the novel cell therapies that are currently under clinical trials for transplant recipients.

An approach to timing selection of emergency operation for acute cholangitis of severe type

This article reports on a retrospective analysis on 121 patients and a prospectivestudy on 21 patients with acute cholangitis of severe type(ACST)for a study on the timing se-lection of emergency operation for ACST.Twenty two clinical,biological,etiologic,pathologicand operative variables were analyzed.Simple regression revealed 11 factors with prognosticsignificance,but multivariate analysis detected only 6 factors with independent significance inpredicting mortality(age,mean blood pressure,generalized peritonitis,serum albumin-globin ra-tio,blood culture,and the number of failed organs and systems).The results indicate that theclinical principles of treatment for ACST should be the combination of medical and surgicaltreatment.Active conservative treatment is practically applicable to the majority of ACST,espe-cially,those with short history and few complication.Prognostic mathematical model of ACSTdoes good for its timing selection of emergency operation.A critical level of 0.40 is determinedto be the discriminant score for emergency bile duct drainage.The model seems to have advan-tages over the traditional method.

Radiofrequency ablation vs surgical resection in elderly patients with hepatocellular carcinoma in Milan criteria

BACKGROUND Surgical resection and radiofrequency ablation(RFA)represent two possible strategy in treatment of hepatocellular carcinoma(HCC)in Milan criteria.AIM To evaluate short-and long-term outcome in elderly patients(>70 years)with HCC in Milan criteria,which underwent liver resection(LR)or RFA.METHODS The study included 594 patients with HCC in Milan criteria(429 in LR group and 165 in RFA group)managed in 10 European centers.Statistical analysis was performed using the Kaplan-Meier method before and after propensity score matching(PSM)and Cox regression.RESULTS After PSM,we compared 136 patients in the LR group with 136 patients in the RFA group.Overall survival at 1,3,and 5 years was 91%,80%,and 76%in the LR group and 97%,67%,and 41%in the RFA group respectively(P=0.001).Diseasefree survival at 1,3,and 5 years was 84%,60%and 44%for the LR group,and 63%,36%,and 25%for the RFA group(P=0.001).Postoperative Clavien-Dindo IIIIV complications were lower in the RFA group(1%vs 11%,P=0.001)in association with a shorter length of stay(2 d vs 7 d,P=0.001).In multivariate analysis,Model for End-stage Liver Disease(MELD)score(>10)[odds ratio(OR)=1.89],increased value of international normalized ratio(>1.3)(OR=1.60),treatment with radiofrequency(OR=1.46),and multiple nodules(OR=1.19)were independent predictors of a poor overall survival while a high MELD score(>10)(OR=1.51)and radiofrequency(OR=1.37)were independent factors associated with a higher recurrence rate.CONCLUSION Despite a longer length of stay and a higher rate of severe postoperative complications,surgery provided better results in long-term oncological outcomes as compared to ablation in elderly patients(>70 years)with HCC in Milan criteria.

Glucose Metabolic Alteration of Cerebral Cortical Subareas in Rats with Renal Ischemia/Reperfusion Based on Small-Animal Positron Emission Tomography

Objective:To investigate glucose metabolic alterations in cerebral cortical subareas using ^(18)F-labeled glucose derivative fluorodeoxyglucose(FDG)micro-positron emission tomography(PET)scanning in a rat renal ischemia/reperfusion(RIR)model.Methods:Small-animal PET imaging in vivo was performed with ^(18)F-labeled FDG as a PET tracer to identify glucose metabolic alterations in cerebral cortical subregions using a rat model of RIR.Results:We found that the average standardized uptake value(SUV_(average))of the cerebral cortical subareas in the RIR group was significantly increased compared to the sham group(P<0.05).We also found that glucose uptake in different cortical subregions including the left auditory cortex,right medial prefrontal cortex,right para cortex,left retrosplenial cortex,right retrosplenial cortex,and right visual cortex was significantly increased in the RIR group(P<0.05),but there was no significant difference in the SUV_(avcrage) of right auditory cortex,left medial prefrontal cortex,left para cortex,and left visual cortex between the two groups.Conclusion:The ^(18)F-FDG PET data suggests that RIR causes a profound shift in the metabolic machinery of cerebral cortex subregions.

