Corrigendum to "Curcumin-loaded nanocomplexes: Acute and chronic toxicity studies in mice and hamsters" [Toxicol. Rep. 8 (2021) 1346-1357/ S221475002100127X].

[This corrects the article DOI: 10.1016/j.toxrep.2021.06.021.].

RET rearrangements are relevant to histopathologic subtypes and clinicopathological features in Thai papillary thyroid carcinoma patients.

Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. The RET gene rearrangements CCDC6::RET and NCOA4::RET are the most common RET gene rearrangements in PTC patients. Different RET::PTC rearrangements are associated with different PTC phenotypes. Methods: Eighty-three formalin-fixed paraffin-embedded (FFPE) PTC samples were examined. The prevalence and expression levels of CCDC6::RET and NCOA4::RET were determined using semi-quantitative polymerase chain reaction (qRT-PCR). The association of these rearrangements with clinicopathological data was investigated. Results: The presence of CCDC6::RET rearrangement was significantly associated with the classic subtype and absence of angio/lymphatic invasion (p < 0.05). While NCOA4::RET was associated with the tall-cell subtype, and presence of angio/lymphatic invasion and lymph node metastasis (p < 0.05). Multivariate analysis demonstrated that an absence of extrathyroidal extension and extranodal extension were independent predictive factors for CCDC6::RET, whereas the tall-cell subtype, large tumor size, angioinvasion, lymphatic invasion and perineural invasion were independent predictive factors for NCOA4::RET (p < 0.05). However, the mRNA expression level of CCDC6::RET and of NCOA4::RET were not significantly associated with clinicopathological data. Conclusion: CCDC6::RET was correlated with an innocent PTC subtype and characteristics, but NCOA4::RET correlated with an aggressive phenotype of PTC. Therefore, these RET rearrangements strongly associated with clinicopathological phenotypes and can be used as predictive markers in PTC patients.

Dense GM-CSFRα-expressing immune infiltration is allied with longer survival of intrahepatic cholangiocarcinoma patients.

Background: Intrahepatic cholangiocarcinoma (iCCA) is a cancer arising from intrahepatic bile duct epithelium. An iCCA incidence is increasing worldwide; however, the outcome of the disease is dismal. The linkage between chronic inflammation and iCCA progression is well established, but the roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) remain unrevealed. Thus, a better understanding of GM-CSF functions in CCA may provide an alternative approach to CCA treatment. Methods: Differential GM-CSF and GM-CSFRα mRNA expressions in CCA tissues were investigated by Gene Expression Profiling Interactive Analysis (GEPIA) based on The Cancer Genome Atlas (TCGA) database. The protein expressions and localizations of GM-CSF and its cognate receptor (GM-CSFRα) in iCCA patients' tissues were demonstrated by the immunohistochemistry (IHC) techniques. The survival analyses were performed using Kaplan-Meier survival analysis with log-rank test and Cox proportional hazard regression model for multivariate analysis. The GM-CSF productions and GM-CSFRα expressions on CCA cells were assessed by ELISA and flow cytometry. The effects of GM-CSF on CCA cell proliferation and migration were evaluated after recombinant human GM-CSF treatment. The relationship between GM-CSF or GM-CSFRα level and related immune cell infiltration was analyzed using the Tumor Immune Estimation Resource (TIMER). Results: GEPIA analysis indicated GM-CSF and GM-CSFRα expressions were higher in CCA tissues than in normal counterparts, and high GM-CSFRα was related to the longer disease-free survival of the patients (p < 0.001). IHC analysis revealed that CCA cells differentially expressed GM-CSF, while GM-CSFRα was expressed on cancer-infiltrating immune cells. The patient whose CCA tissue contained high GM-CSF expressed CCA, and moderate to dense GM-CSFRα-expressing immune cell infiltration (ICI) acquired longer overall survival (OS) (p = 0.047), whereas light GM-CSFRα-expressing ICI contributed to an increased hazard ratio (HR) to 1.882 (95% CI [1.077-3.287]; p = 0.026). In non-papillary subtype, an aggressive CCA subtype, patients with light GM-CSFRα-expressing ICI had shorter median OS (181 vs. 351 days; p = 0.002) and the HR was elevated to 2.788 (95% CI [1.299-5.985]; p = 0.009). Additionally, TIMER analysis demonstrated GM-CSFRα expression was positively correlated with neutrophil, dendritic cell, and CD8+ T cell infiltrations, though it was conversely related to M2-macrophage and myeloid-derived suppressor cell infiltration. However, the direct effects of GM-CSF on CCA cell proliferation and migration were not observed in the current study. Conclusions: Light GM-CSFRα-expressing ICI was an independent poor prognostic factor for iCCA patients. Anti-cancer functions of GM-CSFRα-expressing ICI were suggested. Altogether, the benefits of acquired GM-CSFRα-expressing ICI and GM-CSF for CCA treatment are proposed herein and require elucidation.

