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.

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-β 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 (AU)

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.

miR-21-5p Inhibits Ferroptosis in Hepatocellular Carcinoma Cells by Regulating the AKT/mTOR Signaling Pathway through MELK.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent cancers, and its incidence rate is increasing worldwide. At present, there is no ideal treatment for HCC. In recent years, molecular-targeted therapy has shown significant therapeutic benefits for patients. Ferroptosis is a modality of regulated cell death, and previous studies have found that inducing ferroptosis in liver cancer cells can inhibit the progression of liver cancer. The aim of this study is to investigate the regulatory mechanism of miR-21-5p in regulating ferroptosis in HCC cells. Methods: CCK-8 was used to measure cell viability, EdU and colony formation were used to measure cell proliferation, and Transwell assays were used to measure cell migration and invasion. RT-qPCR was used to detect the level of miR-21-5p, Western blotting was used to detect the protein expression level, a dual-luciferase reporter gene assay was used to determine the targeting relationship between miR-21-5p and MELK, and coimmunoprecipitation was used to determine the interaction between MELK and AKT. Results: Overexpression of miR-21-5p and MELK facilitated the viability, proliferation, colony formation, invasion, and migration of HCC cells. Downregulation of miR-21-5p suppressed the level of MELK and the progression of HCC. MELK regulated the AKT/mTOR signaling pathway, causing changes in the levels of GPX4, GSH, FTH1, xCT, heme oxygenase 1(HO-1), reactive oxygen species, and Fe2+ to regulate the ferroptosis of hepatoma cells. Erastin, an inducer of ferroptosis, attenuated the repressive influence of miR-21-5p on ferroptosis in HCC cells. Conclusion: In summary, this study demonstrates that miR-21-5p inhibits the ferroptosis of HCC cells by regulating the AKT/mTOR signaling pathway through MELK.

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.

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.

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.

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.

Long noncoding RNA NEAT1 promotes cell proliferation and invasion by regulating hnRNP A2 expression in hepatocellular carcinoma cells.

Growing evidence demonstrates that long noncoding RNAs (lncRNAs) are involved in the progression of various cancers, including hepatocellular carcinoma (HCC). The role of nuclear-enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, has not been fully explored in HCC. We aimed to determine the expression, roles and functional mechanisms of NEAT1 in the proliferation and invasion of HCC. Based on real-time polymerase chain reaction data, we suggest that NEAT1 is upregulated in HCC tissues compared with noncancerous liver tissues. The knockdown of NEAT1 altered global gene expression patterns and reduced HCC cell proliferation, invasion and migration. RNA immunoprecipitation and RNA pull-down assays confirmed that U2AF65 binds to NEAT1. Furthermore, the study indicated that NEAT1 regulated hnRNP A2 expression and that this regulation may be associated with the NEAT1-U2AF65 protein complex. Thus, the NEAT1-hnRNP A2 regulation mechanism promotes HCC pathogenesis and may provide a potential target for the prognosis and treatment of HCC.

Mechanism of immune hyporesponsiveness induced by recipient- derived immature dendritic cells in liver transplantation rat.