Protective effect of fu-qi granule on carbon tetrachloride-induced liver fibrosis in rats

AIM： To investigate the effcacy of fu-qi granule （FQG） on carbon tetrachloride （CCl4） induced liver fbrosis in rats and the underlying mechanisms. METHODS： Sixty rats were randomly divided into six groups： normal control group, CCl4 induced liver fbrosis group, AnluoHuaxianWan group and three treatment groups of FQG. Treatment of rats with intraperitoneal injection of carbon tetrachloride solution at 0.3 mL per 100 g body weigh twice a week for 8 wk. The normal control group the rats were given the media （olive oil） at the same time. In the frst 2 wk, rats were raised with feedstuff （80% corn meal, 20% lard, 0.5% cholesterol）. Serum samples were collected for alanine transaminase, aspartate aminotransferase, alkaline phosphatase, albumin, total protein assay and typical histopathological changes was observed in Hematoxylin-eosin staining sections. Smooth muscle alpha actin （α-SMA） was analyzed with immunohistochemistry. Mammalian target of rapamycin （mTOR） and hypoxia-inducible factor-1 （HIF-1α） expressions were detected by Western blot-ting. Tissue inhibitor of matrix metalloproteinases-1 （TIMP-1） and matrix metalloproteinases-9 （MMP-9） were measured with semi-quantitative reverse transcriptase-polymerase chain reaction.RESULTS： FQG significantly reduced the serum levels of alanine transaminase, aspartate aminotransferase, alkaline phosphatase and increased the serum contents of albumin, total protein in rats with liver fibrosis. Moreover, FQG promoted extracellular matrix degradation by increasing MMP-9 and inhibiting TIMP-1 and α-SMA. mTOR and HIF-1α expression in liver significantly decreased in the rats treated with FQG. CONCLUSION： The results indicated that FQG signi-fcantly reverse fbrosis induced by CCl4, which should be developed as a new and promising preparation for the prevention of liver fbrosis.

Specific Patterns of Spinal Metabolite Ratio Underlying a-Me-5-HTevoked Pruritus Compared with Compound 48/80 Based on Proton Nuclear Magnetic Resonance Spectroscopy

Summary:Mechanisms of pruritus are implicated in the dysregulation of the metabolites in the spinal cord.We investigated pruritus behavioral testing in three groups of young adult male C57B1/6 mice,including one group treated with normal saline,while the other groups intradermally injected with a-Me-5-HT(histamine-independent pruritogen),compound 48/80(histaminedependent pruritogen)at the nape skin of the neck,respectively.Proton nuclear magnetic resonance spectroscopy(MRS)was used to compare spinal metabolites from the vertebral cervical among three groups,and to study the association of spinal metabolite ratio and pruritus intensity.The MRS-measured N-acetylaspartate-to-myoinositol ratio(NAA/Ins)was significantly correlated with the number of scratches between normal saline group and 48/80 group or a-Me-5-HT group(both P<0.0001),indicating that NAA/Ins may be a robust surrogate marker of histamine-independent/dependent pruritogen.There was significant difference in Glu/Ins between normal saline group and 48/80 group(P=0.017),indicating that Glu/Ins may be a surrogate marker of histamine-dependent pruritogen,while GABA/Ins was highly significantly different between normal saline group and a-Me-5-HT group(P=0.008),suggesting that GABA/Ins may be a surrogate marker of histamineindependent pruritogen.MRS may reflect the extent of pruritus intensity elicited by a-Me-5-HT and compound 48/80 with sensitivity similar to the number of scratches,and above potential markers need to be further validated in pre-clinical and clinical treatment trials.

Mechanism insights and therapeutic intervention of tumor metastasis:latest developments and perspectives

Metastasis remains a pivotal characteristic of cancer and is the primary contributor to cancer-associated mortality.Despite its significance,the mechanisms governing metastasis are not fully elucidated.Contemporary findings in the domain of cancer biology have shed light on the molecular aspects of this intricate process.Tumor cells undergoing invasion engage with other cellular entities and proteins en route to their destination.Insights into these engagements have enhanced our comprehension of the principles directing the movement and adaptability of metastatic cells.The tumor microenvironment plays a pivotal role in facilitating the invasion and proliferation of cancer cells by enabling tumor cells to navigate through stromal barriers.Such attributes are influenced by genetic and epigenetic changes occurring in the tumor cells and their surrounding milieu.A profound understanding of the metastatic process’s biological mechanisms is indispensable for devising efficacious therapeutic strategies.This review delves into recent developments concerning metastasis-associated genes,important signaling pathways,tumor microenvironment,metabolic processes,peripheral immunity,and mechanical forces and cancer metastasis.In addition,we combine recent advances with a particular emphasis on the prospect of developing effective interventions including the most popular cancer immunotherapies and nanotechnology to combat metastasis.We have also identified the limitations of current research on tumor metastasis,encompassing drug resistance,restricted animal models,inadequate biomarkers and early detection methods,as well as heterogeneity among others.It is anticipated that this comprehensive review will significantly contribute to the advancement of cancer metastasis research.