Diminishing acetyl-CoA carboxylase 1 attenuates CCA migration via AMPK-NF-κB-snail axis.

Cholangiocarcinoma (CCA), a cancer of the biliary tract, is a significant health problem in Thailand. Reprogramming of cellular metabolism and upregulation of lipogenic enzymes have been revealed in CCA, but the mechanism is unclear. The current study highlighted the importance of acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme in de novo lipogenesis, on CCA migration. ACC1 expression in human CCA tissues was determined by immunohistochemistry. The results demonstrated that increased ACC1 was related to the shorter survival of CCA patients. Herein, ACC1-deficient cell lines (ACC1-KD) were generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (cas9) system and were used for the comparative study. The ACC1 levels in ACC1-KD were 80-90 % lower than in parental cells. Suppression of ACC1 significantly reduced intracellular malonyl-CoA and neutral lipid contents. Two-fold growth retardation and 60-80 % reduced CCA cell migration and invasion were observed in ACC1-KD cells. The reduced 20-40 % of intracellular ATP levels, AMPK activation, lowered NF-κB p65 nuclear translocation, and snail expression were emphasized. Migration of ACC1-KD cells was restored by supplementation with palmitic acid and malonyl-CoA. Altogether, the importance of rate-limiting enzyme in de novo fatty acid synthesis, ACC1, and AMPK-NF-κB-snail axis on CCA progression was suggested herein. These might be the novel targets for CCA drug design. (ACC1, AMPK, Cholangiocarcinoma, De novo lipogenesis, NF-κB, Palmitic acid).

Fluorescence in situ hybridization detection of chromosome 7 and/or 17 polysomy as a prognostic marker for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is highly endemic in the Northeast Thailand. Recently, chromosome aberrations provided new insights into pathogenesis of CCA. Therefore, chromosome aberration might be used as a prognostic factor and therapeutic planning of this cancer. This aim of this study is to examine the correlation between an increase of chromosome 7 (C7) and/or 17 (C17) copy number variants (CNVs) with clinicopathological data and the overall survival time (OS) of CCA patients using fluorescence in situ hybridization (FISH) assays. C7 and C17 CNVs were examined using FISH form 157 formalin-fixed paraffin-embedded (FFPE) tissues of CCA patients from Khon Kaen, Thailand between 2011 and 2015. OS was visualized using Kaplan-Meier plot. Univariate and multivariate analyses were used to determine the ability of the clinicopathological parameters to predict OS. C17 > trisomy (odd ratio, 6.944, P < 0.001), C7/17 trisomy (odd ratio; 4.488, P = 0.019), and C7/17 > trisomy (odd ratio; 6.723, P < 0.001) were independently predictive factors for lymph node metastasis. Interestingly, an increase of C7, C17, and C7/17 CNVs in both trisomy and > trisomy was independently correlated with short median OS. An increased of C7 and/or 17 have a potential as a poor prognostic marker in CCA patients.

Chromosomal aberrations, visualized using UroVysion® fluorescence in-situ hybridization assay, can predict poor prognosis in formalin-fixed paraffin-embedded tissues of cholangiocarcinoma patients.

