Dendritic cells originating exosomal miR-193b-3p induces regulatory T cells to alleviate liver transplant rejection.

BACKGROUND: Exosomes exert considerable influence in mediating regulatory T (Treg) cells differentiation, which attach great importance to attenuating acute cellular rejection after liver transplantation (LT). And, miRNAs are known to play essential roles in cell-cell communication delivered by exosomes. However, the function of exosomal miRNAs in regulating Treg cells after LT remains unknown. Here, we performed an expression profiling analysis of exosome-miRNAs from human plasma after LT and investigated their immunoregulatory effects on Treg cells. METHODS: Fifty-eight LT patients and nine donors were included in this report. miRNA profiles in plasma exosomes were analyzed using next-generation sequencing. Flow cytometry, HE and multiplex immunofluorescent staining were used to identify Treg cells in the liver and peripheral blood. A lentiviral vector system was used to overexpress miR-193b-3p in dendritic cells (DCs), and exosomes isolated from these transfected cells were co-cultured with spleen lymphocytesin vitro. A quantitative Real-time PCR and enzyme-linked immunosorbent assay were used to detect the expression of cytokines. RESULTS: Treg cell infiltration was increased in the liver along with Th17 and CD8+ T cell, and it was down-regulated in peripheral blood in the acute rejection group. High-throughput sequencing revealed that miR-193b-3p was markedly up-regulated in plasma exosomes of non-rejection LT patients. The NLRP3 inflammasome was screened as a target for miR-193b-3p based on target prediction and functional enrichment analyses. Exosomal miR-193b-3p derived from DCs increased Treg cells as demonstrated in vitro. miR-193b-3p overexpression down-regulated NLRP3 as well as the inflammatory cytokines IL-1ß and IL-17A while increasing levels of the cytokines IL-10 and TGF-ß. CONCLUSION: DC derived exosomal miR-193b-3p promoted Treg cells by inhibiting NLRP3 expression. These findings not only provide a new perspective on the mechanisms, but also hold great promise for the treatment or prevention of liver allograft rejection.

Domino hepatocyte transplantation using explanted human livers with metabolic defects attenuates D-GalN/LPS-induced acute liver failure.

BACKGROUND: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF). METHODS: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses. RESULTS: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 106 ± 3.4 × 106 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers. CONCLUSION: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF.

Hypoxia inducible factor 1α promotes interleukin-1 receptor antagonist expression during hepatic ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is a major risk associated with liver surgery and transplantation, and its pathological mechanism is complex. Interleukin-1 receptor antagonist (IL-1ra) can protect the liver from IRI. However, the regulatory mechanism of IL-1ra expression is still unclear. AIM: To identify the mechanism that could protect the liver in the early stage of IRI. METHODS: To screen the key genes in hepatic IRI, we performed RNA sequencing and gene enrichment analysis on liver tissue from mice with hepatic IRI. Subsequently, we verified the expression and effect of IL-1ra in hepatic IRI. We also used promoter mutagenesis and chromatin immunoprecipitation assay to search for the transcriptional regulatory sites of hypoxia-inducible factor (HIF)-1α. Finally, to explore the protective mechanism of ischemic preconditioning (IP), we examined the expression of HIF-1α and IL-1ra after IP. RESULTS: We identified IL-1ra as a key regulator in hepatic IRI. The expression of IL-1ra was significantly upregulated after hepatic IRI both in vivo and in vitro. Furthermore, we found that HIF-1α regulated Il-1ra transcription in response to hypoxia. Increased HIF-1α accumulation promoted IL-1ra expression, whereas inhibition of HIF-1α exhibited the opposite effect. We also confirmed a predominant role for hypoxia response element in the regulation of Il1ra transcription by HIF-1α activation. Of note, we demonstrated that IP protects against hepatic IRI by inducing IL-1ra expression, which is mediated through HIF-1α. CONCLUSION: We demonstrated that ischemia or hypoxia leads to increased expression of IL-1ra through HIF-1α. Importantly, IP protects the liver from IRI via the HIF-1α-IL-1ra pathway.

