Exosome-derived circCCAR1 promotes CD8 + T-cell dysfunction and anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND: Circular RNAs (circRNAs) can be encapsulated into exosomes to participate in intercellular communication, affecting the malignant progression of a variety of tumors. Dysfunction of CD8 + T cells is the main factor in immune escape from hepatocellular carcinoma (HCC). Nevertheless, the effect of exosome-derived circRNAs on CD8 + T-cell dysfunction needs further exploration. METHODS: The effect of circCCAR1 on the tumorigenesis and metastasis of HCC was assessed by in vitro and in vivo functional experiments. The function of circCCAR1 in CD8 + T-cell dysfunction was measured by enzyme-linked immunosorbent assay (ELISA), western blotting and flow cytometry. Chromatin immunoprecipitation, biotinylated RNA pull-down, RNA immunoprecipitation, and MS2 pull-down assays were used to the exploration of mechanism. A mouse model with reconstituted human immune system components (huNSG mice) was constructed to explore the role of exosomal circCCAR1 in the resistance to anti-PD1 therapy in HCC. RESULTS: Increased circCCAR1 levels existed in tumor tissues and exosomes in the plasma of HCC patients, in the culture supernatant and HCC cells. CircCCAR1 accelerated the growth and metastasis of HCC in vitro and in vivo. E1A binding protein p300 (EP300) and eukaryotic translation initiation factor 4A3 (EIF4A3) promoted the biogenesis of circCCAR1, and Wilms tumor 1-associated protein (WTAP)-mediated m6A modification enhanced circCCAR1 stability by binding insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). CircCCAR1 acted as a sponge for miR-127-5p to upregulate its target WTAP and a feedback loop comprising circCCAR1/miR-127-5p/WTAP axis was formed. CircCCAR1 is secreted by HCC cells in a heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1)-dependent manner. Exosomal circCCAR1 was taken in by CD8 + T cells and caused dysfunction of CD8 + T cells by stabilizing the PD-1 protein. CircCCAR1 promoted resistance to anti-PD1 immunotherapy. Furthermore, increased cell division cycle and apoptosis regulator 1 (CCAR1) induced by EP300 promoted the binding of CCAR1 and ß-catenin protein, which further enhanced the transcription of PD-L1. CONCLUSIONS: The circCCAR1/miR-127-5p/WTAP feedback loop enhances the growth and metastasis of HCC. Exosomal circCCAR1 released by HCC cells contributes to immunosuppression by facilitating CD8 + T-cell dysfunction in HCC. CircCCAR1 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for HCC patients.

Improving diameter measurement from Fraunhofer diffraction with fringe segment splicing.

Fraunhofer diffraction is an easy but powerful method for measuring the diameter of a thin filament. In practice, however, the diffraction pattern attainable is always subject to limits imposed by various imperfections in real systems, such as small angle approximation and sensor threshold, thus degrading the measurement resolution. In this Letter, we propose a method of fringe segment splicing for improving the diameter measurement from Fraunhofer diffraction. The fringe segment is chosen from a real diffraction pattern and is used to reproduce an ideal diffraction fringe, where the theoretical estimates give the best approximation to the observations. The problem of diameter measurement is solved in the spatial frequency-domain with an ideal diffraction fringe. Our results show that the relative error in this method is less than 0.1% and is far superior to that of previous methods.

Methylated SEPT9 combined with AFP and PIVKA-II is effective for the detection of HCC in high-risk population.

