Promotion of perineural invasion of cholangiocarcinoma by Schwann cells via nerve growth factor.

Background: Cholangiocarcinoma (CCA), a highly lethal tumor of the hepatobiliary system originating from bile duct epithelium, can be divided into the intrahepatic, hilar, and extrahepatic types. Due to its insidious onset and atypical early clinical symptoms, the overall prognosis is poor. One of the important factors contributing to the poor prognosis of CCA is the occurrence of perineural invasion (PNI), but the specific mechanisms regarding how it contributes to the occurrence of PNI are still unclear. The main purpose of this study is to explore the molecular mechanism leading to the occurrence of PNI and provide new ideas for clinical treatment. Methods: CCA cell lines and Schwann cells (SCs) were stimulated to observe the changes in cell behavior. SCs cocultured with tumor supernatant and SCs cultured in normal medium were subjected to transcriptome sequencing to screen the significantly upregulated genes. Following this, the two types of tumor cells were cultured with SC supernatant, and the changes in behavior of the tumor cells were observed. Nonobese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice were injected with cell suspension supplemented with nerve growth factor (NGF) via the sciatic nerve. Four weeks later, the mice were euthanized and the tumor sections were removed and stained. Results: Nerve invasion by tumor cells was common in CCA tissues. SCs were observed in tumor tissues, and the number of SCs in tumor tissues and the degree of PNI were much higher than were those in normal tissues or tissues without PNI. The overall survival time was shorter in patients with CCA with PNI than in patients without PNI. SCs were enriched in CCA tissues, indicating the presence of PNI and associated with poor prognosis in CCA patients. CCA was found to promote NGF secretion from SCs in vitro. After the addition of exogenous NGF in CCA cell culture medium, the proliferation activity and migration ability of CCA cells were significantly increased, suggesting that SCs can promote the proliferation and migration of CCA through the secretion of NGF. NGF, in turn, was observed to promote epithelial-mesenchymal transition in CCA through tropomyosin receptor kinase A (TrkA), thus promoting its progression. Tumor growth in mice shows that NGF can promote PNI in CCA. Conclusions: In CCA, tumor cells can promote the secretion of NGF by SCs, which promotes the progression of CCA and PNI by binding to its high-affinity receptor TrkA, leading to poor prognosis.

A Bayesian Network Prediction Model for Microvascular Invasion in Patients with Intrahepatic Cholangiocarcinoma: A Multi-institutional Study.

BACKGROUND: Microvascular invasion (MVI) has been reported to be an independent prognostic factor of recurrence and poor overall survival in patients with intrahepatic cholangiocarcinoma (ICC). This study aimed to explore the preoperative independent risk factors of MVI and establish a Bayesian network (BN) prediction model to provide a reference for surgical diagnosis and treatment. METHODS: A total of 531 patients with ICC who underwent radical resection between 2010 and 2018 were used to establish and validate a BN model for MVI. The BN model was established based on the preoperative independent variables. The ROC curves and confusion matrix were used to assess the performance of the model. RESULTS: MVI was an independent risk factor for relapse-free survival (RFS) (P < 0.05). MVI has a correlation with postoperative recurrence, early recurrence (< 6 months), median RFS and median overall survival (all P < 0.05). The preoperative independent risk variables of MVI included obstructive jaundice, prognostic nutritional index, CA19-9, tumor size, and major vascular invasion, which were used to establish the BN model. The AUC of the BN model was 78.92% and 83.01%, and the accuracy was 70.85% and 77.06% in the training set and testing set, respectively. CONCLUSION: The BN model established based on five independent risk variables for MVI is an effective and practical model for predicting MVI in patients with ICC.

Prognostic value of lymphadenectomy in node-negative intrahepatic cholangiocarcinoma: A multicenter, retrospectively study.

