Effect evaluation of vascular resection for patients with hilar cholangiocarcinoma: original data and meta-analysis.

BACKGROUND/AIMS: To evaluate the effect of vascular resection (VR) in surgical management of hilar cholangiocarcinoma (HCCA), this report did a clinical analysis and conducted a systematic review, combined other studies, based on meta-analysis. METHODOLOGY: Two hundred and thirty eight HCCA patients underwent hepatectomy in the Eastern Hepatobiliary Surgery Hospital. Binary logistic regression analysis was performed to investigate the potentially complications associated factors. Kaplan-Meier test was employed to compare the long-term survival of patients in four groups (RO + PVR-free, RO + PVR, R1 and R2). Meta-analysis was performed with RevMan 4.3.2 software. RESULTS: The results suggested that hepatectomy and HAR were important negative factors from complications (P < 0.01). Compared with patients in other groups, survival of patients in RO + PVR group was worse than RO + PVR-free group, better than R2 group and similar to R1 group with P = 0.001, 0.047 and 0.606 respectively. The results of meta-analysis suggested patients who underwent VR had higher complications rate and mortality rate than patients who did not. Moreover, patients with vascular resection had lower long-term survival rate. CONCLUSIONS: VR used to be considered effective to the patients with vascular invasion. However our study suggest that the surgical decision of undergoing VR should be made cautiously, since VR could diminish the survival time in some cases.

Prognostic analysis and risk assessment based on RNA editing in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, and prognosis assessment is crucial for guiding treatment decisions. In this study, we aimed to develop a personalized prognostic model for HCC based on RNA editing. RNA editing is a post-transcriptional process that can affect gene expression and, in some cases, play a role in cancer development. By analyzing RNA editing sites in HCC, we sought to identify a set of sites associated with patient prognosis and use them to create a prognostic model. We gathered RNA editing data from the Synapse database, comprising 9990 RNA editing sites and 250 HCC samples. Additionally, we collected clinical data for 377 HCC patients from the Cancer Genome Atlas (TCGA) database. We employed a multi-step approach to identify prognosis-related RNA editing sites (PR-RNA-ESs). We assessed how patients in the high-risk and low-risk groups, as defined by the model, fared in terms of survival. A nomogram was developed to predict the precise survival prognosis of HCC patients and assessed the prognostic model's utility through a receiver operating characteristic (ROC) analysis and decision curve analysis (DCA). Our analysis identified 33 prognosis-related RNA editing sites (PR-RNA-ESs) associated with HCC patient prognosis. Using a combination of LASSO regression and cross-validation, we constructed a prognostic model based on 13 PR-RNA-ESs. Survival analysis demonstrated significant differences in the survival outcomes of patients in the high-risk and low-risk groups defined by this model. Additionally, the differential expression of the 13 PR-RNA-ESs played a role in shaping patient survival. Risk-prognostic investigations further distinguished patients based on their risk levels. The nomogram enabled precise survival prognosis prediction. Our study has successfully developed a highly personalized and accurate prognostic model for individuals with HCC, leveraging RNA editing data. This model has the potential to revolutionize clinical evaluation and medical management by providing individualized prognostic information. The identification of specific RNA editing sites associated with HCC prognosis and their incorporation into a predictive model holds promise for improving the precision of treatment strategies and ultimately enhancing patient outcomes in HCC.

miR-204 inhibits epithelial to mesenchymal transition by targeting slug in intrahepatic cholangiocarcinoma cells.