BACKGROUND: The methylation SEPT9 (mSEPT9) appeared to be effective for hepatocellular carcinoma (HCC) detection. However, its performance in high-risk population has not been validated. We designed a pilot study and aimed to investigate the performance of mSEPT9, AFP, PIVKA-II and their combination in hepatic cirrhosis (HC) population. METHODS: A training cohort was established including 103 HCC and 114 HC patients. 10 ml blood was collected from each patient with K2EDTA tubes, and 3-4 ml plasma was extracted for subsequent tests. The performance of mSEPT9, AFP, PIVKA-II and their combination was optimized by the training cohort. Test performance was prospectively validated with a validation cohort, including 51 HCC and 121 HC patients. RESULTS: At the optimal thresholds in the training cohort, the sensitivity, specificity and area under curve (AUC) was 72.82%, 89.47%, 0.84, and 48.57%, 89.92%, 0.79, and 63.64%, 95.95%, 0.79 for mSEPT9, AFP and PIVKA-II, respectively. The combined test significantly increased the sensitivity to 84.47% (P < 0.05) at the specificity of 86.84% with an AUC of 0.91. Stage-dependent performance was observed with all single markers and their combination in plasma marker levels, positive detection rate (PDR) and AUC. Moderate correlation was found between mSEPT9 and AFP plasma levels (r = 0.527, P < 0.0001). Good complementarity was found between any two of the three markers, providing optimal sensitivity in HCC detection when used in combination. Subsequent validation achieved a sensitivity, specificity and AUC of 65.31%, 92.86%, 0.80, and 44.24%, 89.26%, 0.75, and 62.22%, 95.27%, 0.78 for mSEPT9, AFP and PIVKA-II, respectively. The combined test yielded a significantly increased sensitivity of 84.00% (P < 0.05) at 85.57% specificity, with an AUC at 0.89. CONCLUSIONS: The performance was optimal by the combination of mSEPT9, AFP, PIVKA-II compared with any single marker, and the combination may be effective for HCC opportunistic screening in HC population.

Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common cancer worldwide, and sorafenib is a first-line drug for the treatment of advanced liver cancer. Resistance to sorafenib has become a major challenge in the treatment of hepatocellular carcinoma, however, studies have shown that metformin can promote ferroptosis and sorafenib sensitivity. Therefore, the aim of this study was to investigate the promotion of ferroptosis and sorafenib sensitivity by metformin via ATF4/STAT3 in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma cells Huh7 and Hep3B and induced sorafenib resistance (SR) Huh7/SR and Hep3B/SR cells were used as in vitro cell models. Cells were injected subcutaneously to establish a drug-resistant mouse model. CCK-8 was used to detect cell viability and sorafenib IC50. Western blotting was used to detect the expression of relevant proteins. BODIPY staining was used to analyze the lipid peroxidation level in cells. A scratch assay was used to detect cell migration. Transwell assays were used to detect cell invasion. Immunofluorescence was used to localize the expression of ATF4 and STAT3. RESULTS: Metformin promoted ferroptosis in hepatocellular carcinoma cells through ATF4/STAT3, decreased sorafenib IC50, increased ROS and lipid peroxidation levels, decreased cell migration and invasion, inhibited the expression of the drug-resistant proteins ABCG2 and P-GP in hepatocellular carcinoma cells, and thus inhibited sorafenib resistance in hepatocellular carcinoma cells. Downregulating ATF4 inhibited the phosphorylated nuclear translocation of STAT3, promoted ferroptosis, and increased the sensitivity of Huh7 cells to sorafenib. Metformin was also shown in animal models to promote ferroptosis and sorafenib sensitivity in vivo via ATF4/STAT3. CONCLUSION: Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3, and it inhibits HCC progression.

Exosome-derived miR-142-5p from liver stem cells improves the progression of liver fibrosis by regulating macrophage polarization through CTSB.

BACKGROUND: This study aims to explore the effect of liver stem cells (LSCs)-derived exosomes and the miR-142a-5p carried by them on the process of fibrosis by regulating macrophages polarization. METHODS: In this study, CCL4 was used to establish liver fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB) and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect liver fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and liver fibrosis model were constructed to verify the expression of miR-142a-5p and ctsb. RESULTS: Immunofluorescence of LSCs markers CK-18, epithelial cell adhesion molecule (EpCam), and AFP showed that these markers were up-regulated in LSCs. In addition, we evaluated the ability of LSCs to excrete EVs by labeling LSCs-EVs with PKH67. We found that CCL4 and EVs were simultaneously treated at 50 and 100 µg doses, and both doses of EVs could reduce the degree of liver fibrosis in mice. We tested markers of M1 or M2 macrophage polarization and found that EVs reduced M1 marker expression and promoted M2 marker expression. Further, ELISA was used to detect the secreted factors related to M1 and M2 in tissue lysates, which also verified the above views. Further analysis showed that the expression of miR-142a-5p increased significantly with the increase of EVs treatment concentration and time. Further, in vitro and in vivo LSCs-EVs regulate macrophage polarization through miR-142a-5p/ctsb pathway and affect the process of liver fibrosis. CONCLUSION: Our data suggest that EVs-derived miR-142-5p from LSCs improves the progression of liver fibrosis by regulating macrophage polarization through ctsb.

