N7-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression.

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

Hypoxia-driven tumor stromal remodeling and immunosuppressive microenvironment in scirrhous HCC.

BACKGROUND AND AIMS: Scirrhous HCC (SHCC) is one of the unique subtypes of HCC, characterized by abundant fibrous stroma in the tumor microenvironment. However, the molecular traits of SHCC remain unclear, which is essential to develop specialized therapeutic approaches for SHCC. APPROACH AND RESULTS: We presented an integrative analysis containing single-cell RNA-sequencing, whole-exome sequencing, and bulk RNA-sequencing in SHCC and usual HCC samples from 134 patients to delineate genomic features, transcriptomic profiles, and stromal immune microenvironment of SHCC. Multiplexed immunofluorescence staining, flow cytometry, and functional experiments were performed for validation. Here, we identified SHCC presented with less genomic heterogeneity while possessing a unique transcriptomic profile different from usual HCC. Insulin-like growth factor 2 was significantly upregulated in SHCC tumor cells compared to usual HCC, and could serve as a potential diagnostic biomarker for SHCC. Significant tumor stromal remodeling and hypoxia were observed in SHCC with enrichment of matrix cancer-associated fibroblasts and upregulation of hypoxic pathways. Insulin-like growth factor 2 was identified as a key mediator in shaping the hypoxic stromal microenvironment of SHCC. Under this microenvironment, SHCC exhibited an immunosuppressive niche correlated to enhanced VEGFA signaling activity, where CD4 + T cells and CD8 + T cells were dysfunctional. Furthermore, we found that another hypoxic-related molecule SPP1 from SHCC tumor cells suppressed the function of dendritic cells via the SPP1-CD44 axis, which also probably hindered the activation of T cells. CONCLUSION: We uncovered the genomic characteristics of SHCC, and revealed a hypoxia-driven tumor stroma remodeling and immunosuppressive microenvironment in SHCC.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

Anti-PD-L1 blockade facilitates antitumor effects of radiofrequency ablation by improving tumor immune microenvironment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a complex disease with advanced presentation that significantly affects survival rates. Therefore, novel therapeutic strategies are needed. In this study, we investigate the tumor microenvironment (TME) in HCC by analyzing 13 HCC samples at single cell level. We identified key cell populations, including CD8 + T cells, Tregs, M1/M2 macrophages, and CD4 + memory T cells, and explored their roles and interactions. Our research revealed an early enrichment of CD8 + T cells, which could potentially lead to their exhaustion and facilitate tumor progression. We also investigated the impact of percutaneous radiofrequency ablation (RFA) on the immune microenvironment. Using a dual tumor mouse model, we demonstrated that RFA induces necrosis, enhancing antigen presentation and altering immune responses. Our results indicate that RFA increases PD-L1 expression in residual liver tissue, suggesting potential immune escape mechanisms. Furthermore, the combination of RFA and anti-PD-L1 therapy in the mouse model resulted in significant improvements in immune modulation. This included increased CD8 + T cell efficacy and decreased Treg infiltration. This combination shows promise as an approach to counteract HCC progression by altering the immune landscape. This study highlights the critical interaction within the TME of HCC and suggests the possibility of improving patient outcomes by targeting immune evasion mechanisms through combined therapeutic strategies.

Imatinib facilitates gemcitabine sensitivity by targeting epigenetically activated PDGFC signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with poor prognosis. Gemcitabine-based chemotherapy has become one of the main modalities of its management. However, gemcitabine resistance frequently occurs, leading to failure of PDAC therapy. Platelet-derived growth factors (PDGFs) and their receptors play important roles in cancer progression and chemoresistance. We aimed to investigate the biological function and therapeutic significance of platelet-derived growth factor C (PDGFC) in drug-resistant PDAC. Our study showed that PDGFC was abnormally highly expressed in gemcitabine-resistant PDAC. Silencing PDGFC expression can enhance the therapeutic effect of gemcitabine on PDAC. Mechanistically, the transcription of PDGFC is mediated by H3K27 acetylation, and PDGFC promotes gemcitabine resistance by activating the PDGFR-PI3K-AKT signaling pathway. The PDGFR inhibitor imatinib inhibits the PDGFR pathway. Imatinib and gemcitabine have a synergistic effect on the treatment of PDAC, and imatinib can significantly enhance the anti-tumor effect of gemcitabine in a drug-resistant PDAC patient-derived xenograft model. In conclusion, PDGFC is a potential predictor of gemcitabine-resistant PDAC. Imatinib inhibits PDGFR activation to promote gemcitabine sensitivity in PDAC. Combined modality regimen of imatinib and gemcitabine is likely to translate into clinical trial for the treatment of PDGFC-associated gemcitabine-resistant patients.

Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

DAGLß is the principal synthesizing enzyme of 2-AG and promotes aggressive phenotype of intrahepatic cholangiocarcinoma via AP-1/DAGLß/miR4516 feedforward circuitry.

The endocannabinoid system (ECS) is dysregulated in various liver diseases. Previously, we had shown that the major endocannabinoid 2-arachidonoyl glycerol (2-AG) promoted tumorigenesis of intrahepatic cholangiocarcinoma (ICC). However, biosynthesis regulation and clinical significance of 2-AG remain elusive. In the present study, we quantified 2-AG by gas chromatography/mass spectrometry (GC/MS) and showed that 2-AG was enriched in patients with ICC samples as well as in thioacetamide-induced orthotopic rat ICC model. Moreover, we found that diacylglycerol lipase ß (DAGLß) was the principal synthesizing enzyme of 2-AG that significantly upregulated in ICC. DAGLß promoted tumorigenesis and metastasis of ICC in vitro and in vivo and positively correlated with clinical stage and poor survival in patients with ICC. Functional studies showed that activator protein-1 (AP-1; heterodimers of c-Jun and FRA1) directly bound to the promoter and regulated transcription of DAGLß, which can be enhanced by lipopolysaccharide (LPS). miR-4516 was identified as the tumor-suppressing miRNA of ICC that can be significantly suppressed by LPS, 2-AG, or ectopic DAGLß overexpression. FRA1 and STAT3 were targets of miR-4516 and overexpression of miRNA-4516 significantly suppressed expression of FRA1, SATA3, and DAGLß. Expression of miRNA-4516 was negatively correlated with FRA1, SATA3, and DAGLß in patients with ICC samples. Our findings identify DAGLß as the principal synthesizing enzyme of 2-AG in ICC. DAGLß promotes oncogenesis and metastasis of ICC and is transcriptionally regulated by a novel AP-1/DAGLß/miR4516 feedforward circuitry.NEW & NOTEWORTHY Dysregulated endocannabinoid system (ECS) had been confirmed in various liver diseases. However, regulation and function of 2-arachidonoyl glycerol (2-AG) and diacylglycerol lipase ß (DAGLß) in intrahepatic cholangiocarcinoma (ICC) remain to be elucidated. Here, we demonstrated that 2-AG was enriched in ICC, and DAGLß was the principal synthesizing enzyme of 2-AG in ICC. DAGLß promotes tumorigenesis and metastasis in ICC via a novel activator protein-1 (AP-1)/DAGLß/miR4516 feedforward circuitry.

Renin-angiotensin system inhibitors improve the survival of cholangiocarcinoma: a propensity score-matched cohort study.