BACKGROUND/AIMS: MicroRNAs (miRNAs) play critical roles during carcinogenesis and cancer progression. Down-regulation of miR-204 has been frequently observed in various cancers. In this study, we investigated the roles and mechanisms of miR-204 in human intrahepatic cholangiocarcinoma (ICC). METHODS: The relative expression of miR-204 in ICC tissues and cell lines was monitored by qRT-PCR. Effects of miR-204 were studied in human ICC cell lines HuH28 and HuCCT1, and cells were analyzed for proliferation, migration and invasion. Expression levels of miR-204 target gene Slug and EMT markers (E-cadherin and vimentin) in ICC cell lines and tissues were measured by qRT-PCR, western blotting and immunofluorescence. RESULTS: miR-204 was frequently downregulated in human ICC, and the low-level expression of miR-204 was significantly associated with lymph node metastasis. Overexpression of miR-204 dramatically suppressed ICC cell migration and invasion, as well as the epithelial-mesenchymal transition process (EMT). Slug was identified as a direct target of miR-204, and its downregulation by miR-204 in HuH28 cells reversed EMT, as shown by the increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker vimentin. CONCLUSION: These findings suggest that miR-204 plays negative roles in the invasive and/or metastatic potential of ICC, and that its suppressive effects are mediated by repressing Slug expression.

Sorafenib sensitizes hepatocellular carcinoma cell to cisplatin via suppression of Wnt/ß-catenin signaling.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In this study, we found that nuclear accumulation of ß-catenin was higher in cisplatin-resistant Huh7 cells than in Huh7 cells, indicating that Wnt signaling was activated in cisplatin-resistant cells. Wnt signaling inhibition increased cisplatin-induced growth inhibition in hepatoma cell. We further demonstrated that sorafenib could inhibit Wnt signaling in Huh7 cells and cisplatin-resistant Huh7 cells. Co-treatment with cisplatin and sorafenib was more effective in inhibiting cancer cell proliferation than cisplatin alone in vitro and in vivo, whereas Wnt3a (Wnt activator) treatment abrogated sorafenib-induced growth inhibition. These data demonstrated that sorafenib sensitizes human HCC cell to cisplatin via suppression of Wnt/ß-catenin signaling, thus offering a new target for chemotherapy of HCC.

Characterization of cancer-related fibroblasts (CAF) in hepatocellular carcinoma and construction of CAF-based risk signature based on single-cell RNA-seq and bulk RNA-seq data.

Background: Cancer-associated fibroblasts (CAFs) are involved in tumor growth, angiogenesis, metastasis, and resistance to therapy. We sought to explore the CAFs characteristics in hepatocellular carcinoma (HCC) and establish a CAF-based risk signature for predicting the prognosis of HCC patients. Methods: The signal-cell RNA sequencing (scRNA-seq) data was obtained from the GEO database. Bulk RNA-seq data and microarray data of HCC were obtained from the TCGA and GEO databases respectively. Seurat R package was applied to process scRNA-seq data and identify CAF clusters according to the CAF markers. Differential expression analysis was performed to screen differentially expressed genes (DEGs) between normal and tumor samples in TCGA dataset. Then Pearson correlation analysis was used to determine the DEGs associated with CAF clusters, followed by the univariate Cox regression analysis to identify CAF-related prognostic genes. Lasso regression was implemented to construct a risk signature based on CAF-related prognostic genes. Finally, a nomogram model based on the risk signature and clinicopathological characteristics was developed. Results: Based on scRNA-seq data, we identified 4 CAF clusters in HCC, 3 of which were associated with prognosis in HCC. A total of 423 genes were identified from 2811 DEGs to be significantly correlated with CAF clusters, and were narrowed down to generate a risk signature with 6 genes. These six genes were primarily connected with 39 pathways, such as angiogenesis, apoptosis, and hypoxia. Meanwhile, the risk signature was significantly associated with stromal and immune scores, as well as some immune cells. Multivariate analysis revealed that risk signature was an independent prognostic factor for HCC, and its value in predicting immunotherapeutic outcomes was confirmed. A novel nomogram integrating the stage and CAF-based risk signature was constructed, which exhibited favorable predictability and reliability in the prognosis prediction of HCC. Conclusion: CAF-based risk signatures can effectively predict the prognosis of HCC, and comprehensive characterization of the CAF signature of HCC may help to interpret the response of HCC to immunotherapy and provide new strategies for cancer treatment.