OBJECTIVE: To investigate the mechanism of immune hyporesponsiveness induced by donor-antigen- unloaded recipient-derived immature dendritic cell (imDC) of liver grafts in rats. METHODS: Forty Sprague-Dawley rats (donor) and forty male Wistar rats (recipient) were randomly divided into 4 groups: control, cyclosporine A (CsA), mature DC (mDC), and imDC groups respectively, with 10 donor rats and 10 recipient rats in each group. Recipient rats in CsA group were treated with 10 mg•kg⁻¹â€¢d⁻¹ CsA starting day 2 after the transplantation. Recipients in the mDC or imDC groups were given Wistar rat derived mDCs (1 × 106/rat) or imDCs (1 × 106/rat) via dorsal vein of the penis respectively 1 day before the transplantation. In each group, 5 recipients were kept for determination of survival time and the other 5 rats were executed at day 10 after transplantation. Blood samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBIL), interleukin 2 (IL-2), interferon gamma (IFN-γ), IL-4, and IL-10 levels. Liver tissue was harvested for HE staining and acute rejection evaluation. Expression levels of Fas-L/Fas in the grafts were detected by immunohistochemical staining; and Western blot was used to detect the expression level of Scurfin. RESULTS: The survival time of CsA and imDC groups was significantly longer than that of control and mDC groups (all P < 0.05). The levels of serum ALT and TBIL in the control group (2072.20 ± 217.93 IU/L and 147.42 ± 22.02 µmol/L) and mDC group (2117.00 ± 285.13 IU/L and 141.58 ± 20.82 µmol/L) were significantly higher than those in the CsA group (59.68 ± 13.48 IU/L and 15.40 ± 2.13 µmol/L) or imDC group (50.80 ± 9.63 IU/L and 14.44 ± 3.49 µmol/L) (all P < 0.05). In the CsA and imDC groups, the levels of IL-2 (22.52 ± 3.75 pg/mL and 22.12 ± 3.90 pg/mL) and IFN-γ (309.20 ± 25.19 pg/mL and 321.00 ± 21.64 pg/mL) were significantly lower, but the levels of IL-4 (297.60 ± 25.07 pg/mL and 277.00 ± 22.47 pg/mL) and IL-10 (1226.00 ± 140.49 pg/mL and 1423.00 ± 106.39 pg/mL) were higher than those of the control (IL-2: 147.78 ± 12.80 pg/mL, IFN-γ: 1758.60 ± 106.22 pg/mL, IL-4: 17.40 ± 4.77 pg/mL, IL-10: 81.00 ± 9.47 pg/mL) and mDC groups (IL-2: 142.34 ± 9.29 pg/mL, IFN-γ: 1835.00 ± 82.63 pg/mL, IL-4: 15.60 ± 3.96 pg/mL, IL-10: 68.80 ± 11.23 pg/mL) (all P < 0.01). The expression level of Scurfin protein on CD4+ CD25+ T cells of the imDC group (1.34 ± 0.29) was significantly higher than that in the control (0.72 ± 0.13), CsA (0.37 ± 0.11), and mDC groups (0.78 ± 0.17) (all P < 0.05). CONCLUSION: Donor-antigen-unloaded recipient-derived imDC is an effective treatment in inducing immune hyporesponsiveness through induction of T cell apoptosis, shift in Thl/Th2 balance, and proliferation of regulatory T cell.

[A causal analysis of intra-abdominal hemorrhage after reduced-size liver transplantation in rats].

OBJECTIVE: To analyze the cause of abdominal hemorrhage after reduce-size liver transplantation in rats. METHODS: Healthy female SD rats were used as the donors and male SD rats as the recipients (weighing 260-280 g), with the recipients weighing 10 g more than that of the donors. The donor operation was performed by the same surgeon under direct vision, and the liver graft size reduction procedure was completed in the donor operation. The recipient operation was performed by two surgeons under direct vision. RESULTS: A total of 270 SD rats received reduce-size liver transplantation successfully, and 44 of the rats died from intra-abdominal hemorrhage. The abdominal hemorrhages, listed in the order of incidences, included anastomotic hemorrhage of the inferior vena cava of the superior liver (28 cases), subcapsular hemorrhage of the liver (9 cases), ligation hemorrhage of the left outboard lobe, the nipple lobe and the triangle lobe of the liver (9 , 7 and 7 cases, respectively), hemorrhage of the right suprarenal vein and lumbar veins (5 cases), hemorrhage of the mechanical injury (4 cases), cuff hemorrhage of the portal vein and inferior vein cava of the inferior liver (both 4 cases). Eight rats had anastomotic hemorrhage of the inferior vena cava of the superior liver and ligation hemorrhage of the left outboard lobe, 5 had hemorrhage of the two ligation points of the reduce-size liver; for management of the hemorrhage, 10 rats received suture or/and ligature, and 6 had washing and hot water bath. CONCLUSION: The most common cause of hemorrhage after reduce-size liver transplantation in rats is the anastomotic hemorrhage of the inferior vena cava of the superior liver, and this finding may provide clues for improving the success rate of reduced size liver transplantation in rats.