IL-17 Activates the IL-6/STAT3 Signal Pathway in the Proliferation of Hepatitis B Virus-Related Hepatocellular Carcinoma.

BACKGROUND/AIMS: We performed this study to determine the role of IL-17 in the immune microenvironment of hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). METHODS: HepG2 cells were treated with IL-17, STAT3 inhibitor S31-201 or IL-6 neutralizing monoclonal antibody (IL-6 mAb). Cell proliferation and migration were compared using the Cell Counting kit-8 (CCK-8) and Transwell assays, respectively. Real-time quantitative PCR (RT-qPCR), Western Blot, ELISA, immunofluorescence and histological staining were used for determining the expression levels of IL-17, IL-6, MCP-1, CCL5, VEGF, STAT3 and p-STAT3. HCC xenograft models were constructed in wild type and IL-17 knockout mice to clarify the effects of IL-17 on HCC in vivo. RESULTS: Exogenous IL-17 enhanced the proliferation and migration of HepG2 cells, and it activated the phosphorylation of STAT3. RT-qPCR and ELISA showed that IL-17 promoted the expression of IL-6. The CCK-8 and Transwell assays showed that S31-201 or IL-6 mAb remarkably reversed the promotion effects of proliferation and migration by exogenous IL-17 in HepG2 cells. Additionally, IL-6 could promote the phosphorylation of STAT3, while IL-6 mAb acted as an inhibitor, and exogenous IL-17 could neutralize the inhibitory effects of IL-6 mAb. In vivo, compared to the wild type mice, the tumor volume, weight, density and size were decreased in IL-17 knockout mice. Additionally, the expression levels of p-STAT3, IL-6, MCP-1, CCL5 and VEGF decreased in IL-17 knockout mice. CONCLUSIONS: IL-17 can enhance the proliferation of HepG2 cells in vitro and in vivo via activating the IL-6/STAT3 pathway. Therefore, the IL-17/IL-6/STAT3 signaling pathway is a potential therapeutic target for HBV-related HCC.

miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3ß-involved Wnt/ß-catenin signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the most lethal cancers. MicroRNA-155 (miR-155) and collagen triple helix repeat containing 1 (CTHRC1) were found to be involved in hepatocarcinogenesis, but their detailed functions in HCC are unclear. Here, we aimed to investigate the underlying role of miR-155-5p and CTHRC1 in HCC. METHODS: miR-155-5p and CTHRC1 expression levels were detected by qRT-PCR, IHC and WB in HCC patients and cell lines. Dual-luciferase assay, qRT-PCR and WB were used to validate the target interaction between miR-155-5p and CTHRC1. Biological behaviors, including apoptosis, cell cycle progression, and cell proliferation, invasion and migration, were measured by flow cytometry, CCK-8 assay and Transwell tests. A xenograft model was established to examine the effects of miR-155-5p and CTHRC1 on tumor formation. WB was finally utilized to identify the role of GSK-3ß-involved Wnt/ß-catenin signaling in HCC growth and metastasis. RESULTS: Our results showed that miR-155-5p and CTHRC1 were down-regulated and up-regulated, respectively, in HCC patients and cell lines. Dual-luciferase assay verified that CTHRC1 was the direct target of miR-155-5p. Moreover, elevated miR-155-5p expression promoted apoptosis but suppressed cell cycle progression and cell proliferation, invasion and migration in vitro and facilitated tumor formation in vivo; elevated CTHRC1 expression abolished these biological effects. Additionally, miR-155-5p overexpression increased metastasis- and anti-apoptosis-related protein expression and decreased pro-apoptosis-related protein expression, while forced CTHRC1 expression conserved the expression of these proteins. CONCLUSION: Altogether, our data suggested that miR-155-5p modulated the malignant behaviors of HCC by targeting CTHRC1 and regulating GSK-3ß-involved Wnt/ß-catenin signaling; thereby, miR-155-5p and CTHRC1 might be promising therapeutic targets for HCC patients.