Chimeric transcripts observed in non-canonical FGFR2 fusions with partner genes' breakpoint located in intergenic region in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. FGFR family fusion have been identified in many diseases, and FGFR2 fusion is a validated oncogenic driver in ICC. At present, a variety of fusion forms have been reported, including gene-gene, gene-intergenic, and intergenic-intergenic fusion. Here, by performing RNA- and DNA-sequencing analysis, FGFR2 fusions were found in 10.1% of ICC, including 4 gene-intergenic fusions. We confirmed that the non-canonical rearrangements can generate chimeric transcripts, and used conventional splicing mechanism to explain the event. Our study provides possible target therapy for these 4 patients and possibility analysis scheme for similar situation.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

Long Noncoding RNA NEAT1 Upregulates Survivin and Facilitates Gallbladder Cancer Progression by Sponging microRNA-335.

BACKGROUND: Gallbladder cancer (GBC) is the most common cancer of the biliary tract, but molecularly targeted therapies are not available for GBC. Loss of microRNA (miR)-335 expression may be a useful predictor of clinical outcomes and the reversal of its loss of expression may be a useful treatment strategy for GBC. In this study, we investigated whether a long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) sponges miR-335 in GBC cells. MATERIALS AND METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry were used to determine the expression of miR-335; NEAT1; survivin; and Ki67 in GBC cell lines (GBC-SD and SGC-996) and tissue samples from patients (n = 25). Cell Counting Kit-8, colony-formation, and Transwell migration and invasion assays were performed to measure cell proliferation, migration, and invasion. Bioinformatic analysis and dual-luciferase reporter assays were utilized to analyze correlativity. RESULTS: miR-335 overexpression resulted in inhibition of GBC cell proliferation and invasion. In addition, knockdown of NEAT1 resulted in downregulation of survivin expression. As NEAT1 competitively "sponges" miR-335, NEAT1 knockdown resulted in inhibited GBC cell proliferation and invasion in vitro and GBC tumor growth in vivo. Furthermore, NEAT1 was found to be upregulated in GBC samples, and its expression was inversely correlated with miR-335 levels, but positively correlated with survivin levels. CONCLUSION: These findings indicate that NEAT1 promotes survivin expression by functioning as a competitive endogenous RNA for miR-335 in GBC cells; thus, we have identified a potential biomarker and target for GBC diagnosis and therapy.

Bile cell­free DNA as a novel and powerful liquid biopsy for detecting somatic variants in biliary tract cancer.

Tissue sampling of biliary tract carcinomas (BTCs) for molecular characterization is challenging. The aim of this study was to investigate the possibility of identifying individual actionable mutations derived from bile cell­free DNA (cfDNA) using targeted deep sequencing. Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in the present study. Using targeted deep sequencing with a panel of 150 tumor­related genes, paired bile cfDNA and tumor DNA were analyzed for mutational variants individually and then compared. The present study, to the best of our knowledge, is the first to reveal that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case for plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. When comparing bile cfDNA and tumor DNA for single nucleotide variation (SNV)/insertion and deletion (Indel), the results using targeted deep sequencing revealed high sensitivity (94.7%) and specificity (99.9%). Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. The present study revealed that targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. These preliminary results may shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

Downregulation of A20 increases the cytotoxicity of IFN-γ in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, effective therapeutic strategy. Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, antiviral, and antitumor effects. Although IFN-γ is a promising antitumor agent, its application is limited by resistance in tumor cells. A20 is a zinc-finger protein that was initially identified as a gene product induced by tumor necrosis factor α in human umbilical vein endothelial cells. In this study, we found that silencing of A20 combined with IFN-γ significantly represses cell viability, and induces apoptosis and cell-cycle arrest in HCC cells. By investigating mechanisms implicated in A20 and IFN-γ-mediated signaling pathways, we revealed that the phosphoinositide 3-kinase/Akt signaling pathway and antiapoptotic B-cell lymphoma 2 proteins were repressed. Moreover, we also found that phosphorylation of STAT1 and STAT3 was significantly enhanced after the downregulation of A20 in combination with treatment of IFN-γ. Inhibitor of STAT1 but not STAT3 could block the antitumor effect of IFN-γ. Therefore, targeting A20 enhances the cytotoxicity of IFN-γ against HCC cells and may present a promising therapeutic strategy for HCC.