BACKGROUND: This study aimed to evaluate the prognostic value of lymph node dissection (LND) in node-negative intrahepatic cholangiocarcinoma (ICC) and identify the appropriately total number of lymph nodes examined (TNLE). METHODS: Data from node-negative ICC patients who underwent curative intent resection in ten Chinese hepatobiliary centers from January 2010 to December 2018 were collected. Overall survival (OS), relapse-free survival (RFS) and postoperative complications were analyzed. Propensity score matching (PSM) was performed to reduce the bias due to confounding variables in LND group and non-lymph node dissection (NLND) group. The optimal TNLE was determined by survival analysis performed by the X-tile program using the enumeration method. RESULTS: A total of 637 clinically node-negative ICC patients were included in this study, 74 cases were found lymph node (LN) positive after operation. Among the remaining 563 node-negative ICC patients, LND was associated with longer OS but not RFS before PSM (OS: 35.4 vs 26.0 months, p = 0.047; RFS: 15.0 vs 15.4 months, p = 0.992). After PSM, patients in LND group had better prognosis on both OS and RFS (OS: 38.0 vs 23.0 months, p < 0.001; RFS: 15.0 vs 13.0 months, p = 0.029). There were no statistically differences in postoperative complications. When TNLE was greater than 8, OS (48.5 vs 31.1 months, p = 0.025) and RFS (21.0 vs 13.0 months, p = 0.043) were longer in the group with more dissected LNs. CONCLUSION: Routinely LND for node-negative ICC patients is recommended for it helps accurate tumor staging and associates with better prognosis. The optimal TNLE is more than 8.

Exosome biogenesis: machinery, regulation, and therapeutic implications in cancer.

Exosomes are well-known key mediators of intercellular communication and contribute to various physiological and pathological processes. Their biogenesis involves four key steps, including cargo sorting, MVB formation and maturation, transport of MVBs, and MVB fusion with the plasma membrane. Each process is modulated through the competition or coordination of multiple mechanisms, whereby diverse repertoires of molecular cargos are sorted into distinct subpopulations of exosomes, resulting in the high heterogeneity of exosomes. Intriguingly, cancer cells exploit various strategies, such as aberrant gene expression, posttranslational modifications, and altered signaling pathways, to regulate the biogenesis, composition, and eventually functions of exosomes to promote cancer progression. Therefore, exosome biogenesis-targeted therapy is being actively explored. In this review, we systematically summarize recent progress in understanding the machinery of exosome biogenesis and how it is regulated in the context of cancer. In particular, we highlight pharmacological targeting of exosome biogenesis as a promising cancer therapeutic strategy.

A comparative study of robotics and laparoscopic in minimally invasive pancreatoduodenectomy: A single-center experience.

Objective: To retrospectively compare the short-term benefits of robotic surgery and laparoscopic in the perioperative period of minimally invasive pancreatoduodenectomy (MIPD). Methods: This retrospective analysis evaluated patients who underwent laparoscopic pancreatoduodenectomy (LPD) or robotic pancreatoduodenectomy (RPD) from March 2018 to January 2022 in the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Perioperative data, including operating time, complications, morbidity and mortality, estimated blood loss (EBL), and postoperative length of stay, were analysed. Result: A total of 190 cases of MIPD were included, of which 114 were LPD and 76 were RPD. There was no significant difference between the two groups in gender, age, previous history of upper abdominal operation, jaundice (>150 µmol/L), or diabetes (P > 0.05). The conversion rate to laparotomy was similar in the LPD and RPD groups (5.3% vs. 6.6%, P = 0.969). A total of 179 cases of minimally invasive pancreatoduodenectomy were successfully performed, including 108 cases of LPD and 71 cases of RPD. There were significant differences between the laparoscopic and robotic groups in operation time [mean, 5.97 h vs. 5.42 h, P < 0.05] and postoperative length of stay [mean, 15.3 vs. 14.6 day, P < 0.05]. No significant difference was observed between the two groups in terms of EBL, intraoperative transfusion, complication rate, mortality rate, or reoperation rate (P > 0.05). There were no significant differences in pathological type, number of lymph nodes harvested, or positive lymph node rate (P > 0.05). Conclusion: RPD had an advantage compared to LPD in reduced operation time and postoperative length of stay, technical feasibility, and safety.

NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma.

RNA modification affects many biological processes and physiological diseases. The 5-methylcytosine (m5C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m5C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that a major function of the hypermethylated genes participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m5C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.

A Nomogram Model to Predict Early Recurrence of Patients With Intrahepatic Cholangiocarcinoma for Adjuvant Chemotherapy Guidance: A Multi-Institutional Analysis.