ILC2s expanded by exogenous IL-33 regulate CD45+CD11b+F4/80high macrophage polarization to alleviate hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is an adverse consequence of hepatectomy or liver transplantation. Recently, immune mechanisms involved in hepatic IRI have attracted increased attention of investigators working in this area. In specific, group 2 innate lymphoid cells (ILC2s), have been strongly implicated in mediating type 2 inflammation. However, their immune mechanisms as involved with hepatic IRI remain unclear. Here, we reported that the population of ILC2s is increased with the development of hepatic IRI as shown in a mouse model in initial stage. Moreover, M2 type CD45+CD11b+F4/80high macrophages increased and reached maximal levels at 24 h followed by a significant elevation in IL-4 levels. We injected exogenous IL-33 into the tail vein of mice as a mean to stimulate ILC2s production. This stimulation of ILC2s resulted in a protective effect upon hepatic IRI along with an increase in M2 type CD45+CD11b+F4/80high macrophages. In contrast, depletion of ILC2s as achieved with use of an anti-CD90.2 antibody substantially abolished this protective effect of exogenous IL-33 and M2 type CD45+CD11b+F4/80high macrophage polarization in hepatic IRI. Therefore, this exogenous IL-33 induced potentiation of ILC2s appears to regulate the polarization of CD45+CD11b+F4/80high macrophages to alleviate IRI. Such findings provide the foundation for the development of new targets and strategies in the treatment of hepatic IRI.

Potential correlation of allograft infiltrating group 2 innate lymphoid cells with acute rejection after liver transplantation.

Accumulating evidence indicates the critical roles of group 2 innate lymphoid cells (ILC2s) in immunoregulation. However, the role of ILC2s in acute rejection after liver transplantation (LT) remains elusive. In this study, we analyzed the frequency, counts, and signature cytokines of ILC2s in liver transplant recipients by flow cytometric analysis and multiplex immunofluorescence assay. We also assessed the spatial distribution and correlation between hepatic ILC2s and Treg cells. The changes of ILC2s were dynamically monitored in the mouse LT model. We found that the frequencies of circulating ILC2s were comparable in liver transplant recipients with either rejection or non-rejection compared with the control group. The hepatic ILC2s counts were significantly increased in the rejection group than in the non-rejection and control groups, and a similar trend was observed for Treg cells. In the mouse LT model, allograft infiltrating ILC2s dramatically increased within 14 days post-transplant. The frequency of ILC2s in bone marrow significantly increased at 7 days post-transplant and rapidly decreased at 14 days after LT. Similarly, there was a significant increase in the frequency of splenic ILC2s within two weeks post-transplant. Multiplex immunofluorescence assay showed a close correlation between hepatic ILC2s and Treg cells by analyzing their spatial distribution and distance. In conclusion, the number of allograft infiltrating ILC2s was closely related to rejection after LT. Allograft infiltrating ILC2s may play inhibitory roles in posttransplant immune homeostasis, favoring resolution of liver allograft rejection by interacting with Treg cells or promoting the migration of Tregs cells into the liver allograft.

Characterization and Proteomic Analyses of Proinflammatory Cytokines in a Mouse Model of Liver Transplant Rejection.