LXRalpha gene downregulation by lentiviral-based RNA interference enhances liver function after fatty liver transplantation in rats.

BACKGROUND: Steatotic liver grafts, although accepted, increase the risk of poor posttransplantation liver function. However, the growing demand for adequate donor organs has led to the increased use of so-called marginal grafts. Liver X receptor alpha (LXRalpha) is important in fatty acid metabolism and interrelated with the specific ischemia-reperfusion injury in fatty liver transplantation. This study aimed to investigate whether LXRalpha RNA interference (RNAi) could improve the organ function of liver transplant recipients. METHODS: Fifty Sprague-Dawley rats were fed with a high-fat diet and 56% alcohol. The livers of these animals had greater than 60% macrovesicular steatosis and were used as liver donors. The experimental donors were treated with 7X107 TU LXRalpha-RNAi-LV of a mixture injection and control donors with negative control-LV vector injection into the portal vein 72 hours before the operation. The effects of LXRalpha-RNAi-LV were assessed by serum aminotransferases, histology, immunostaining, and protein levels. The transcription of LXRalpha mRNA was assessed by reverse transcription-polymerase chain reaction. RESULTS: Compared with controls, LXRalpha RNAi inhibited the expression of LXRalpha at the mRNA (0.53+/-0.03 vs 0.94+/-0.02, P<0.05) and protein levels (0.51+/-0.08 vs 1.09+/-0.12, P<0.05). LXRalpha RNAi also decreased the expressions of sterol regulatory element-binding protein 1c (SREBP-1c) and CD36. LXRalpha RNAi consequently reduced fatty acid accumulation in hepatocytes. Compared with control animals, LXRalpha RNAi-treated group had lower serum alanine aminotransferase, aspartate aminotransferase, interleukin-1beta, and tumor necrosis factor-alpha levels and milder pathologic damages. TUNEL analysis revealed a significant reduction of apoptosis in the livers of rats treated with LXRalpha-RNAi-LV, and overall survival as determined by the Kaplan-Meier method was improved among rats treated with LXRalpha-RNAi-LV (P<0.05). CONCLUSION: LXRalpha-RNAi-LV treatment significantly downregulated LXRalpha expression and improve steatotic liver graft function and recipient survival after a fatty liver transplantation in rats.

Unraveling the Heterogeneity of CD8+ T-Cell Subsets in Liver Cirrhosis: Implications for Disease Progression.

Background/Aims: : Liver cirrhosis involves chronic inflammation and progressive fibrosis. Among various immune cells, CD8+ T cells are considered a major contributor to hepatic inflammation and fibrosis. However, the exact molecular pathways governing CD8+ T-cell-mediated effects in cirrhosis remain unclear. Methods: : This study analyzed transcriptomic and single-cell sequencing data to elucidate CD8+ T-cell heterogeneity and implications in cirrhosis. Results: : Weighted gene co-expression analysis of bulk RNA-seq data revealed an association between cirrhosis severity and activated T-cell markers like HLA and chemokine genes. Furthermore, single-cell profiling uncovered eight CD8+ T-cell subtypes, notably, effector memory (Tem) and exhausted (Tex) T cells. Tex cells, defined by PDCD1, LAG3, and CXCL13 expression, were increased in cirrhosis, while Tem cells were decreased. Lineage tracing and differential analysis highlighted CXCL13+ Tex cells as a terminal, exhausted subtype of cells with roles in PD-1 signaling, glycolysis, and T-cell regulation. CXCL13+ Tex cells displayed T-cell exhaustion markers like PDCD1, HAVCR2, TIGIT, and TNFRSF9. Functional analysis implicated potential roles of these cells in immunosuppression. Finally, a CXCL13+ Tex-cell gene signature was found that correlated with cirrhosis severity and poorer prognosis of liver cancer. Conclusions: : In summary, this comprehensive study defines specialized CD8+ T-cell subpopulations in cirrhosis, with CXCL13+ Tex cells displaying an exhausted phenotype associated with immune dysregulation and advanced disease. Key genes and pathways regulating these cells present potential therapeutic targets.

Chronic steatosis aggravates cold-storage induced acute ischemic injury in rat donor livers through the perturbation of lipophagy.