Background: The influence of different postoperative recurrence times on the efficacy of adjuvant chemotherapy (ACT) for intrahepatic cholangiocarcinoma (ICC) remains unclear. This study aimed to investigate the independent risk factors and establish a nomogram prediction model of early recurrence (recurrence within 1 year) to screen patients with ICC for ACT. Methods: Data from 310 ICC patients who underwent radical resection between 2010 and 2018 at eight Chinese tertiary hospitals were used to analyze the risk factors and establish a nomogram model to predict early recurrence. External validation was conducted on 134 patients at the other two Chinese tertiary hospitals. Overall survival (OS) and relapse-free survival (RFS) were estimated by the Kaplan-Meier method. Multivariate analysis was conducted to identify independent risk factors for prognosis. A logistic regression model was used to screen independent risk variables for early recurrence. A nomogram model was established based on the above independent risk variables to predict early recurrence. Results: ACT was a prognostic factor and an independent affecting factor for OS and RFS of patients with ICC after radical resection (p < 0.01). The median OS of ICC patients with non-ACT and ACT was 14.0 and 15.0 months, and the median RFS was 6.0 and 8.0 months for the early recurrence group, respectively (p > 0.05). While the median OS of ICC patients with non-ACT and ACT was 41.0 and 84.0 months, the median RFS was 20.0 and 45.0 months for the late recurrence group, respectively (p < 0.01). CA19-9, tumor size, major vascular invasion, microvascular invasion, and N stage were the independent risk factors of early recurrence for ICC patients after radical resection. The C-index of the nomogram was 0.777 (95% CI: 0.713~0.841) and 0.716 (95%CI: 0.604~0.828) in the training and testing sets, respectively. Conclusion: The nomogram model established based on the independent risk variables of early recurrence for curatively resected ICC patients has a good prediction ability and can be used to screen patients who benefited from ACT.

Recurrence and prognosis in intrahepatic cholangiocarcinoma patients with different etiology after radical resection: a multi-institutional study.

OBJECTIVE: We aimed to evaluate the prognosis and adjuvant chemotherapy (ACT) in intrahepatic cholangiocarcinoma (ICC) patients with different etiology after radical resection. METHODS: A total of 448 patients with ICC who underwent radical resection between 2010 and 2018 at ten Chinese tertiary hospitals were analyzed in the study. These patients were divided into conventional ICC (Con-ICC, n = 261, 58.2%), hepatitis B virus ICC (HBV-ICC, n = 102, 22.8%) and hepatolithiasis (Stone-ICC, n = 85,19.0%) subtypes according to different etiology. Propensity score matching (PSM) was conducted to mitigate the baseline differences between Con-ICC and HBV-ICC, Con-ICC and Stone-ICC, HBV-ICC and Stone-ICC subtypes. RESULTS: Univariate and multivariate analysis showed that different etiology was a prognostic factor for overall survival and relapse-free survival, and different etiology was an independent risk factor for overall survival in ICC patients, respectively (P < 0.05). In addition, there was a statistical difference for overall survival in early recurrence patients among the three etiological subtypes (P < 0.05). After PSM, the overall survival of patients with Stone-ICC was worse than those of Con-ICC and HBV-ICC subtypes (P < 0.05), while the relapse-free survival of patients with Stone-ICC was equivalent to patients with Con-ICC and HBV-ICC (P > 0.05). In Stone-ICC patients, the median overall survival was 16.0 months and 29.7 months, and the median relapse-free survival was 9.0 months and 20.0 months for non-ACT and ACT patients, respectively (P < 0.05). CONCLUSION: The prognosis of Stone-ICC patients was significantly worse than those of Con-ICC and HBV-ICC patients. Interestingly, postoperative adjuvant chemotherapy can improve the prognosis of Stone-ICC patients effectively.

Single-cell transcriptomics analysis reveals intratumoral heterogeneity and identifies a gene signature associated with prognosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) tumors exhibit high heterogeneity. However, current understanding of tumor cell heterogeneity of HCC and the association with prognosis remains very limited. In the present study, we collected and examined tumor tissue from one HCC patient by single-cell RNA sequencing (scRNA-seq). We identified 5753 cells and 16 clusters including hepatocytes/cancer cells, T cells, macrophages, endothelial cells, fibroblasts, NK cells, neutrophils, and B cells. In six tumor cell subclusters, we identified a cluster of proliferative tumor cells associated with poor prognosis. We downloaded scRNA-seq data of GSE125449 from the NCBI-GEO as validation dataset, and found that a cluster of hepatocytes exhibited high proliferation activity in HCC. Furthermore, we identified a gene signature related to the proliferation of HCC cells. This gene signature is efficient to classify HCC patients into two groups with distinct prognosis in both TCGA and ICGC database cohorts. Our results reveal the intratumoral heterogeneity of HCC at single cell level and identify a gene signature associated with HCC prognosis.