BACKGROUND: Hypertension is a risk factor for cholangiocarcinoma (CCA). The effect of anti-hypertensive drugs on the prognosis of CCA is not clear. METHODS: This is a retrospective study of 102 patients (56.9% males, median age 66 years) diagnosed with CCA and hypertension concurrently and received radical surgery (R0), with a median follow-up of 36.7 months. Kaplan-Meier analysis, Cox regressions, and propensity score (PS) matching were applied for statistical analysis. RESULTS: Results of multivariable cox analysis showed that renin-angiotensin system inhibitors (RASis) usage was a protective factor for progression-free survival (PFS) (hazard ratio [HR] = 0.55, 95% confidence interval [95% CI]: 0.32-0.96) and overall survival (OS) (HR = 0.40, 95% CI: 0.20-0.79), respectively. Calcium channel blockers, diuretics, and ß-blockers didn't show significant associations. The association of RASis usage and PFS and OS was derived by PS matching, with a cohort of 28 RASis users and 56 RASis non-users. The median PFS and OS of RASis users (PFS, 17.6 months (9.2-34.4); OS, 24.8 months (16.5-42.3)) were longer than RASis non-users (PFS, 10.5 months (4.1-24.1); OS, 14.6 months (10.6-28.4)). The 1 year, 2 years, and 3 years' survival rates of RASis users (89.1%, 77.0%, and 65.5%) were higher than RASis non-users (70.9%, 54.0%, and 40.0%). CONCLUSIONS: RASis usage improves the survival of patients with CCA and hypertension concurrently.

Three-day postoperative antibiotics reduces post-hepatectomy infection rate in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND AND AIM: The evidences for use of postoperative antibiotics (POA) in hepatocellular carcinoma (HCC) patients who underwent hepatectomy are controversial. We aimed to explore the relationship between POA and hepatectomy-related infection in a hepatitis B virus (HBV)-related HCC population. METHODS: We retrospectively collected 934 HCC patients who underwent hepatectomy for curative intent from three tertiary hospitals in China. The incidences of postoperative infection including surgical site infection and remote site infection were recorded and calculated. Univariable and multivariable logistic regression analyses were performed to explore related factors of postoperative infection and POA. And the relationship between infection rates with different durations of POA was investigated. RESULTS: The overall infection rate was 8.2% (77/934), including 6.5% (61/934) of surgical site infection and 2.0% (19/934) of remote site infection. Multivariable analysis revealed that the administration of POA was negatively related with the incidence of postoperative infection significantly (odds ratio = 0.50, 95% confidence interval = 0.30 to 0.83; P = 0.008). Albumin-bilirubin score, Barcelona Clinic Liver Cancer (BCLC) stage and extent of hepatectomy were independently related to the POA. And 3-day regimen seemed to be the shortest duration of POA to gain the lowest incidence of postoperative infection. CONCLUSIONS: Postoperative antibiotic is necessary for HBV-related HCC patients to prevent postoperative infection, especially for those with higher albumin-bilirubin score, at BCLC stage B-C, or who underwent major hepatectomy. For HBV-related HCC patients, postoperative second-generation cephalosporins, or ceftriaxone for 3 days after surgery might be proper.

Prognostic Role of Time to Surgery in Hepatocellular Carcinoma at Barcelona Clinic Liver Cancer Stage 0-A.

BACKGROUND: Postsurgical recurrence is common in early-stage hepatocellular carcinoma (HCC). Prolonged time to surgery (TTS) may lead to tumor progression. However, the impact of TTS on HCC prognosis is controversial in Western studies and unknown in China. We aim to investigate the impact of TTS on the prognosis of Chinese HCC patients at Barcelona Clinic Liver Cancer (BCLC) stage 0-A who underwent surgery. PATIENTS AND METHODS: We retrospectively enrolled 967 BCLC 0-A HCC patients who underwent surgery at three tertiary centers in China. Primary outcomes were recurrence-free survival (RFS) and overall survival (OS). Restricted cubic spline (RCS) was used to select the cutoff value of TTS. Propensity score matching (PSM) was performed to reduce confounding bias, and a time-dependent Cox model was utilized to investigate factors influencing TTS. RESULTS: The median TTS of BCLC 0-A HCC patients was 13 days (interquartile range: 10-21 days). For patients with TTS ≤ 70 days, the cutoff value of TTS was 13 days according to RCS. After PSM, corresponding 1-, 3-, and 5-year RFS of the TTS > 13 days and TTS ≤ 13 days groups were 75.6%, 55.3%, 46.4% and 71.2%, 52.3%, 38.8%, respectively (P = 0.103). Corresponding 1-, 3-, and 5-year OS of TTS > 13 days and TTS ≤ 13 days groups were 93.7%, 82.8%, 69.6% and 92.4%, 78.5%, 68.4%, respectively (P = 0.580). Time-dependent Cox analysis revealed that age and tumor size were factors influencing TTS. CONCLUSIONS: Our study suggests that, for patients with TTS ≤ 70 days, prolonged TTS had no impact on BCLC 0-A Chinese HCC patients receiving surgery.