Overexpression of the X-linked ribosomal protein S4 predicts poor prognosis in patients with intrahepatic cholangiocarcinoma.

X-linked ribosomal protein S4 (RPS4X) has previously been reported to be associated with cisplatin resistance and clinical outcome in bladder and ovarian cancer. However, the value of RPS4X as a diagnostic and prognostic marker in intrahepatic cholangiocarcinoma (ICC) has not yet been investigated. The present study evaluated the expression pattern, and diagnostic and prognostic value of RPS4X in patients with ICC. Retrospective analysis was performed for a total of 201 patients with intrahepatic cholangiocarcinoma, and 8 patients with inflammation of the bile duct. Immunohistochemistry was performed using tissue microarrays to characterize the expression profile of RPS4X. Receiver operating characteristic (ROC) curves, the Kaplan-Meier estimator and Cox regression analysis were applied to evaluate the potential diagnostic and prognostic value of RPS4X in ICC. RPS4X was significantly upregulated in ICC tissues compared with the inflamed bile duct tissues. When differentiating ICC from normal controls, ROC analysis of RPS4X gave an area under the curve value of 0.9030 (sensitivity, 82.59%; specificity, 100%). RPS4X expression was significantly positively correlated with serum alkaline phosphatase levels. Survival analysis demonstrated that RPS4X expression levels were an independent prognostic factor for overall survival. Therefore, RPS4X expression levels may serve as a novel diagnostic and prognostic marker in ICC.

Effect evaluation of vascular resection for patients with hilar cholangiocarcinoma: original data and meta-analysis.

To evaluate the effect of vascular resection (VR) in surgical management of hilar cholangiocarcinoma (HCCA), this report is used in a clinical analysis and conducted a systematic review, combined other studies, based on meta-analysis. 238 HCCA patients underwent hepatectomy in the Eastern Hepatobiliary Surgery Hospital. Binary logistic regression analysis was performed to investigate the potentially complicated associated factors. Kaplan-Meier test was employed to compare the long-term survival of patients in four groups (R0+PVR-free, R0+PVR, R1, and R2). Meta-analysis was performed using RevMan 4.3.2 software. The results suggested that hepatectomy and HAR were important negative factors from complications (p < 0.01). Compared with patients in other groups, survival of patients in R0+PVR group was worse than R0+PVR-free group, better than R2 group, and similar to R1group with p = 0.001, 0.047, and 0.606, respectively. The results of meta-analysis suggested patients who underwent VR had higher complications rate and mortality rate than patients who did not. Moreover, patients with vascular resection had lower long-term survival rate. VR used to be considered effective to the patients with vascular invasion. However, our study suggests that the surgical decision of undergoing VR should be made cautiously, since VR could diminish the survival time in some cases.

TGF-ß upregulates miR-182 expression to promote gallbladder cancer metastasis by targeting CADM1.

UNLABELLED: Transforming growth factor ß (TGF-ß) plays important roles in tumor metastasis by regulating miRNAs expression. miR-182 is an important molecule in the regulation of cancer progression. The aim of the study is to assess the role of miR-182 in TGF-ß-induced cancer metastasis. In the present study, we found that miR-182 levels are significantly upregulated in GBC tissues compared with normal controls, and miR-182 expression is remarkably increased in primary tumors that subsequently metastasized, when compared to those primary tumors that did not metastasize. TGF-ß induces miR-182 expression in GBC cells, and overexpression of miR-182 promotes GBC cell migration and invasion, whereas miR-182 inhibition suppresses TGF-ß-induced cancer cell migration and invasion. The blockage of miR-182 by a specific inhibitor effectively inhibits pulmonary metastases in vivo. We further identified that the cell adhesion molecule1 (CADM1) is a new target gene of miR-182. miR-182 negatively regulates CADM1 expression in vitro and in vivo. Importantly, re-expression of CADM1 in GBC cells partially abrogates miR-182-induced cell invasion. CONCLUSIONS: miR-182 is an important mediator of GBC metastasis, thus offering a new target for the development of therapeutic agents against GBC.