Cholangiocarcinoma (CCA) is a highly aggressive malignant tumor that has highest incidence in northeastern Thailand. The survival rate of CCA patients after receiving surgical treatment is quite low. Recently, genetic alterations including chromosome abnormalities have been studied as predictive factors and to aid planning for further treatment. This study aims to investigate the association between chromosomal aberrations, clinical data, and overall survival time of CCA patients. Formalin-fixed paraffin-embedded (FFPE) tissues from 194 CCA patients were examined. The copy numbers of chromosomes 3, 7, 17 and 9p21 were investigated using the UroVysion® fluorescence in-situ hybridization (FISH) assay. The overall survival time (OS) of CCA patients with or without polysomy of chromosomes 3, 7, 17 and/or loss of 9p21 were statistically analyzed in association with their clinicopathological parameters. Kaplan-Meier analysis was performed. The OS of patients with polysomy of chromosomes 3 + 7 was significantly shorter than those without this polysomy (log-rank P = 0.006; median OS 14.79 vs. 19.62 months). Moreover, patients with polysomy of chromosomes 3 + 7+17 and heterozygous for 9p21 loss have significantly shorter survival time than those without such chromosomal aberrations (log-rank P = 0.001; median OS 15.74 vs. 37.57 months). Interestingly, multivariate analysis revealed that polysomy of chromosomes 3 + 7 and of chromosomes 3 + 7+17 with 9p21 heterozygous loss were independent predictive factors of a poor OS (P = 0.027; P = 0.008, respectively).The chromosomal aberrations patterns which we evaluated using FISH; 1) polysomy of chromosomes 3 + 7 and 2) polysomy of chromosomes 3 + 7+17 with 9p21 heterozygous loss, have strong potential as indicators of poor prognosis in CCA patients.

Adherence of Helicobacter pylori to Opisthorchis viverrini gut epithelium and the tegument mediated via L-fucose binding adhesin.

Recent reports implicate both the liver fluke Opisthorchis viverrini as a reservoir of Helicobacter pylori within the human gastrointestinal tract and H. pylori in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma. We postulated that adherence of bacterial ligands to host receptors initiates colonization of the live fluke by H. pylori and here we aimed to assess the molecular interaction between O. viverrini and H. pylori by investigating host receptors for H. pylori in the fluke. Several known receptors of H. pylori including Lewis B, sialyl-Lewis X, Toll-like receptor 4 and L-fucose were detected immunohistochemically and histochemically by focusing analysis on the gut epithelium and tegument of the adult stage of the fluke. The frequency of detection of Lewis B, sialyl-Lewis X, TLR4 and L-fucose in 100 individual worms was 3, 3, 19 and 70%, respectively. Detection of H. pylori by a diagnostic ureA gene-based PCR assay revealed the presence of H. pylori in individual O. viverrini worms in 41 of 49 (79%) worms examined. In addition, numbers of bacteria decreased in a dose- and time-dependent fashion following exposure to fucosidase. These findings suggested that L-fucose represents a tractable receptor for H. pylori that can mediate bacterial colonization of the gut of O. viverrini.

Curcumin-loaded nanocomplexes: Acute and chronic toxicity studies in mice and hamsters.

We recently developed a modified solid dispersion of curcumin-loaded nanocomplexes (CNCs) in gums which promoted the prolonged and sustained release of curcumin. However, its safety assessment has not yet been investigated. Here, acute and chronic toxicities of CNCs were assayed using mice and hamsters. CNCs were orally administered to the animals. Doses of CNCs used for acute toxicity testing were 0.1, 1.1, 11.0 g/kg body weight for mice and 0.2, 2.1 and 21.4 g/kg body weight for hamsters. Doses of CNCs for chronic toxicity testing were 0.09, 0.27, 0.8 g/kg body weight/day for mice and 0.18, 0.54 and 1.61 g/kg body weight/day for hamsters. This regimen was followed daily for 6 months. Low and medium doses of CNCs did not induce any side effects in acute and chronic toxicity tests in either animal species. However, in acute toxicity testing, the organ-weight to body-weight ratio of spleen was significantly increased in mice treated with 11 g/kg body weight along with elevated levels of some biochemical parameters. There was a significant increase in organ-weight to body-weight ratios of stomach, liver and heart in hamsters treated with 21.4 g/kg body weight, but no elevated levels of biochemical parameters. Oral LD50 of CNCs in mice and hamsters were 8.9 and 16.8 g/kg body weight (equivalent to 2.5 and 4.7 g curcumin/kg body weight), respectively. Daily CNCs high-dose treatment for 6 months significantly increased organ-weight to body-weight ratios of stomach and intestine in mice and of lung and heart in hamsters. Elevated levels of glucose, total protein, ALT, AST and globulin in mice, and increased levels of AST, but decrease in cholesterol, in hamsters were concurrently observed with inflammation in liver and lung. These abnormalities were resolved within 28 days after cessation of treatment. The no-observed-adverse-effect level of CNCs was determined at 0.27 and 0.54 g/kg body weight/day in mice and hamsters. In conclusion, toxicity of high-dose CNCs treatment was graded as very low, possibly due to the components of the nanocomplex.

CagA+ Helicobacter pylori infection and N-nitrosodimethylamine administration induce cholangiocarcinoma development in hamsters.