The application of real-time indocyanine green fluorescence cholangiography in laparoscopic living donor left lateral sectionectomy

Background:The judgment of the division point of the bile duct has always been one of the difficulties of laparoscopic left lateral sectionectomy(LLLS).The purpose of this study was to assess the effects of indocyanine green(ICG)fluorescence cholangiography during LLLS on the occurrence of biliary complications in both donors and recipients.The optimal dose and injection time of ICG were also investigated.Methods:This is a retrospective cohort study.From October 2016 to December 2022,the clinical data of 103 donors who underwent LLLS and relevant recipients were retrospectively analyzed.According to whether ICG fluorescence cholangiography was used,they were divided into a non-ICG group(n=46)and an ICG group(n=57).Biliary complications were observed and the optimal dose and injection time of ICG were explored.Results:Three donors in the non-ICG group suffered from bile leakage.Four grafts had multiple bile duct openings and biliary complications were observed in the relevant recipients who received these grafts in the non-ICG group.Two recipients had bile leakage,and the other two had biliary stenosis.There was no biliary complications both in donors and recipients in the ICG group.The fluorescence intensity of the liver was 108.1±17.6 at a dose of 0.004 mg/kg 90 minutes after injection,significantly weaker than that at 0.05 mg/kg 30 minutes(200.3±17.6,P=0.001)and 90 minutes after injection(140.2±15.4,P=0.001).The fluorescence intensity contrast value at a dose of 0.004 mg/kg was stronger than that at 0.05 mg/kg,both measured 90 minutes after injection(0.098±0.032 vs.0.078±0.022,P=0.021).Conclusions:ICG fluorescence cholangiography is safe and feasible in LLLS.It reduces biliary complications in both donors and recipients.The optimal ICG dose was 0.004 mg/kg,and 90 minutes after injection was the best observation time.ICG fluorescence cholangiography is recommended for routine use in LLLS.

RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma

Background and Aims:Chronic hepatitis caused by hepatitis B virus(HBV)infection is a leading cause of hepatocellular carcinoma(HCC).We investigated the roles of oncogenic HBV infection-associated long noncoding RNAs in HCC.Methods:Bioinformatics analysis of data from the Cancer Genome Atlas(TCGA)was performed to screen potential oncogenic HBV-related lncRNAs.Next,we assessed their expression in clinical samples and investigated their correlation with clinical characteristics.The detailed oncogenic effects were analyzed by performing in vitro and in vivo studies.Results:RP11-40C6.2,an HBV infection-related lncRNA,was identified by analysis of the TCGA–Liver Hepatocellular Carcinoma database.Gene Set Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of differentially expressed genes revealed a strong association of RP11-40C6.2 with the Hippo signaling pathway.RP11-40C6.2 was overexpressed in HCC patients with HBV infection compared to those without HBV infection.RP11-40C6.2 transcription showed a positive association with HBV-X protein(HBx),but not HBV core protein(HBc)expression,both of which are carcinogenic proteins.Luciferase gene reporter and ChIP assays revealed that YAP1/TAZ/TEADs complex enhanced RP11-40C6.2 transcription by binding to its promoter area.RP11-40C6.2 showed oncogenic characteristics in HCC cell lines and in animal models that were mediated via activation of YAP1.In vitro ubiquitylation assay revealed that RP11-40C6.2 can promote the stabilization of YAP1 by stopping phosphorylation at its s127 residue and further stopping its degradation through binding to 14-3-3.Conclusions:RP11-40C6.2 is an HBV infection-related lncRNA that exerts its oncogenic effects by targeting the Hippo signaling pathway.

Inhibitory effect of retroviral vector containing anti-sense Smad_4 gene on Ito cell line, LI90