Liver transplantation (LT) is an effective strategy for the treatment of end-stage liver disease, but immune rejection remains a significant detriment to the survival and prognosis of these LT patients. While immune rejection is closely related to cytokines, the cytokines investigated within previous studies have been limited and have not included a systematic analysis of proinflammatory cytokines. In the present study, we used a protein chip system and proteomics to detect and analyze serum proinflammatory cytokines and differentially expressed proteins in liver tissue in a mouse model of liver transplantation. In addition, bioinformatics analysis was employed to analyze the proinflammatory cytokines and differential changes in proteins in response to this procedure. With these analyses, we found that serum contents of GC-CSF, CXCL-1, MCP-5, and CXCL-2 were significantly increased after liver transplantation, while IL-5, IL-10, and IL-17 were significantly decreased. Results from Gene Ontology (GO) and KEGG pathway analyses revealed that the cytokine-cytokine receptor, Th1/Th2 cell differentiation, and JAK-STAT signaling pathway were enriched in a network associated with the activation of immune response. Results from our proteomic analysis of liver tissue samples revealed that 470 proteins are increased and 50 decreased, including Anxa1, Anxa2, Acsl4, Sirpa, S100a8, and S100a9. KEGG pathway analysis indicated that the neutrophil extracellular trap formation, NOD-like receptor signaling pathway, and leukocyte transendothelial migration were all associated with liver transplant rejection in these mice. Bioinformatics analysis results demonstrated that CXCL-1/CXCL-2 and S100a8/S100a9 were the genes most closely related to the functions of neutrophils and the mononuclear phagocyte system. These findings provide new insights into some of the critical factors associated with liver transplant rejection and thus offer new targets for the treatment and prevention of this condition.

Characteristics of changes in double positive CD4+CD8+ T cells in liver transplantation.

Although double positive CD4+CD8+ T (DPT) cells has been reported to be involved in some diseases, their trajectory and function as associated with liver transplantation (LT) remain unclear. In the present study, we found that the number of DPT cells was increased in the blood and liver tissue of LT patients. Meanwhile, we compared the distribution of DPT cells in peripheral blood samples and in penetrating liver tissue between liver rejection versus non-rejection patients, as well as the proportion of DPT cells as a function of the extent of liver rejection. The number of DPT cells in the rejection group was significantly increased. An analysis of the spatial distance and correlations between DPT and Treg cells, revealed that these cells showed a high degree of contiguity. In a mouse liver transplant model, the number of DPT cells were significantly increased in liver tissue, and the number of CD8+ T cells gradually increased, while CD4+ T cells decreased as a function of time post-transplantation. Expression level of PD-1 in DPT cells also increased in a temporally-dependent manner post liver transplantation and the changes of PD-1+ DPT cells were related to the degree of liver transplant rejection. In DPT cells interacting with Treg, there was an increased expression of PD-1, which enhanced cellular exhaustion. In conclusion, the capacity for DPT cells to induce immune tolerance may represent a new and important protocol for use in targeting treatments for the prevention of liver transplant rejection.

CD80+ dendritic cell derived exosomes inhibit CD8+ T cells through down-regulating NLRP3 expression after liver transplantation.

BACKGROUND: Graft-infiltrated dendritic cells (DCs) and CD8+ T lymphocytes are closely related with immune regulation after liver transplantation (LT). An additional factor which appears to play an important role with regard to transplantation immunity are exosomes, which are membrane-derived small vesicles released by various cells. However, the regulation of CD80+ DCs derived exosomes on CD8+ T cells in response to LT remains unclear. METHODS: Ten LT patients and two donors were included in this study. Multiple immunofluorescencewas performed to identify infiltratedCD8+ Tcells and DCs in human liver samples. The expression of NLRP3 and Ki-67 were measured usingimmunohistochemistry and immunofluorescence staining.Changes in CD80+ DCs and CD8+ T cells within the liver and blood of a mouse model of LT were detected with flow cytometry. After coculture with CD80+ DCs derived exosomes, the proliferation, adhesion, and transmigration ofCD8+ T cells were determined with the use of CCK-8, Adhesion and Transmigration assay in vitro.CD8+ T cells related cytokines were measured using Western blot, qRT-PCR and ELISA. RESULTS: In human liver samples, there was an increase in intrahepatic CD8+ T cell infiltration in the acute LT rejection group, an effect which was accompanied by high expressions of NLRP3 and Ki-67 index and a decrease in DCs. Similarly, lower levels of CD80+ DCs were observed with acute rejection in an LT mouse model, along with increased numbers of CD8+ T cells in the liver and blood. In vitro, CD80+ DCs derived exosomes modulated the secretion of CD8+ T cells from cytokines by down-regulating NLRP3 expression, combined with a synchronous inhibition in the adhesion, invasion, and proliferation of CD8+ T cells. CONCLUSION: CD80+ DCs derived exosomes negatively regulate CD8+ T cells via inhibition of NLRP3 expression, a series of events which are essential to attenuate acute LT rejection. These results reveal a new role for CD80+ DCs exosomes as related to tolerance induction in LT.