BACKGROUND: Helicobacter pylori (HP) has been detected in the hepatobiliary tract of cholangiocarcinoma (CCA) patients in regions both endemic and non-endemic for Opisthorchis viverrini (OV) infection. However, whether H. pylori infection promotes CCA development remains unknown. We investigated CCA development in hamsters induced by a combination of infection with H. pylori and administration of N-nitrosodimethylamine (NDMA) and compared findings with those in an OV plus NDMA group. MATERIALS AND METHODS: Eighty-five hamsters were divided into four groups: (1) normal, (2) administered NDMA, (3) infected with cagA+ H. pylori and administered NDMA (HN group), and (4) infected with OV and administered NDMA (ON group). Animals were euthanized at 3 and 6 months post-infection. Histopathological changes of liver and the expression of markers associated with carcinogenesis were studied. RESULTS: At 3 months post-infection (p.i.), cholangitis and lymphoid follicles without tumor appearance were noted in the HN group, whereas extensive fibrosis was seen in members of the ON group, 10% of which had developed tumors. At 6 months p.i., 10% of hamsters administered NDMA alone had developed CCA, whereas in the HN and ON groups, 20% and 60% of hamsters, respectively, had developed CCA. Cytokeratin-19 (CK19) expression was observed in the CCA tissues of both the HN and the ON groups, confirming the bile duct origin of the CCA cells. CCA development in the HN group might be inflammation-mediated, as suggested by overexpression of HMGB1, PCNA, IL-8, and 8-OxodG in CCA tissues. CONCLUSION: cagA+ H. pylori infection and carcinogen intake can induce CCA development with slow progression.

Profiling of Bile Microbiome Identifies District Microbial Population between Choledocholithiasis and Cholangiocarcinoma Patients.

OBJECTIVE: Choledocholithiasis (CDL), a potential risk for cholangiocarcinoma (CCA) development, is often a consequence of bacterial infection. Thus, the microbial population that contributes to CDL might also be involved in CCA development. We compared the microbiome in bile fluid of CDL patients and CCA patients. METHODS: Bile samples were collected from CDL (n = 30) and CCA (n =30) patients. Microbial profiling was performed individually by the sequencing of V3-V4 regions of the 16S rRNA gene. RESULTS: Enterobacter, Pseudomonas, and Stenotrophomonas species were much more abundant in bile samples from CCA compared to CDL (p.

High Monopolar Spindle 1 Is Associated with Short Survival of Cholangiocarcinoma Patients and Enhances the Progression Via AKT and STAT3 Signaling Pathways.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium. The major problems of this cancer are late diagnosis and a high rate of metastasis. CCA patients in advanced stages have poor survival and cannot be cured with surgery. Therefore, targeting molecules involved in the metastatic process may be an effective CCA treatment. Monopolar spindle 1 (MPS1) is a kinase protein that controls the spindle assemble checkpoint in mitosis. It is overexpressed in proliferating cells and various cancers. The functional roles of MPS1 in CCA progression have not been investigated. The aims of this study were to examine the roles and molecular mechanisms of MPS1 in CCA progression. Immunohistochemistry results showed that MPS1 was up-regulated in carcinogenesis of CCA in a hamster model, and positive expression of MPS1 in human CCA tissues was correlated to short survival of CCA patients (n = 185). Small interfering RNA (siRNA)-induced knockdown of MPS1 expression reduced cell proliferation via G2/M arrest, colony formation, migration, and invasion. Moreover, MPS1 controlled epithelial to mesenchymal transition (EMT)-mediated migration via AKT and STAT3 signaling transductions. MPS1 was also involved in MMPs-dependent invasion of CCA cell lines. The current research highlights for the first time that MPS1 has an essential role in promoting the progression of CCA via AKT and STAT3 signaling pathways and could be an attractive target for metastatic CCA treatment.

Infection with Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Human Cholangiocytes.

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, Helicobacter pylori strain NCTC 11637, and the congeneric bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte of the H69 cell line induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Analysis to quantify cellular proliferation, migration, and invasion in real-time by both H69 cholangiocytes and CC-LP-1 line of cholangiocarcinoma cells using the xCELLigence approach and Matrigel matrix revealed that exposure to 10 H. pylori bacilli per cell stimulated migration and invasion by the cholangiocytes. In addition, 10 bacilli of H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection by H. pylori contributes to the malignant transformation of the biliary epithelium.

Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.