In this study, we explored the effects of cold ischemia on chronic steatosis and lipid signaling in vivo. Sprague Dawley (SD) rat models of chronic steatosis were established. Pathological observations and liver indices were assessed through hematoxylin-eosin (HE)- and Oil Red O staining. Autophagy and metabolism in adipose tissue were analyzed under post-ischemia and hypoxic conditions via western blotting and immunofluorescent analysis. We found that cold ischemia treatment exacerbated hepatic steatosis and reduced lipid phagocytosis. This manifested as a loss of Microtubule-associated protein 1A/1B-light chain 3 (LC3) and Perilipin 2 (PLIN2), and lower levels of autophagy. Cold ischemia also inhibited lipophagy in transplanted rat livers, most notably in moderate-to-severe steatosis models. Ischemia and hypoxia inhibited lipid phagocytosis and increased lipid accumulation. Collectively, these data show that chronic steatosis aggravates cold storage induced acute ischemic injury in rat donor livers through the inhibition of lipophagy. Moderate-to-severe steatosis therefore influences the postoperative recovery of liver transplant recipients, which should be immediately transplanted to reduce the risk of cold ischemia.

Identification of hub genes in heart failure by integrated bioinformatics analysis and machine learning.

Objective: To screen feature genes of heart failure patients through machine learning methods, in order to identify characteristic genes driving heart failure and investigate the progression of heart failure. Methods: Heart failure patient samples were downloaded from the public database GEO (Gene Expression Omnibus), including the datasets GSE116250, GSE120895, and GSE59867. GSE116250 and GSE120895 were used as the testing set, while GSE59867 was used as the validation set. LASSO regression analysis and SVM-RFE were utilized to identify feature genes. Results: Analysis showed that among the differentially expressed genes between normal and heart failure patients, 9 genes were upregulated and 10 genes were downregulated. ROC curve analysis in the training set showed that TAGLN and SGPP2 had AUC values greater than 0.7. Moreover, SDSL and SMTNL2 had even higher AUC values of greater than 0.9. However, further analysis in the validation set showed that only SDSL had an AUC value greater than 0.7. Western blot experiments, RT-PCR, and ISO-induced experiments confirmed that SDSL was highly expressed in heart failure patients and promoted heart failure progression. In addition, SDSL promoted PARP1 expression and knockdown of SDSL expression led to decreased Cleaved-PARP1 expression and reduced cardiomyocyte apoptosis. Conversely, overexpression of SDSL resulted in increased PARP1 expression and myocardial cell apoptosis. These results suggest that elevated expression of SDSL in cardiomyocytes from heart failure patients may be an important factor promoting the occurrence and development of heart failure. Conclusions: Using machine learning methods and experimental validation, it has been demonstrated that SDSL is a driving gene in patients with heart failure, providing a new treatment direction for clinical treatment.

MiR-21-5p promotes sorafenib resistance and hepatocellular carcinoma progression by regulating SIRT7 ubiquitination through USP24.

OBJECTIVE: To validate the mechanism by which miR-21-5p mediates autophagy in drug-resistant cells in hepatocellular carcinoma (HCC), aggravating sorafenib resistance and progression of HCC. METHODS: HCC cells were treated with sorafenib to establish sorafenib-resistant cells, and nude mice were subcutaneously injected with hepatoma cells to establish animal models. RT-qPCR was used to determine the level of miR-21-5p, and Western blotting was used to determine the level of related proteins. Cell apoptosis, cell migration, the level of LC3 were accessed. Immunohistochemical staining was used for detection of Ki-67 and LC3. A dual-luciferase reporter assay certified that miR-21-5p targets USP42, and a co-immunoprecipitation assay validated the mutual effect between USP24 and SIRT7. RESULTS: miR-21-5p and USP42 were highly expressed in HCC tissue and cells. Inhibition of miR-21-5p or knockdown of USP42 inhibited cell proliferation and cell migration, upregulated the level of E-cadherin, and downregulated the level of vimentin, fibronectin and N-cadherin. Overexpression of miR-21-5p reversed the knockdown of USP42. Inhibition of miR-21-5p downregulated the ubiquitination level of SIRT7, downregulated the levels of LC3II/I ratio and Beclin1, and upregulated the expression of p62. The tumor size in the miR-21-5p inhibitor group was smaller, and Ki-67 and LC3 in tumor tissue were reduced, while the overexpression of USP42 reversed the effect of the miR-21-5p inhibitor. CONCLUSION: miR-21-5p promotes deterioration and sorafenib resistance in hepatocellular carcinoma by upregulating autophagy levels. Knockdown of miR-21-5p inhibits the development of sorafenib-resistant tumors by USP24-mediated SIRT7 ubiquitination.