The presence of microvascular invasion guides treatment strategy in recurrent HBV-related HCC.

OBJECTIVES: We used the status of microvascular invasion (MVI) at primary resection to help treatment selection for hepatitis B virus-positive (HBV+) recurrent hepatocellular carcinoma (rHCC) patients in Barcelona Clinic Liver Cancer (BCLC) stage B-C. METHODS: From 2009 to 2017, we enrolled 221 consecutive HBV+ rHCC patients at BCLC stage B-C who underwent re-resection (RR), radiofrequency ablation (RFA), or transarterial chemoembolization (TACE). Post recurrence survival (PRS) and overall survival (OS) were compared between RR/RFA and TACE according to MVI status. A one-to-one propensity score matching analysis was performed. RESULTS: For MVI(-) patients, the median PRS was 62.3 months for the RR/RFA group and 21.1 months for the TACE group (p = 0.039). The corresponding OS was 71.4 months and 26.6 months, respectively (p = 0.010). For MVI(+) patients, the median PRS in the RR/RFA group and TACE group was 14.7 months and 10.1 months (p = 0.115). The corresponding OS was 23.4 months and 16.4 months, respectively (p = 0.067). After matching, the dominance of RR/RFA over TACE remained in MVI(-) patients for both PRS (62.3 months vs 15.3 months, p = 0.019) and OS (98.1 months vs 33.4 months, p = 0.046). No significant difference was found in MVI(+) patients for either PRS (14.7 months vs 11.8 months, p = 0.593) or OS (23.4 months vs 28.1 months, p = 0.662). CONCLUSIONS: MVI status definitely helps select treatment options in HBV+ rHCC patients. For MVI(-) patients, RR/RFA provided better survival than TACE while for MVI(+) patients, TACE shared similar survival outcomes. KEY POINTS: • This study aimed at the determination of the optimal treatment options (ablation /resection vs TACE) in case of recurrent HBV-related HCC. • It showed that MVI status, established at primary resection of HCC, was a powerful marker for selecting the best treatment option in these patients. • In MVI(-) patients, RR/RFA achieved a better survival than TACE. In MVI(+) patients, TACE shared similar survival.

Hepatic resection versus transarterial chemoembolization in infiltrative hepatocellular carcinoma: A multicenter study.

BACKGROUND AND AIM: Prognosis of infiltrative hepatocellular carcinoma (iHCC) is poor, and the treatments selection based on efficacy is unclear. We performed this multicenter study to compare the efficacy of hepatic resection and transarterial chemoembolization (TACE) in treating patients with iHCC. METHODS: We retrospectively analyzed the overall survivals (OS) in 319 patients with iHCC who were initially treated by hepatic resection (n = 133) or TACE (n = 186) at four tertiary centers. Fifty-eight patients in the TACE group were assessed as resectable and compared with the hepatic resection group in subgroup analysis. A propensity score matched (PSM) analysis was performed to reduce selection bias. Cox regression was performed to identify significant factors associated with OS. RESULTS: The median OS time was significantly longer in the hepatic resection group than that in the TACE group, before and after PSM (before PSM, 17.5 vs 7.3 months, P < 0.0001; after PSM, 14.0 vs 7.3 months, P < 0.0001). The multivariable analysis indicated TACE as a risk factor of OS (hazard ratio = 2.233, 95% confidence interval = 1.492 to 3.341, P < 0.0001), as well as portal venous tumor thrombosis grades 3-4 and alpha fetal protein (AFP) > 400 ng/mL. In the subgroup analysis, the better efficacy of hepatic resection over TACE persisted regardless of the grade of portal venous tumor thrombosis and the level of AFP. As for resectable patients, hepatic resection still showed significant survival benefit (before PSM, 17.5 vs 11.2 months, P = 0.0013; after PSM, 14.0 vs 10.9 months, P = 0.0304). CONCLUSION: Hepatic resection might be the better choice for patients with iHCC due to its better survival benefit than TACE.