Three cholangiocarcinoma (CCA) cell line-formerly named, M156, M213 and M214 have been intensively used with discrepancy of their tumor origins. They were assumed to be originated from three different donors without authentication. To verify the origins of these cell lines, the short tandem repeat (STR) analysis of the currently used cell lines, the cell stocks from the establisher and the primary tumor of a CCA patient were performed. Their phenotypic and genotypic originality were compared. The currently used 3 CCA cell lines exhibited similar STR as CCA patient ID-M213 indicating the same origin of these cells. The cell stocks from the establisher, however, revealed the same STR of M213 and M214 cells, but not M156. The misidentification of M214 and M156 is probably due to the mislabeling and cross-contamination of M213 cells during culture. These currently used cell lines were renamed as KKU-213A, -213B and -213C, for the formerly M213, M214 and M156 cells, respectively. These cell lines were established from a male with an intrahepatic mass-forming CCA stage-4B. The tumor was an adenosquamous carcinoma with the liver fluke ova granuloma in evidence. All cell lines had positive CK19 with differential CA19-9 expression. They exhibited aneuploidy karyotypes, distinct cell morphology, cell growth, cytogenetic characteristic and progressive phenotypes. KKU-213C formed a adenosquamous carcinoma, whereas KKU-213A and KKU-213B formed poorly- and well-differentiated squamous cell carcinomas in xenografted mice. mRNA microarray revealed different expression profiles among these three cell lines. The three cell lines have unique characteristics and may resemble the heterogeneity of tumor origin.

Establishment of Highly Transplantable Cholangiocarcinoma Cell Lines from a Patient-Derived Xenograft Mouse Model.

Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2-/-/Jak3-/- mice. A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.

Prognostic biomarkers for cholangiocarcinoma and their clinical implications.

INTRODUCTION: Cholangiocarcinoma (CCA) is a poorly prognostic cancer with limited treatment options. Most patients have unresectable tumors when they are diagnosed and the chemotherapies provided are of limited benefit. Prognostic markers are therefore necessary to predict the disease outcome, risk of relapse, or to suggest the best treatment option. Areas covered: This article provides an up-to-date review of biomarkers with promising characteristics to be prognostic markers for CCA reported in the past 5 years. The biomarkers are sub-classified into tissue and serum markers. Proteins, RNAs, peripheral blood cells etc., that are associated with aggressive phenotypes, signal pathways, chemo-drug resistance, and those that reflect the survival time of CCA patients are evaluated for their prognostic prediction values. Expert commentary: CCAs are heterogeneous tumors of different histo-pathological subtypes and genetic influences and, therefore, potential markers should be validated in larger collectives with varied epidemiological backgrounds. A systematic review and meta-analysis should be done to clarify the impact of the reported biomolecules for their potential prognostic values. Non- or low-invasive sample collections, as well as the simple and affordable determination methods, should be constructed to make the prognostic biomarkers available in clinical practice.

Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand.

Cholangiocarcinoma is a rare type of cancer which is an increasingly discernible health threat. The disease is usually very difficult in diagnosis and various treatment modalities are typically not effective. Cholangiocarcinoma is a complex and very heterogeneous malignancy characterized by tumor location, different risk factors, molecular profiling, and prognosis. Cancer cell lines represent an important tool for investigation in various aspects of tumor biology and molecular therapeutics. We established two cell lines, KKU-452 and KKU-023, which were derived from patients residing in the endemic area of liver fluke infection in Thailand. Both of tumor tissues have gross pathology of perihilar and intrahepatic mass-forming cholangiocarcinoma. Two cell lines were characterized for their biological, molecular and genetic properties. KKU-452 and KKU-023 cells are both adherent cells with epithelium morphology, but have some differences in their growth pattern (a doubling time of 17.9 vs 34.8 h, respectively) and the expression of epithelial bile duct markers, CK7 and CK19. Cytogenetic analysis of KKU-452 and KKU-023 cells revealed their highly complex karyotypes; hypertriploid and hypotetraploid, respectively, with multiple chromosomal aberrations. Both cell lines showed mutations in p53 but not in KRAS. KKU-452 showed a very rapid migration and invasion properties in concert with low expression of E-cadherin and high expression of N-cadherin, whereas KKU-023 showed opposite characters. KKU-023, but not KKU-452, showed in vivo tumorigenicity in xenografted nude mice. Those two established cholangiocarcinoma cell lines with unique characters may be valuable for better understanding the process of carcinogenesis and developing new therapeutics for the patients.