Analysis of angiogenesis-related subtypes of hepatocellular carcinoma and tumor microenvironment infiltration feature in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly vascularized tumor, and angiogenesis plays an important role in its progression. However, the role of angiogenesis in cell infiltration in the tumor microenvironment (TME) remains unclear. METHODS: We evaluated the associations of 35 angiogenesis-related genes (ARGs) with the clinicopathological features of 816 HCC patients. In addition, we assessed the associations between the ARGs and TME cell infiltration. A nomogram was constructed to determine the prognostic value of ARGs for HCC. The ARG score was used to distinguish angiogenic subtypes of HCC, and its usefulness for predicting the prognosis and treatment response of HCC patients was evaluated. RESULTS: We distinguished three ARG clusters differing in terms of TME cell infiltration, immune cell activation status, clinicopathological features, and clinical outcomes. There were significant associations of ARG expression with tumor immunity, the epithelial-mesenchymal transition (EMT), and transforming growth factor-ß expression. An ARG score model was constructed to generate a risk score for each patient based on differentially expressed genes between clusters. Furthermore, a high ARG score was associated with high expression of CTLA-4 and PD-L1/PD-1, and a low Tumor Immune Dysfunction and Exclusion score, indicating the usefulness of the ARG score for selecting patients for immunotherapy. Considering the relationship between ARGs and tumor immunity, immunotherapy combined with vascular-targeted therapy may be the best treatment for HCC. CONCLUSIONS: ARGs play an important role in TME diversity and complexity in HCC patients. The ARG score of HCC predicts TME invasion and can guide immunotherapy.

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

Objectives: Abdominal aortic aneurysm (AAA) has a high risk of rupture of the aorta and is one of the leading causes of death in older adults. This study is aimed at confirming the influence and mechanism of the abnormally expressed ANXA6 gene in AAA. Methods: Clinical samples were collected for proteome sequencing to screen for differentially expressed proteins. An Ang II-induced vascular smooth muscle cell (VSMC) aging model as well as an AAA animal model was used. Using RT-qPCR to detect the mRNA levels of EZH2, ANXA6, IK-6, and IL-8 in cells and tissues were assessed. Western blotting and immunohistochemistry staining were used apply for the expression of associated proteins in cells and tissues. SA-ß-gal staining, flow cytometry, and DHE staining were used to detect senescent cells and the level of ROS. The cell cycle was assessed by flow cytometry. Arterial pathology was observed by HE staining. The aging of VSMCs in arterial tissue was assessed by coimmunofluorescence for α-SMA and p53. Results: There were 24 differentially expressed proteins in the AAA clinical samples, including 10 upregulated protein and 14 downregulated protein, and the differential expression of ANXA6 was associated with vascular disease. Our study found that ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced VSMC aging model. Knockdown of ANXA6 or overexpression of EZH2 inhibited Ang II-induced ROS, inhibited cell senescence, decreased Ang II evoked G1 arrest, and increased cells in G2 phase, while overexpression of ANXA6 played the opposite role. Overexpression of EZH2 inhibited ANXA6 expression by increasing H3K27me3 modification at the ANXA6 promoter. Simultaneous overexpression of EZH2 and the protective effect of EZH2 on cell senescence were partially reversed by ANXA6. Similarly, ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced AAA animal model. Knockdown of ANXA6 and overexpression of EZH2 alleviated Ang II-induced VSMC senescence and inhibited AAA progression, while simultaneous overexpression of EZH2 and ANXA6 partially reversed the protective effect of EZH2 on AAA. Conclusion: EZH2 regulates the ANXA6 promoter H3K27me3 modification, inhibits ANXA6 expression, alleviates Ang II-induced VSMC senescence, and inhibits AAA progression.

Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation.