WAVE3 Induces EMT and Promotes Migration and Invasion in Intrahepatic Cholangiocarcinoma.

BACKGROUND: Wiskott-Aldrich syndrome protein family verprolin-homologous protein 3 (WAVE3) plays a critical role in cancer progression and metastasis. However, the specific role of WAVE3 in intrahepatic cholangiocarcinoma (ICC) has not been studied. AIMS: This study aimed to explore the role and mechanism of WAVE3 in the progression and metastasis of ICC. METHODS: The expression of WAVE3 in ICC tissues and adjacent non-cancerous tissues was detected by immunohistochemistry. Western blot analysis was utilized to detect the expression of WAVE3 in ICC cells. A transwell assay was used to assess the potential for migration and invasion. The expression of WAVE3 in CC-LP-1 cells was knocked down by small interfering RNA (siRNA) interference. RESULTS: The expression of WAVE3 in ICC tissues was significantly higher than that in adjacent non-cancerous tissues. The overall survival was lower in the subgroup of ICC patients with higher WAVE3 expression compared to the subgroup with a lower level of WAVE3 expression. WAVE3 expression was an adverse prognostic factor for ICC patients. CC-LP-1 cells expressed higher levels of WAVE3 protein compared to RBE cells and human intrahepatic biliary epithelial cells, which correlated with greater migration and invasion capabilities compared with the RBE cells. After the transfection of CC-LP-1 cells with WAVE3 siRNA, the level of WAVE3 protein was significantly decreased, accompanied by a marked reduction in migration, invasion and proliferation. Moreover, after the knockdown of WAVE3 expression in CC-LP-1 cells, the protein levels of Slug and Vimentin were significantly decreased, while that of E-cadherin was significantly increased. CONCLUSIONS: WAVE3 may represent a new adverse prognostic factor for patients with ICC. This protein enhances migration and invasion capabilities in ICC, most likely through the induction of an epithelial-mesenchymal transition.

Regulation of the extrinsic apoptotic pathway by microRNA-21 in alcoholic liver injury.

IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5'-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.

Secretin stimulates biliary cell proliferation by regulating expression of microRNA 125b and microRNA let7a in mice.

BACKGROUND & AIMS: Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. METHODS: Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. RESULTS: Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. CONCLUSIONS: After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Serum CYFRA 21-1 in Biliary Tract Cancers: A Reliable Biomarker for Gallbladder Carcinoma and Intrahepatic Cholangiocarcinoma.

BACKGROUND: Biliary tract cancers encompass gallbladder carcinoma, and intrahepatic, perihilar and distal cholangiocarcinoma. Upregulated serum CYFRA 21-1 has been reported in intrahepatic cholangiocarcinoma. AIMS: The present study aimed to explore the clinical significance of serum CYFRA 21-1 in all biliary tract cancer subtypes. METHODS: Serum CYFRA 21-1, carbohydrate antigen 19-9 and carcinoembryonic antigen were quantitated preoperatively, postoperatively and during follow-up in 134 malignant and 52 benign patients. Receiver operator characteristic curves of biomarkers were analyzed. Level of CYFRA 21-1 was correlated with patients' clinicopathological features and follow-up data. RESULTS: CYFRA 21-1 was significantly upregulated in biliary malignancies, and expressional difference existed between these subtypes. Based on the maximal Youden's index, cutoff values were selected (ng/mL): 2.61 for biliary tract cancers (sensitivity 74.6 % and specificity 84.6 %); 3.27 for intrahepatic cholangiocarcinoma (75.6 and 96.2 %) and gallbladder carcinoma (93.7 and 96.2 %); 2.27 for perihilar cholangiocarcinoma (71.0 and 71.2 %); and 2.61 for distal cholangiocarcinoma (63.3 and 84.6 %). CYFRA 21-1 showed better diagnostic performance than other biomarkers in gallbladder carcinoma and intrahepatic cholangiocarcinoma; its performance was not inferior to that of the combination of these three biomarkers and declined after curative resection and re-elevated when tumor recurred, which was correlated with tumor aggressiveness and TNM stage; it was an independent predictor for 1-year recurrence-free survival and overall survival on multivariate analysis. CONCLUSION: Serum CYFRA 21-1 represents a reliable biomarker for gallbladder carcinoma and intrahepatic cholangiocarcinoma.