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy

HIF-1α-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation

N6-methyladenosine(m6A),and its reader protein YTHDF1,play a pivotal role in human tumorigenesis by affecting nearly everystage of RNA metabolism.Autophagy activation is one of the ways by which cancer cells survive hypoxia.However,the possibleinvolvement of m6A modification of mRNA in hypoxia-induced autophagy was unexplored in human hepatocellular carcinoma(HCO).In this study,specific variations in YTHDF1 expression were detected in YTHDF1-overexpressing,knockout,and-knockdownHCC cells,HCC organoids,and HCC patient-derived xenograft(PDX)murine models.YTHDF1 expression and hypoxia inducedautophagy were significantly correlated in vitro;signifhcant overexpression of YTHDF1 in HCC tissues was associated with poorprognosis,Multivariate cox regression analysis identihed YTHDF1 expression as an independent prognostic factor in patients withHCC.Multiple HC models conhrmed that YTHDF1 deficiency inhibited HCC autophagy,growth,and metastasis.Luciferase reporterassays and chromatin immunoprecipitation demonstrated that HlIF-1a regulated YTHDF1 transcription by directly binding to itspromoter region under hypoxia.The results of methylated RNA immunoprecipitation sequencing,proteomics,and polysomeprofling indicated that YTHDF1 contibuted to the translation of autophagy-related genes ATG2A and ATG14 by binding to m6A-modifhed ATG2A and ATG14 mRNA,thus facilitating autophagy and autophagy-related malignancy of HCC.Taken together,HlE-1d-induced YTHDF1 expression was associated with hypoxia-induced autophagy and autophagy-related HCC progression via promoting translation of autophagy-related genes ATG2A and ATG14 in a m6A-dependent manner.Our fndings suggest thatYTHDF1 is a potential prognostic biomarker and therapeutic target for patients with HCC.

Icariside Ⅱ, a main compound in Epimedii Folium, induces idiosyncratic hepatotoxicity by enhancing NLRP3 inflammasome activation

Idiosyncratic drus-induced liver injury(IDILI)is an intrequent but potentially serious disease that develops the main reason for post-marketing safety warnings and withdrawals of drugs.Epimedii Folium(EF),the widely used herbal medicine,has shown to cause idiosyncratic liver injury,but the underlying mechanisms are poorly understood.Increasing evidence has indicated that most cases of IDILI are immune mediated.Here,we report that icarisideⅡ(ICSⅡ),the major active and metabolic constituent of EF,causes idiosyncratic liver injury by promoting NLRP3 inflammasome activation.ICSⅡexacerbates NLRP3 inflammasome activation triggered by adenosine triphosphate(ATP)and nigericin,but not silicon dioxide(SiO2),monosodium urate(MSU)crystal or cytosolic lipopolysaccharide(LPS).Additionally,the activation of NLRC4 and AIM2 inflammasomes is not affected by ICSⅡ.Mechanistically,synergistic induction of mitochondrial reactive oxygen species(mtROS)is a crucial contributor to the enhancing effect of ICSⅡon ATP-or nigericin-induced NLRP3 inflammasome activation.Importantly,in vivo data show that a combination of non-hepatotoxic doses of LPS and ICSⅡcauses the increase of aminotransferase activity,hepatic inflammation and pyroptosis,which is attenuated by Nlrp3 deficiency or pretreatment with MCC950(a specific NLRP3 inflammasome inhibitor).In conclusion,these findings demonstrate that ICSⅡcauses idiosyncratic liver injury through enhancing NLRP3 inflammasome activation and suggest that ICSⅡmay be a risk factor and responsible for EF-induced liver injury.

Exosome-transported circHDAC1_004 Promotes Proliferation, Migration, and Angiogenesis of Hepatocellular Carcinoma by the miR-361-3p/NACC1 Axis

Background and Aims:Hepatocellular carcinoma(HCC)is among the most common malignant tumors globally.Circular RNAs(circRNAs),as a type of noncoding RNAs,reportedly participate in various tumor biological processes.However,the role of circHDAC1_004 in HCC remains unclear.Thus,we aimed to explore the role and the underlying mechanisms of circHDAC1_004 in the development and progression of HCC.Methods:Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect circHDAC1_004 expression(circ_0005339)in HCC.Sanger sequencing and agarose gel electrophoresis were used to determine the structure of circHDAC1_004.In vitro and in vivo experiments were used to determine the biological function of circHDAC1_004 in HCC.Herein,qRT-PCR,RNA immunoprecipitation,western blotting,and a luciferase reporter assay were used to explore the relationships among circHDAC1_004,miR-361-3p,and NACC1.Results:circHDAC1_004 was upregulated in HCC and significantly associated with poor overall survival.circHDAC1_004 promoted HCC cell proliferation,stemness,migration,and invasion.In addition,circHDAC1_004 upregulated human umbilical vein endothelial cells(HUVECs)and promoted angiogenesis through exosomes.circHDAC1_004 promoted NACC1 expression and stimulated the epithelialmesenchymal transition pathway by sponging miR-361-3p.Conclusions:We found that circHDAC1_004 overexpression enhanced the proliferation,stemness,and metastasis of HCC via the miR-361-3p/NACC1 axis and promoted HCC angiogenesis through exosomes.Our findings may help develop a possible therapeutic strategy for HCC.