Prediction Efficacy of Prognostic Nutritional Index and Albumin-Bilirubin Grade in Patients With Intrahepatic Cholangiocarcinoma After Radical Resection: A Multi-Institutional Analysis of 535 Patients.

BACKGROUND: The preoperative nutritional status and the immunological status have been reported to be independent prognostic factors of patients with intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate whether prognostic nutritional index (PNI) + albumin-bilirubin (ALBI) could be a better predictor than PNI and ALBI alone in patients with ICC after radical resection. METHODS: The prognostic prediction evaluation of the PNI, ALBI, and the PNI+ALBI grade was performed in 373 patients with ICC who underwent radical resection between 2010 and 2018 at six Chinese tertiary hospitals, and external validation was conducted in 162 patients at four other Chinese tertiary hospitals. Overall survival (OS) and relapse-free survival (RFS) were estimated using the Kaplan-Meier method. Multivariate analysis was conducted to identify independent prognostic factors. A time-dependent receiver operating characteristic (ROC) curve and a nomogram prediction model were further constructed to assess the predictive ability of PNI, ALBI, and the PNI+ALBI grade. The C-index and a calibration plot were used to assess the performance of the nomogram models. RESULTS: Univariate analysis showed that PNI, ALBI, and the PNI+ALBI grade were prognostic factors for the OS and RFS of patients with ICC after radical resection in the training and testing sets (p < 0.001). Multivariate analysis showed that the PNI+ALBI grade was an independent risk factor for OS and RFS in the training and testing sets (p < 0.001). Analysis of the relationship between the PNI+ALBI grade and clinicopathological characteristics showed that the PNI+ALBI grade correlated with obstructive jaundice, alpha-fetoprotein (AFP), cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA125), PNI, ALBI, Child-Pugh grade, type of resection, tumor size, major vascular invasion, microvascular invasion, T stage, and N stage (p < 0.05). The time-dependent ROC curves showed that the PNI+ALBI grade had better prognostic predictive ability than the PNI, ALBI, and the Child-Pugh grade in the training and testing sets. CONCLUSION: Preoperative PNI+ALBI grade is an effective and practical predictor for the OS and RFS of patients with ICC after radical resection.

Melatonin inhibits proliferation, migration, and invasion by inducing ROS-mediated apoptosis via suppression of the PI3K/Akt/mTOR signaling pathway in gallbladder cancer cells.

BACKGROUND: Melatonin is an indolic compound mainly secreted by the pineal gland and plays a vital role in the regulation of circadian rhythms and cancer therapy. However, the effects of melatonin in gallbladder cancer (GBC) and the related mechanism remain unknown. METHODS: In this study, the antitumor activity of melatonin on gallbladder cancer was explored both in vitro and in vivo. After treatment with different concentrations of melatonin, the cell viability, migration, and invasion of gallbladder cancer cells (NOZ and GBC-SD cells) were evaluated by CCK-8 assay, wound healing, and Transwell assay. RESULTS: The results showed that melatonin inhibited growth, migration, and invasion of gallbladder cancer cells. Subsequently, the assays suggested that melatonin significantly induced apoptosis in gallbladder cancer cells and altered the expression of the apoptotic proteins, including Bax, Bcl-2, cytochrome C, cleaved caspase-3, and PARP. Besides, the intracellular reactive oxygen species (ROS) was found to be upregulated after melatonin treatment in gallbladder cancer cells. Melatonin was found to suppress the PI3K/Akt/mTOR signaling pathway in a time-dependent manner by inhibiting the phosphorylation of PI3K, Akt, and mTOR. Treatment with N-acetyl-L-cysteine (NAC) or 740 Y-P remarkably attenuated the antitumor effects of melatonin in NOZ and GBC-SD cells. Finally, melatonin suppressed the growth of GBC-SD cells in an athymic nude mice xenograft model in vivo. CONCLUSIONS: Our study revealed that melatonin could induce apoptosis by suppressing the PI3K/Akt/mTOR signaling pathway. Therefore, melatonin might serve as a potential therapeutic drug in the future treatment of gallbladder cancer.