Background: To establish an animal model of pre-sensitization following liver transplantation either with or without immunosuppressors. To study whether accelerated liver rejection or acute antibody-mediated rejection (AMR) occurred and study the characteristics and potential mechanism in the animal model. Methods: Lewis (LEW) rats were subjected to liver [liver graft of Brown Norway (BN) rat] transplantation 2 weeks after lymphocyte injection (lymphocytes of BN rat; pre-sensitization). At 2 weeks after transplantation, serum samples of recipients were collected for antibody analysis to identify donor-specific alloantibody (DSA) level. The recipients were treated with or without a low dose of immunosuppressor (2 mg/kg). The liver grafts of each group were analyzed by hematoxylin and eosin (HE) stain, Masson stain, CK19, C4d, and CD20 immunohistochemical (IHC) stain, CD3, CD68, and CD86 immunofluorescence and transmission electron microscope (TEM) to study the characteristics of liver rejection. Moreover, cytotoxin-associated genes, M1 macrophages conversion-related proteins, and interleukin-6 (IL-6) signaling pathway proteins were detected by western blotting. Results: High level of DSA and accelerated liver rejection occurred in the pre-sensitized rat models following liver transplant. Accelerated liver graft rejection occurred in the pre-sensitized, post liver transplant rats regardless of whether a low dose immunosuppressor had been applied. Severe injury of the interlobular bile ducts and accelerated fibrosis could be observed. Moreover, evidence of endothelial injury, such as capillary inflammation, was found in the pre-sensitized, post-transplant rats. In addition, C4d deposition and M1 macrophages recruitment were also found in this sensitized followed transplant model, indicating that complement activation might occur in this model. The levels of IL-6, JAK1, STAT3, SHP2, and ERK1-2 were increased in the pre-sensitized, post-transplant rats. Conclusions: Pre-sensitized post liver transplant rats might be potential AMR models for further study.

Long noncoding RNA NEAT1 changes exosome secretion and microRNA expression carried by exosomes in hepatocellular carcinoma cells.

BACKGROUND: This study aimed to investigate the roles and functions of nuclear-enriched abundant transcript 1 (NEAT1) in exosome secretion and exosomal microRNA (miRNA) changes in hepatocellular carcinoma (HCC) cells. METHODS: HepG2 and HuH-7 cells were divided into two groups: Lv-control (which were infected with lentivirus without NEAT1 expression) and Lv-NEAT1 (which were infected with lentivirus with NEAT1 overexpression). Each group was used to study cell function (proliferation, invasion, and apoptosis) and exosome secretion by nanoparticle tracking analysis (NTA), electron microscopy, and nanoflow cytometry (nanoFCM). Different levels of messenger RNA (mRNA), miRNA, and exosomal miRNA were detected by RNA sequencing. Next, potential target RNAs were verified by reverse transcription polymerase chain reaction (RT-PCR). Changed exosomal miRNAs were found and miRNA mimics were used to study cell function in NEAT1-overexpression and NEAT1-knockdown HCC cells. RESULTS: The data showed that NEAT1-overexpression promoted exosome secretion. The overexpression of NEAT1 altered global genes, including exosome-related genes. Compared with the control group, we observed that several miRNAs changed in the exosomes secreted by NEAT1-overexpressing cells. Our study found that these changed exosomal miRNAs played a suppressor role in HCC. Transfection of miR-634, miR-638, and miR-3960 reversed the enhanced invasion and proliferation in HCC cells with a high level of NEAT1 expression. CONCLUSIONS: These results suggested that NEAT1 regulates exosome-related genes, which might be associated with increasing exosome secretion by NEAT1-overexpressing cells. Furthermore, NEAT1 promotes cell invasion and proliferation via downregulation of miR-634, miR-638, and miR-3960 in exosomes. This study may provide potential targets for exosome-mediated miRNA transfer in HCCs with a high level of NEAT1 expression therapy.

A new scheme with infusion of hepatitis B immunoglobulin combined with entecavir for prophylaxis of hepatitis B virus recurrence among liver transplant recipients.