DNA methylation profiling identifies EYA4 gene as a prognostic molecular marker in hepatocellular carcinoma.

BACKGROUND: DNA hypermethylation plays important roles in carcinogenesis by silencing key genes. This study aims to identify pivotal genes in hepatocellular carcinoma (HCC) by DNA methylation microarray and to assess their prognostic values. MATERIALS AND METHODS: DNA methylation microarray was performed in 45 pairs of HCC and adjacent nontumorous tissues and six normal liver tissues to identify hypermethylated genes in HCC. Potential prognosis-related genes were selected among hypermethylated genes by analyzing influences of methylation levels on disease-free survival (DFS) and overall survival (OS) in 45 patients. Their prognostic values were validated in 154 patients with HCC (including the initial 45 patients) to determine the independent prognostic gene. RESULTS: Altogether, 54 CpG islands in 44 genes were hypermethylated in HCC compared with liver tissues. Among them, methylation levels of ERG and HOXA11 were inversely associated with DFS (both P < 0.050), and methylation levels of EYA4 were inversely related to DFS and OS (both P < 0.050). EYA4 expression was inversely related to tumor size (P < 0.050). Lower EYA4 expression and larger tumor size were independent predictors of both shorter DFS and OS, and higher Barcelona Clinic Liver Cancer (BCLC) staging was an independent predictor of shorter OS (all P < 0.050). CONCLUSIONS: EYA4 functions as a prognostic molecular marker in HCC. Its aberrant hypermethylation and subsequent down-regulation may promote tumor progression.

[Experimental study on concentrations and pharmacokinetics of antibiotics in bile and evaluation of their microbicidal potential].

OBJECTIVE: To study the concentrations and pharmacokinetics of 6 different kinds of antibiotics in rabbit bile, and evaluate their microbicidal potential. METHODS: Thirty-six health rabbits were randomly divided into 6 groups, and each group was 6 rabbits. After anaesthesia, the common bile duct of rabbit was isolated and cumulated with a silicone tube. The rabbits were administered intravenously with the equal-effect dose of antibiotics. Bile (1.5 ml) was collected at different time points after administration, and the concentration of antibiotics of bile was assayed by high performance liquid chromatography. The bile drug concentration-time data were processed by software to figure out the pharmacokinetic parameters such as maximum concentration (C(max)), peak time (T(max)), half-life time (T(1/2)), clearance (CL) and apparent volume of distribution (VD). The bile antibiotics concentration contrasted to the minimum inhibitory concentration (MIC), and attained the bactericidal index (C(max)/MIC) and the time when the drug concentration exceeded the MIC (T(>MIC)). RESULTS: The C(max) and T1/2 of each antibiotic were as the followings: piperacillin (7 950 ± 3 023) mg/L and (1.97 ± 1.23) h, ceftriaxone (1 104 ± 248) mg/L and (3.14 ± 0.57) h, cefoperazone (5 215 ± 2 225) mg/L and (0.89 ± 0.13) h, meropenem (31.97 ± 12.44) mg/L and (0.36 ± 0.11) h, levofloxacin (66.3 ± 36.9) mg/L and (3.32 ± 2.57) h, metronidazole (28.2 ± 10.2) mg/L and (0.81 ± 0.33) h, respectively. Piperacillin/tazobactam and cefoperazone/sulbactam had the largest bactericidal index and the longest T(>MIC), and their bactericidal indexes were (62.1 ± 23.6) - (993.8 ± 377.9) and (164.8 ± 69.0) - (659.3 ± 275.9), their T(>MIC) were (6.00 ± 2.53) - (8.00 ± 0.00) h and (6.33 ± 1.97) - (8.00 ± 0.00) h. The bactericidal index and T(>MIC) of levofloxacin were the smallest, which were (2.1 ± 1.2) - (8.3 ± 4.6) and (0.54 ± 0.25) - (2.67 ± 1.03) h . Ceftriaxone and meropenem were as the medium, and their bactericidal indexes and T(>MIC) were (4.3 ± 1.0) - (69.2 ± 15.5) , (1.42 ± 0.65) - (8.00 ± 0.00) h and (2.0 ± 0.8) - (1 031.3 ± 401.4) , (0.29 ± 0.10) - (1.83 ± 0.26) h. The bactericidal index of metronidazole to anaerobic ranged from 7.4 to 294.9, and the T(>MIC) ranged from 1.88 to 5.00 h. CONCLUSIONS: The bile concentrations of six antibiotics all exceed their effective bactericidal concentrations. The concentration-time curves of piperacillin, cefoperazone, meropenem and metronidazole conformed to one-compartment model, and ceftriaxone and levofloxacin are conformed to two-compartment model. Piperacillin/tazobactam and cefoperazone/sulbactam have the largest bactericidal index and the longest T(>MIC), so they can be chosen as the first choice for the therapy of hepatobiliary infection.For the anaerobic, the microbicidal potential of metronidazole is high.