Lenvatinib induces anticancer activity in gallbladder cancer by targeting AKT.

Gallbladder cancer (GBC) is characterized by poor prognosis, early metastasis, and high recurrence rates, which seriously threaten human health. The effect of lenvatinib, a widely used drug in anti-hepatocellular carcinoma in China, on GBC progress, as well as its underlying molecular mechanism, remains unclear. Therefore, the present study investigated the effect of lenvatinib on human GBC GBC-SD and NOZ cells and its underlying mechanisms. A series of experiments, including cell proliferation, clone formation, wound healing, and cell migration and invasion assays, as well as flow cytometry, were performed to investigate the anticancer effect of lenvatinib on GBC. Western blotting was used to detect alterations in protein expression of CKD2, CKD4, cyclin D1, caspase-9, matrix metalloproteinase (MMP)-2, cell migration-inducing protein (CEMIP) and phospho-AKT (p-AKT). In addition, the chemosensitivity of lenvatinib-treated GBC cells to gemcitabine (GEM) and whether the activation of phosphoinositide 3 kinase (PI3K)/AKT contributed to the chemoresistance were determined. Finally, the anticancer effect of lenvatinib in vivo was detected using a xenograft mouse model. These data showed that treatment with lenvatinib inhibited cell proliferation, colony formation ability, migration, induced apoptosis, regulated cell cycle and resulted in decreased resistance to GEM. Treatment with lenvatinib decreased the expression of MMP-2, CEMIP, CDK2, CDK4 and cyclin D1, and increased the expression of cleaved caspase-9, which was mediated by the inactivation of the PI3K/AKT pathway in vitro. In addition, lenvatinib inhibited autophagy in GBC-SD and NOZ cells. Besides, Lenvatinib suppressed GBC cell growth in vivo by targeting p-AKT. In combination, the present data indicated that lenvatinib plays a potential anticancer role in GBC by downregulating the expression of p-AKT.

An Apoptotic Gene Signature for the Prognosis of Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major public health burden worldwide owing to high incidence and poor prognosis. Although numerous apoptotic genes were disclosed in HCC, the prognostic value and clinical utility of the genes remained unclear. METHODS: The differentially expressed genes (DEGs) were identified in the microarray and RNA sequencing data from public databases. The apoptosis-related differentially expressed genes (AR-DEGs) were selected to construct a Lasso-penalized Cox regression model. The signature including five apoptotic genes was used to calculate risk score. Then, the receiver operating characteristic (ROC) and survival analysis were conducted based on the signature. A nomogram containing the signature and clinical characteristics was plotted to visualized the prognosis prediction. Finally, the enrichment analysis was performed in the Gene Ontology (GO) to investigate the potential mechanism. RESULTS: Patients with high risk scores were related to worse overall survival than those with low risk. The 3-year and 5-year area under curve (AUC) values of the signature were above 0.7 in databases. And the nomogram presented reliable net benefits for the survival prediction. The nomogram was also tested by probability calibration curves and Decision Curve Analysis (DCA). Furthermore, the five differentially expressed genes were verified again in the HCC clinical specimens with real-time PCR and Western Blot. CONCLUSION: Collectively, the present study formed a novel signature based on five apoptotic genes, and this possibly predicted prognosis and strengthened the communication with HCC patients about the likely treatment.

Calreticulin: a potential diagnostic and therapeutic biomarker in gallbladder cancer.

Recent studies suggested that calreticulin (CRT) has an important role in the progression of various types of cancer. Our previous study suggested that CRT was upregulated and acted as an oncogene in hepatocellular carcinoma. However, the role of CRT in gallbladder cancer (GBC) remains unclear. The expression level of CRT was upregulated in GBC tissues in comparison with adjacent non-tumor tissues and chronic cholecystitis tissues. Moreover, CRT expression was found to be correlated with the tumor size. Knockdown of CRT inhibited cell proliferation, induced apoptosis, arrested cell cycle and resulted in decreased resistance to gemcitabine, which was mediated by the inactivation of the PI3K/Akt pathway. Collectively, the present results suggested a potential role of CRT in GBC progression and provided novel insights into the mechanism underlying the CRT-mediated chemosensitivity in GBC cells.