OBJECTIVE: Liver transplantation significantly increases recurrence of hepatitis B virus (HBV) among high-risk patients. Hepatitis B immunoglobulin (HBIG) and antiviral nucleotide analogues are effective prophylaxis reagents in preventing HBV recurrence. However, HBV recurrence still occurs with these treatments. METHODS: To explore a more cost-effective prophylaxis protocol in patients after liver transplantation, we treated patients with an initial high dose of 10 000 IU HBIG during the anhepatic phase and a second high dose of HBIG at an optimal time point during surgery. The patients were treated with the traditional European protocol as a control, in which one dose of 10 000 IU HBIG was infused during the anhepatic phase and multiple doses of 10 000 IU HBIG were administered daily for 1 week after liver transplantation. RESULTS: There were two mortalities among 50 patients treated with the new protocol and nine mortalities among 52 patients treated with the European protocol within 3 years after liver transplantation. The new prophylaxis method markedly improved the 3-year survival without HBV recurrence in 50 treated patients. However, there were five recurrences in 52 patients treated with the European protocol. High-risk factors such as HBV DNA+, positive hepatitis B e antigen, and hepatocellular carcinoma were all detected among five patients with HBV recurrence. The suppressed HBV recurrence was associated with significantly lower serum alanine aminotransferase and aspartate aminotransferase in the new protocol-treated patients tested at 1 month and 1 week after liver surgery compared with those treated with the European protocol. CONCLUSION: Infusion of two high doses of HBIG during surgery in combination with entecavir significantly prevented HBV recurrence and improved the 3-year survival after liver transplantation.

[Lentiviral-mediated RNA interference of LXRα gene in donor rats with fatty liver enhances liver graft function after transplantation].

OBJECTIVE: To investigate whether RNA interference (RNAi) of LXRα gene in donor rats with fatty liver improves liver graft function after transplantation. METHODS: Fifty donor SD rats were fed a high-fat diet and 56% alcohol to induce macrovesicular steatosis exceeding 60% in the liver. The donor rats were injected via the portal veins with 7 × 107 TU LXRα-RNAi-LV mixture (n=25) or negative control-LV (NC-LV) vector (n=25) 72 h before orthotopic liver transplantation. At 2, 24, and 72 h after the transplantation, the recipient rats were sacrificed to examine liver transaminases, liver graft histology, immunostaining (TUNEL), and protein and mRNA levels of LXRα. RESULTS: Lentivirus-LXRα RNAi inhibited LXRα gene expression at both the mRNA and protein levels in the liver graft and reduced the expressions of SREBP-1c and CD36 as compared with the controls, resulting also in reduced fatty acid accumulation in the hepatocytes. The recipient rats receiving RNAi-treated grafts showed more obvious reduction in serum ALT, AST, IL-1ß and TNF-α levels, and exhibited milder hepatic pathologies than the control rats after the transplantation. TUNEL assay demonstrated a significant reduction in cell apoptosis in LXRα-RNAi-LV-treated liver grafts, and the rats receiving treated liver grafts had a prolonged mean overall survival time. CONCLUSION: LXRα-RNAi-LV treatment of the donor rats with fatty liver can significantly down-regulate LXRα gene expression in the liver graft and improve the graft function and recipient rat survival after liver transplantation.

[Effect of different approaches of lentiviral vector transfection on target gene expression in rat liver].

OBJECTIVE: To investigate the optimal approach of lentiviral vector transfection for effective delivery of exogenous gene into the liver. METHODS: The lentiviral vector was delivered via the ileocolic vein of the ileocecus (portal vein group) or via the caudal vein of SD rats. The effect gene transfection into the liver was assessed by observing the expression of green fluorescence protein expression carried by the lentiviral vector, silencing of LXRα mRNA expression mediated by RNA interference, and liver transaminase changes. The efficiency and safety of the two approaches of transfection were evaluated. RESULTS: All the rats receiving lentiviral transfection survived. In the portal vein group, abundant green fluorescence was detected in the liver at 96 h following the transfection and lasted till 14 days, whereas only weak fluorescence was observed in the caudal vein group. The results of RT-PCR demonstrated a significant higher rate of LXRα knock-down in portal vein group than in caudal vein group (0.135∓0.002 vs 0.713∓0.036, P<0.05). No significant difference in ALT levels found between the two groups. CONCLUSIONS: Infusion via the potal vein is effective for gene transfection into the liver, and puncture from the ileocolic vein of ileocecus can guarantee the survival of rats and improve the transfection efficiency without causing liver injury.