Multiplex Immunofluorescence for Detection of Spatial Distributions of Infiltrating T Cells Within Different Regions of Hepatic Lobules During Liver Transplantation Rejection.

It remains unclear as to whether there are differences that exist in the types and functional status of immune cells within different areas of the liver lobules after rejection of liver transplantation. The composition of infiltrating T cells in liver allografts during liver transplantation rejection is indistinct and difficult to visualize within the same biopsy slide. In an attempt to rectify this problem, we applied multiplex immunofluorescent assays to assess the spatial distribution of various types of infiltrating T cells in different areas of the liver lobules after liver transplantation. In identical areas of the hepatic lobules, the percentage of CD4+ T, CD8+ T, and regulatory T (Treg) cells in the rejection group was greater than that observed in the non-rejection and normal groups. Within all three groups, the percentage of CD4+ T, CD8+ T, and Treg cells from the periportal to perivenous zones initially increased and then decreased. In the rejection group, the percentage of CD8+ T cells gradually increased from the periportal to perivenous zones, with maximal levels in the perivenous as compared with that in the transitional and periportal zones. In conclusion, levels of CD8+ T cells within different regions of liver lobules are closely related to levels of rejection after liver transplantation. Liver transplantation rejection may be linked with increases in CD8+ T cells within the perivenous zone. Although the regional percent of increase in CD4+ T cells may not reflect level of the rejection, the overall numbers of both of CD4+ and CD8+ T cells within different regions were closely related to rejection levels.

Characteristics of Changes in Inflammatory Cytokines as a Function of Hepatic Ischemia-Reperfusion Injury Stage in Mice.

Liver ischemia-reperfusion injury (IRI) can severely compromise the prognosis of patients receiving liver surgery. While inflammation contributes to the damage resulting from IRI, only a limited number of inflammation biomarkers have been identified as being associated with the different stages of hepatic IRI. As an approach to identify some of these inflammatory cytokines and the molecular mechanisms involved within different stages of hepatic IRI, we used an advanced antibody array assay to detect multiple proteins. With this technology, we observed specific differences in the content of inflammatory cytokines between ischemic and sham controls, as well as a function of the different reperfusion stages in a hepatic IRI mouse model. For example, while liver tissue content of IL-12p40/p70 was significantly increased in the ischemic stage, it was significantly decreased in the reperfusion stage as compared with that of the sham group. For other inflammatory cytokines, no changes were obtained between the ischemic and reperfusion stages with levels of IL-17, Eotaxin-2, Eotaxin, and sTNF RII all being consistently increased, while those of TIMP-1, TIMP-2, BLC, and MCSF consistently decreased as compared with that of the sham group at all reperfusion stages examined. Results from protein function annotation Gene Ontology and the KEGG pathway revealed that inflammatory cytokines are enriched in a network associated with activation of inflammatory response signaling pathways such as TLR, TNF, and IL-17 when comparing responses of the IR versus sham groups. The identification of cytokines along with their roles at specific stages of IRI may reveal important new biological markers for the diagnosis and prognosis of hepatic IRI.

MiR-330-3p suppresses phosphoglycerate mutase family member 5 -inducted mitophagy to alleviate hepatic ischemia-reperfusion injury.