Diagnostic and prognostic values of AKR1C3 and AKR1D1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common histological type of primary liver cancer and the majority of patients are diagnosed at an advanced stage and have a poor prognosis. AKR1C3 (Aldo-keto reductase family 1 member C3) and AKR1D1 (Aldo-keto reductase family 1 member D1) catalyze the conversion of aldehydes and ketones to alcohols and play crucial roles in multiple cancers. However, the functions of AKR1C3 and AKR1D1 in HCC remain unclear. In our study, data from the public databases were selected as training and validation sets, then 76 HCC patients in our center were chosen as a test set. Bioinformatics methods suggested AKR1C3 was overexpressed in HCC and AKR1D1 was down-regulated. The receiver operating characteristic curve (ROC) analysis was performed and the area under curve (AUC) values of AKR1C3 and AKR1D1 were above 0.7 (0.948, 0.836, respectively). Also, the high expression of AKR1C3 and low expression of AKR1D1 predicted poor prognosis and short median survival time. Then, the knockdown of AKR1C3 and overexpression of AKR1D1 in HCC cells were achieved with lentivirus. And both decreased cell proliferation, restrained cell viability, and inhibited tumorigenesis. Moreover, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted and the results showed that AKR1C3 and AKR1D1 might participate in the MAPK/ERK and androgen receptor (AR) signaling pathway. Furthermore, the AR and phosphorylated ERK1/2 were significantly reduced after the suppression of AKR1C3 or overexpression of AKR1D1. Collectively, AKR1C3 and AKR1D1 might serve as candidate diagnostic and prognostic biomarkers for HCC and provide potential targets for HCC treatment.

Analysis of bile acid profile in plasma to differentiate cholangiocarcinoma from benign biliary diseases and healthy controls.

Bile acids (BAs) are currently considered as causative agents for Cholangiocarcinoma (CCA). However, the profile of circulating BAs in CCA have not been well characterized. The aim of this study was to describe the alterations of BAs metabolism in patients with CCA compared to benign biliary diseases (BBD) and healthy controls (HC), and to discover the specific BAs as biomarkers for CCA diagnosis. The concentrations of 15 BAs in plasma were measured in a total of 329 subjects, including patients with BBD, CCA, gallbladder cancer (GC), hepatocellular carcinoma (HCC), and healthy subjects, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Binary logistic regression analysis was used to build a diagnostic model for CCA. An imbalance in the ratio of conjugated to unconjugated BAs was observed in CCA patients compared to BBD and HC groups, with higher conjugated BAs and lower unconjugated BAs. A panel of 2 BA metabolites consisting of CDCA and TCDCA showed high diagnostic performance for CCA versus BBD and CCA versus HC, with higher AUC, sensitivity and specificity than carbohydrate antigen 19-9 (CA 199), clinically employed CCA biomarker. Importantly, HCC and GC samples were also included to confirm specificity of the BA biomarkers for CCA diagnosis. In summary, specific changes in plasma concentrations of BAs may serve as diagnostic biomarkers for distinguishing CCA from BBD and HC, with higher performance than CA199.

Interleukin 17A promotes gallbladder cancer invasiveness via ERK/NF-κB signal pathway mediated epithelial-to-mesenchymal transition.

As a pro-inflammatory cytokine, Interleukin 17A (IL-17A) plays an important role in pathology of tumor microenvironment and inflammatory diseases. In this study, we intend to investigate the role of IL-17A on the metastasis of gallbladder cancer (GBC) and related mechanisms. The serum levels of IL-17A were associated with node metastasis and advanced stage. We also found the pro-invasion effect of IL-17A on GBC cells. When treated with IL-17A, the protein level of epithelial marker E-cadherin in GBC cells was significantly down-regulated, while the protein level of the mesenchymal phenotype marker vimentin was significantly increased. IL-17A increased the expression of transcription factor slug, the phosphorylation of ERK1/2 and the nuclear translocation of NF-κB/p50 and p65 in a concentration-dependent manner. Pretreatment of cells with U0126 could reverse the nuclear translocation of NF-κB/p50 and p65 and EMT induced by IL-17A. IL-17A promotes gallbladder cancer invasiveness via ERK/NF-κB signal pathway mediated epithelial-to-mesenchymal transition. As a new therapeutic targets and diagnostic marker, IL-17A may play an important role in the treatment of GBC.