5'-UTR and 3'-UTR Regulation of MICB Expression in Human Cancer Cells by Novel microRNAs.

The treatment of cancer through the induction of natural killer group 2, member D (NKG2D) ligands is of interest, but understanding of mechanisms controlling expression of individual ligand is limited. The major histocompatibility complex (MHC) class I chain related protein B (MICB) is a member of NKG2D ligands. We aimed to investigate the role of 3'-untranslated (3'-UTR) and 5'-untranslated regions (5'-UTR) in post-transcriptional regulation of MICB. Nine novel microRNAs (miRNAs) predicted to interact with 3'-UTR and 5'-UTR using TargetScan, RNAhybrid and miBridge were identified. Their regulation of 3'-UTR, 5'-UTR and both 3'- and 5'-UTR sequences of MICB were indicated by the reduction of luciferase activities of luciferase reporter constructs. Mutations of miRNA binding sites at 3'- and 5'-UTRs resulted in increased luciferase activities confirming the regulation of nine candidate miRNAs. In addition, overexpression of candidate miRNAs also down-regulated the expression of reporter constructs. Consequently, the overexpression and inhibition of candidate miRNAs lead to the decreased and increased. MICB protein expressions on the cells tested, respectively. This study has identified a new role of miRNAs in regulation of MICB expression via both 3'-UTR and 5'-UTR sequences applicable for cancer immunotherapy.

A comparative proteomic analysis of bile for biomarkers of cholangiocarcinoma.

Cholangiocarcinoma is a primary malignant tumor of the bile duct epithelium. Cholangiocarcinoma is usually detected at an advanced stage when successful treatment is no longer possible. As the tumor originates from the bile duct epithelium, bile is an ideal source of tumor biomarkers for cholangiocarcinoma. In this study, we used a quantitative proteomics approach to identify potential tumor-associated proteins in the bile fluid of six cholangiocarcinoma patients. Three different gross-appearance tumor types were used in the analysis: mass-forming type ( n = 2), periductal infiltrating type ( n = 2), and intraductal growth type ( n = 2). Two bile samples from non-cancerous patients were used as controls. Isobaric labeling, coupled with Tandem mass spectrometry, was used to quantify protein levels in the bile of cholangiocarcinoma and control patients. In all, 63 proteins were significantly increased in cholangiocarcinoma bile compared to normal bile. Alpha-1-antitrypsin was one of the overexpressed proteins that increased in cholangiocarcinoma bile samples. Immunohistochemical analysis revealed that alpha-1-antitrypsin was detected in 177 (50%) of 354 cholangiocarcinoma tissues from our Tissue Bank. Immunoblotting of 54 cholangiocarcinoma bile samples showed that alpha-1-antitrypsin was positive in 38 (70%) samples. Fecal enzyme-linked immunosorbent assay showed that alpha-1-antitrypsin level was able to distinguish cholangiocarcinoma patients from normal individuals. In conclusion, alpha-1-antitrypsin is a potential marker for early diagnosis of cholangiocarcinoma.

Elevated prevalence of Helicobacter species and virulence factors in opisthorchiasis and associated hepatobiliary disease.

Recent reports suggest that Opisthorchis viverrini serves as a reservoir of Helicobacter and implicate Helicobacter in pathogenesis of opisthorchiasis-associated cholangiocarcinoma (CCA). Here, 553 age-sex matched cases and controls, 293 and 260 positive and negative for liver fluke O. viverrini eggs, of residents in Northeastern Thailand were investigated for associations among infection with liver fluke, Helicobacter and hepatobiliary fibrosis. The prevalence of H. pylori infection was higher in O. viverrini-infected than uninfected participants. H. pylori bacterial load correlated positively with intensity of O. viverrini infection, and participants with opisthorchiasis exhibited higher frequency of virulent cagA-positive H. pylori than those free of fluke infection. Genotyping of cagA from feces of both infected and uninfected participants revealed that the AB genotype accounted for 78% and Western type 22%. Participants infected with O. viverrini exhibited higher prevalence of typical Western type (EPIYA ABC) and variant AB'C type (EPIYT B) CagA. Multivariate analyses among H. pylori virulence genes and severity of hepatobiliary disease revealed positive correlations between biliary periductal fibrosis during opisthorchiasis and CagA and CagA with CagA multimerization (CM) sequence-positive H. pylori. These findings support the hypothesis that H. pylori contributes to the pathogenesis of chronic opisthorchiasis and specifically to opisthorchiasis-associated CCA.