Mitochondrial dysfunction plays a central role in hepatic ischemia-reperfusion injury (IRI). The significance of mitophagy in hepatic IRI remains poorly understood. The mechanisms that cause IRI are complex, and many factors are involved in the injury formation process. The miR-330-3p mediates cell proliferation, cell death, and metabolism in various organisms. In this study, the levels of miR-330-3p were significantly downregulated in hepatic IRI, and the number of autophagosomes was increased in response to IRI as obtained under both in vivo and in vitro conditions. These results demonstrate that a reduction in miR-330-3p expression represents an important factor involved with promoting hepatic IRI. Moreover, we found that miR-330-3p interacted with phosphoglycerate mutase family member 5 (PGAM5) to regulate mitophagy. In specific, an overexpression of miR-330-3p diminished PGAM5 levels, which promoted mitophagy in response to IRI. In contrast, a downregulation of miR-330-3p was associated with increased PGAM5 levels leading to increased mitophagy. In conclusion, miR-330-3p suppresses PGAM5-induced mitophagy to alleviate hepatic IRI. Such findings not only reveal some of the mechanistic basis for this microRNA in liver injury, but also provide a foundation for new therapeutic approaches in the treatment of this condition.

MiR-27a-5p regulates apoptosis of liver ischemia-reperfusion injury in mice by targeting Bach1.

Ischemia-reperfusion (I/R) injury causes cellular dysfunction and a series of immune or apoptotic reactions. Bach1 is a mammalian transcription factor that represses Hmox1, which encodes heme oxygenase-1 (HO-1) that can degrade heme into free iron, carbon monoxide, and biliverdin, to play an important role in antioxidant, anti-inflammatory, and antiapoptotic activities. MicroRNAs (miRNAs) can be found in a variety of eukaryotic cells and viruses, a class of noncoding small RNAs that are encoded by endogenous genes. The aims of this study were to determine whether miR-27a-5p targets Bach1 and regulates cellular death; the dual-luciferase reporter assay was used to detect this and the results showed that miR-27a-5p significantly decreased the luciferase activity of the Bach1 3'-untranslated region. MiR-27a-5p was increased in mice during hepatic I/R and Bach1 was decreased. By transfecting the AML12 cells with the mimic, inhibitor miR-27a-5p in hypoxia/reoxygenation (H/R) models showed that overexpression of miR-27a-5p decreased Bach1 messenger RNA, upregulated HO-1 expression, and promoted antiapoptotic Bcl-2 and downregulated proapoptotic caspase-3 gene expression. In contrast, the miR-27a-5p inhibitor yielded the opposite results. Meanwhile, transfection with Bach1 small interference RNA obviously upregulated the protein levels of HO-1 and resulted in an increase in Bcl-2 and a decrease in caspase-3 protein levels. Thus, we can conclude that miR-27a-5p is relevant to liver I/R injury and overexpression of miR-27a-5p may alleviate apoptosis in H/R injury by targeting Bach1 in vitro.

Effect of graft size matching on pediatric living-donor liver transplantation at a single center.

We retrospectively analyzed 252 patients with end-stage liver disease who had undergone LDLT from January 2009 to September 2015. Of these, 25 had a GRWR of <2.0% (Group A), 204 had a GRWR of ≥2.0% or <4.0% (Group B), and 23 had a GRWR ≥4.0% (Group C). The three GRWR groups demonstrated similar characteristics, except for recipient age and recipient BMI. The overall 1-, 2-, and 3-year graft survival rates were 95.1%, 93.5%, and 93.5%, respectively. However, among the three groups, graft survival rates at 1 year, 2 years, and 3 years were significantly different (P = .0009). Hepatic artery stenosis/thrombosis was more frequently observed in Group C than in Groups A and B (P = .001). Wound infection was also more frequently observed in Group C than in Group A and B (P = .002). However, intestinal fistula/bile leakage/biliary-enteric anastomotic fistula was more frequently observed in Group A than in Groups B and C (P = .001). In addition, reoperation more frequently occurred in Group A and C than in Group B (P = .001). Recipients with a GRWR between 2.0% and 4.0% had significantly better graft survival rates.