S100A4 promotes hepatocellular carcinogenesis by intensifying fibrosis-associated cancer cell stemness.

A cancer-promoting role of fibrogenesis in the liver has long been speculated; however, the molecular mechanisms regarding this phenomenon are largely unknown. We demonstrated in our previous study that macrophage-derived S100A4 promotes liver fibrosis via activation of hepatic stellate cells; however, whether and how S100A4 directly contributes to the development of fibrosis-associated liver cancer remains elusive. High expression of S100A4 in the fibrotic region was observed in human liver tumor tissues which associated with advanced disease severity. Through an established hepatocarcinogenesis model involving apparent liver fibrogenesis, we found that S100A4-deficient mice developed significantly less and smaller liver tumor nodules, with no change in the liver inflammation but decreased liver fibrosis and expression of stem cell markers in hepatocellular carcinoma (HCC) tissues. Mechanistically, S100A4 directly promoted stem cell-associated genes signatures in a way synergistic with its interacting protein, extracellular matrix component collagen I. This process is dependent on the receptor of advanced glycation end products (RAGE) and ß-catenin signaling. Furthermore, the liver tumor sphere formation in vitro and tumor growth in vivo were greatly enhanced only when the cancer cells were pretreated with both S100A4 and collagen I. Our work firstly demonstrated a key role of S100A4 in synergy with extracellular matrix in the promotion of hepatocellular carcinoma by affecting the stemness of cancer cells.

Identification of novel diagnostic and prognostic biomarkers for hepatocellular carcinoma.

The differential expression of a featured set of genes may serve as a diagnostic biomarker in hepatocellular carcinoma (HCC) patients. The aim of this study was to identify prognostic biomarkers for the diagnosis and survival of HCC based on the analysis of a large cohort of patients. Clinical and RNA­seq data were obtained from The Cancer Genome Atlas (TCGA) database. A transcriptomics analysis was conducted to detect differentially expressed genes (DEGs). Samples from 53 tumors and 20 normal tissues of HCC patients were obtained to further analyze the connection between overall survival (OS) and DEG levels. Based on the OS and progression­free survival (PFS), 4 DEGs (GABRR1, SOX11, COL24A1 and MYLK2) were identified from the TCGA dataset. Using gene ontology (GO) analysis, it was demonstrated that the DEGs were associated with several biological processes, including multicellular organismal and single­multicellular organism processes, which are involved in the development and migration of HCC. In addition, the four genes were significantly upregulated in tumor tissues. Notably, the mRNA expression of the four genes had a negative association with OS and PFS in HCC patients determined using a Kaplan­Meir analysis. The four­gene signature is a potential novel biomarker for the prediction of HCC patient survival.

TGF-ß- and lipopolysaccharide-induced upregulation of circular RNA PWWP2A promotes hepatic fibrosis via sponging miR-203 and miR-223.

Both transforming growth factor-beta (TGF-ß) and lipopolysaccharide (LPS) can activate hepatic stellate cells (HSCs), thus increasing expressions of alpha smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1α1) and promoting liver fibrosis. However, whether TGF-ß and LPS have a common downstream reactor remains unclear. Recently, a strong relationship of circular RNAs (circRNAs) and fibrogenesis has been elucidated. In this study, we compared the expressions of several circRNAs in TGF-ß- and LPS-activated HSCs, and found that circ-PWWP2A was upregulated in both TGF-ß- and LPS-activated HSCs and in mouse fibrotic liver tissues. Meanwhile, circ-PWWP2A was positively correlated with HSC activation and proliferation. Two microRNAs, miR-203 and miR-223, were identified to be the downstream targets of circ-PWWP2A using luciferase reporter assay and pull-down interaction assay. Circ-PWWP2A was suggested to promote HSC activation and proliferation via sponging miR-203 and miR-223, and subsequently increasing Fstl1 and TLR4, respectively. Furthermore, downregulating circ-PWWP2A was indicated to alleviate hepatic fibrosis in vivo. In conclusion, our findings indicated that circ-PWWP2A is the common downstream reactor of TGF-ß and LPS in HSC activation, and that circ-PWWP2A plays a critical role in hepatic fibrogenesis via sponging miR-203 and miR-223.