Fatty acid metabolism affects hepatocellular carcinoma progression via the PPAR-γ signaling pathway and fatty acid ß-oxidation.

PURPOSE: This study aimed to explore novel targets for hepatocellular carcinoma (HCC) treatment by investigating the role of fatty acid metabolism. METHODS: RNA-seq and clinical data of HCC were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatic analyses were employed to identify differentially expressed genes (DEGs) related to prognosis. A signature was then constructed using the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression to classify HCC patients from the TCGA database into low-risk and high-risk groups. The predictive performance of the signature was evaluated through principal components analysis (PCA), Kaplan Meier (KM) survival analysis, receiver operating characteristics (ROC) curves, nomogram, genetic mutations, drug sensitivity analysis, immunological correlation analysis, and enrichment analysis. Single-cell maps were constructed to illustrate the distribution of core genes. Immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR), and western blot were employed to verify the expression of core genes. The function of one core gene was validated through a series of in vitro assays, including cell viability, colony formation, wound healing, trans-well migration, and invasion assays. The results were analyzed in the context of relevant signaling pathways. RESULTS: Bioinformatic analyses identified 15 FAMGs that were related to prognosis. A 4-gene signature was constructed, and patients were divided into high- and low-risk groups according to the signature. The high-risk group exhibited a poorer prognosis compared to the low-risk group in both the training (P < 0.001) and validation (P = 0.020) sets. Furthermore, the risk score was identified as an independent predictor of OS (P < 0.001, HR = 8.005). The incorporation of the risk score and clinicopathologic features into a nomogram enabled the effective prediction of patient prognosis. The model was able to effectively predict the immune microenvironment, drug sensitivity to chemotherapy, and gene mutation for each group. Single-cell maps demonstrated that FAMGs in the model were distributed in tumor cells. Enrichment analyses revealed that the cell cycle, fatty acid ß oxidation and PPAR signaling pathways were the most significant pathways. Among the four key prognostically related FAMGs, Spermine Synthase (SMS) was selected and validated as a potential oncogene affecting cell cycle, PPAR-γ signaling pathway and fatty acid ß oxidation in HCC. CONCLUSIONS: The risk characteristics based on FAMGs could serve as independent prognostic indicators for predicting HCC prognosis and could also serve as evaluation criteria for gene mutations, immunity, and chemotherapy drug therapy in HCC patients. Meanwhile, targeted fatty acid metabolism could be used to treat HCC through related signaling pathways.