Stat3-Atg5 signal axis inducing autophagy to alleviate hepatic ischemia-reperfusion injury.

In performing our experiment, impaired autophagy increased hepatocellular damage during the reperfusion period. It was demonstrated by the effect of blocking autophagy using bafilomycin A1 or knocking Atg5 gene out reduces the anti-apoptotic effect of Stat3. Here we focus on the role of signal transducer and activator of transcription 3 (Stat3) in regulating autophagy to alleviate hepatic IRI. We found that Stat3 was up-regulated during hepatic IRI and was associated with an activation of the autophagic signaling pathway. This increased Stat3 expression, which was allied with high autophagic activity, alleviated liver damage to IR, an effect which was abrogated by Stat3 epletion as demonstrated in both in vivo and in vitro methods. The levels of Atg5 protein were decreased when Stat3 was inhibited by HO 3867 or siStat3. We conclude that Stat3 appeared to exert a pivotal role in hepatic IRI, by activating autophagy to alleviate hepatic IRI, and Atg5 was required for this process. The identification of this novel pathway, that links expression levels of Stat3 with Atg5-mediated autophagy, may provide new insights for the generation of novel protective therapies directed against hepatic IRI.

Vitexin suppresses autophagy to induce apoptosis in hepatocellular carcinoma via activation of the JNK signaling pathway.

Vitexin, a flavonoids compound, is known to exhibit broad anti-oxidative, anti-inflammatory, analgesic, and antitumor activity in many cancer xenograft models and cell lines. The purpose of this study was to investigate the antitumor effects and underlying mechanisms of vitexin on hepatocellular carcinoma. In this study, we found that vitexin suppressed the viability of HCC cell lines (SK-Hep1 and Hepa1-6 cells) significantly. Vitexin showed cytotoxic effects against HCC cell lines in vitro by inducing apoptosis and inhibiting autophagy. Vitexin induced apoptosis in a concentration-dependent manner, and caused up-regulations of Caspase-3, Cleave Caspase-3, and a down-regulation of Bcl-2. The expression of autophagy-related protein LC3 II was significantly decreased after vitexin treatment. Moreover, western blot analysis presented that vitexin markedly up-regulated the levels of p-JNK and down-regulated the levels of p-Erk1/2 in SK-Hep1 cells and Hepa1-6 cells. Cotreatment with JNK inhibitor SP600125, we demonstrated that apoptosis induced by vitexin was suppressed, while the inhibition of autophagy by vitexin was reversed. The results of colony formation assay and mouse model confirmed the growth inhibition role of vitexin on HCC in vitro and in vivo. In conclusion, vitexin inhibits HCC growth by way of apoptosis induction and autophagy suppression, both of which are through JNK MAPK pathway. Therefore, vitexin could be regarded as a potent therapeutic agent for the treatment of HCC.

LncRNA HULC enhances epithelial-mesenchymal transition to promote tumorigenesis and metastasis of hepatocellular carcinoma via the miR-200a-3p/ZEB1 signaling pathway.

Highly upregulated in liver cancer (HULC), a lncRNA that is considered a key molecule in human liver cancer, has recently been revealed to be involved in hepatocellular carcinoma (HCC) development and progression [1, 2]. It has been reported that HULC can promote tumor invasion and metastasis of HCC, but its function and mechanism of action in HCC have not been elucidated. In this study, we found that HULC was aberrantly up-regulated in HCC tissues and associated with TNM stage, intrahepatic metastases, HCC recurrence, and postoperative survival. HULC depletion inhibited the growth and metastasis of HCC cell lines in vitro and in vivo. Moreover, HULC contributes to ZEB1-induced epithelial-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis that plays a key role in cancer progression. This effect of ZEB1 was inhibited by HULC siRNA. We conclude that the HULC functioned as a competing endogenous RNA (ceRNA) to mediate EMT via up-regulating ZEB1. In this way, it sequesters the miR-200a-3p signaling pathway to facilitate HCC metastasis. HULC comes into play as an oncogene in HCC, acting mechanistically by inducing HCC cells to activate EMT. Such an effect promotes tumor progression and metastasis through the miR-200a-3p/ZEB1 signaling pathway. The identification of this novel pathway that links high expression levels of HULC with EMT in HCC cells may serve as the foundation for the development of novel anti-tumor therapeutics.

miR-30b inhibits autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.

AIM: To explore the role and potential mechanism of miR-30b regulation of autophagy in hepatic ischemia-reperfusion injury (IRI). METHODS: An animal model of hepatic IRI was generated in C57BL/6 mice. For in vitro studies, AML12 cells were immersed in mineral oil for 1 h and then cultured in complete Dulbecco's Modified Eagle's Medium (DMEM)/F12 to simulate IRI. Mice and cells were transfected with miR-30b agomir/mimics or antagomir/inhibitor to examine the effect of miR-30b on autophagy to promote hepatic IRI. The expression of miR-30b was measured by real-time polymerase chain reaction. Apoptotic cells were detected by terminal uridine nick-end labeling (TUNEL) staining, and cell viability was detected by methylthiazole tetrazolium assay. The expression of light chain 3, autophagy-related gene (Atg)12, Atg5, P62, and caspase-3 were detected by western blotting analysis. RESULTS: miR-30b levels were significantly downregulated after hepatic IRI, and the numbers of autophagosomes were increased in response to IRI both in vivo and in vitro. These findings demonstrate that low levels of miR-30b could promote hepatic IRI. Furthermore, we found that miR-30b interacted with Atg12-Atg5 conjugate by binding to Atg12. Overexpression of miR-30b diminished Atg12 and Atg12-Atg5 conjugate levels, which promoted autophagy in response to IR. In contrast, downregulation of miR-30b was associated with increased Atg12-Atg5 conjugate levels and increased autophagy. CONCLUSION: miR-30b inhibited autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate.

Bi-directional roles of IRF-1 on autophagy diminish its prognostic value as compared with Ki67 in liver transplantation for hepatocellular carcinoma.

The prognostic values of IRF-1 and Ki-67 for liver transplantation (LT) of hepatocellular carcinoma (HCC) were investigated, as well as the mechanisms of IRF-1 in tumor suppression. Adult orthotropic liver transplantation cases (N = 127) were involved in the analysis. A significant decreased recurrence free survival (RFS) was found in the Ki-67 positive groups. Ki-67, tumor microemboli, the Milan and UCSF criteria were found to be independent risk factors for RFS. In LT for HCC beyond the Milan criteria, a significant decrease in RFS was found in the IRF-1 negative groups. In SK-Hep1 cells, an increase in apoptosis and decrease in autophagy were observed after IFN-γ stimulation, which was accompanied with increasing IRF-1 levels. When IRF-1 siRNA or a caspase inhibitor were used, reductions in LC3-II were diminished or disappeared after IFN-γ stimulation, suggesting that IFN-γ inhibited autophagy via IRF-1 expression and caspase activation. However, after IRF-1 siRNA was introduced, a reduction in LC3-II was found. Thus basic expression of IRF-1 was also necessary for autophagy. IRF-1 may be used as a potential target for HCC treatment based on its capacity to affect apoptosis and autophagy. Ki-67 shows great promise for the prediction of HCC recurrence in LT and can be used as an aid in the selection of LT candidates.

N'-(3-Fluoro-benzyl-idene)-2-methyl-benzohydrazide.

The asymmetric unit of the title compound, C(15)H(13)FN(2)O, contains two independent mol-ecules with different conformations; the two aromatic rings in the independent mol-ecules form dihedral angles of 85.3 (2) and 10.0 (2)°. In the crystal, N-H⋯O hydrogen bonds link the mol-ecules into chains along [100].