Gallbladder Carcinoma Cell Line Research Articles

Aldehyde oxidase 1 promotes gallbladder carcinogenesis through ROS-mediated activation of the Wnt/β-catenin pathway

BackgroundAldehyde oxidase 1 (AOX1) is associated with various pathophysiological processes, including cancer. Specifically, AOX1 has been demonstrated to have a close relationship with the progression of certain cancers. However, the expression, function, and mechanisms of action of AOX1 in gallbladder cancer (GBC) remain unclear. MethodsUtilizing immunohistochemistry, the study quantified the prevalence of AOX1 within tissues of gallbladder carcinoma and those of the surrounding non-cancerous regions. In vitro assays using gallbladder carcinoma cell lines with modulated AOX1 expression levels were performed to assess the protein's role in cell proliferation, migration, and invasion. Furthermore, flow cytometry techniques were harnessed to determine the influence of AOX1 on the content of reactive oxygen species (ROS) in these cells. Additionally, the expression of epithelial-mesenchymal transition (EMT) markers and the Wnt/β-catenin signaling pathway markersin cells with varied AOX1 expression, detected through Western blot analyses. An in vivo xenograft model involving athymic mice was implemented to explore the influence of AOX1 on gallbladder tumor growth, with Western blot analysis applied to measure EMT marker expression in the resulting tumours. ResultsElevated AOX1 protein levels have been observed in gallbladder carcinoma tissues, with such upregulation linked to a negative prognostic outlook for patients. In vitro analyses demonstrate that enhanced AOX1 expression facilitates gallbladder carcinoma cell proliferation, migration, and invasion, while AOX1 suppression yields an inhibitory effect on these cellular behaviors. Western blot results reveal an inverse relationship between AOX1 and E-cadherin levels, yet was positively correlation with N-cadherin, Vimentin, and Snail within both gallbladder cancer cells and in vivo xenograft tumours. Further mechanistic investigation indicates that AOX1 elevation augments reactive oxygen species (ROS) production and initiates the Wnt/β-catenin signaling pathway in these cells. The application of N-acetylcysteine (NAC) and/or KY1797K attenuates the proliferative, migratory, and invasive enhancements imparted by AOX1 overexpression and reinforces these effects when AOX1 is silenced—achieved through ROS mitigation and the obstruction of the Wnt/β-catenin pathway. In vivo studies corroborate these findings, showing AOX1 overexpression to amplify xenograft tumor growth and mesenchymal marker expression, whereas AOX1 interference did the opposite. ConclusionsThe study indicates that AOX1 functions as a carcinogenic factor in gallbladder carcinoma, enhancing cell proliferation, migration, invasion, and the EMT. These effects are driven by the activation of the Wnt/β-catenin pathway mediated by reactive oxygen species (ROS). Therefore,AOX1 presents potential as a valuable prognostic and diagnostic marker as well as a target for therapeutic intervention in the gallbladder cancer.

Microcystin-leucine-arginine promotes the development of gallbladder carcinoma via regulating ELAC2

Gallbladder carcinoma (GBC) is the most prevalent cancer of the bile tract, with unexpected GBC accounting for almost half of all GBC cases in some tertiary medical centers. Although the involvement of microcystin-leucine-arginine (MC-LR) in the development of intrahepatic cholangiocarcinoma has been established, there is a paucity of data regarding its association with GBC. The present study aims to investigate whether MC-LR level in the gallbladder of patients is associated with GBC development and, if so, to characterize the underlying mechanism in GBC cells. Our clinical data revealed that MC-LR level was significantly increased in GBC patients compared to patients with gallbladder stones only (P = 0.009). Moreover, our findings demonstrated that MC-LR could promote the proliferation and metastasis of human GBC cell lines. Furthermore, ELAC2 was identified as a critical mRNA involved in GBC progression through RNA sequencing. Collectively, our study suggests that MC-LR might be involved in the development of GBC by modulating the expression of ELAC2.

Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Recognition of potential therapeutic role of 2-hydroxy-3-methylanthraquinones in the treatment of gallbladder carcinoma: A proteomics analysis.

Gallbladder carcinoma (GBC), with early metastasis and high recurrence rates, is an enormous threat to health. As an anthraquinones monomer of traditional Chinese medicine Hedyotis diffusa, 2-hydroxy-3-methylanthraquinone (HMA) has been reported to inhibit the growth of several cancers. But in our preliminary study, HMA could only weakly induce GBC cell apoptosis. To explore other possible mechanism underlying the inhibition effect of HMA on GBC, this proteomics analysis was performed. A proteomics analysis was performed on one GBC cell line bought from the China Life Science Cell Bank. Several computational techniques were merged to develop analysis for those differently expressed proteins. A comparative protein-protein interaction network analysis was carried out among the differently expressed proteins to identify the proteins potentially inhibiting GBC. Thus, a GO and KEGG analysis was performed to identify the signaling pathways underlying a potential therapeutic role for HMA. A total of 285 proteins were affected by HMA, including 187 upregulated and 98 downregulated. The subcellular localization of differently expressed proteins were identified, including 142 in nuclear, 67 in cytoplasm, 67 in extracellular matrix, 46 in plasma membrane, 13 in mitochondrion, 3 in lysosome, and 1 in cytoskeleton. HMA could regulate EGFR, FN1, PLG, PLAUR, LAMA3, HRG, THBS1, PLAT, KNG1, ENAM, SERPINE1, ECM1, interleukin-8, and trypsin in GBC. Most of the regulated proteins involve in cell migration. Pathways including PI3K-Akt, Wnt, HIF-1, focal adhesion, microRNAs were regulated by HMA. HMA was shown to be an inhibition agent for GBC development, and this analysis would contribute to the development of new anti-GBC drugs.

Long non-coding RNA TMPO-AS1 promotes cell proliferation, migration, invasion and epithelial-to-mesenchymal transition in gallbladder carcinoma by regulating the microRNA-1179/E2F2 axis.

Gallbladder carcinoma (GBC), which is a common tumor of the biliary system, poses a serious threat to human life and health. The present study aimed to investigate the molecular mechanism of the long non-coding (lnc)RNA thymopoietin antisense transcript 1 (TMPO-AS1)/microRNA (miRNA/miR)-1179/E2F transcription factor 2 (E2F2) axis in GBC. The viability, proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT) of GBC cell lines were assessed via the Cell Counting Kit-8, colony formation, Transwell migration and invasion, immunofluorescence and western blot assays. In the present study, lncRNA TMPO-AS1 was significantly upregulated in clinical GBC tissues and cell lines, and was highly expressed in stage III+IV patients with GBC compared with stage I+II patients with GBC. In addition, the overall survival rate of patients with low TMPO-AS1 expression levels was higher than those with high TMPO-AS1 expression levels. Furthermore, TMPO-AS1 knockdown inhibited the viability, proliferation, migration, invasion and EMT of GBC cell lines. In addition, miR-1179 expression was downregulated in clinical GBC tissues and cell lines, and negatively correlated with TMPO-AS1 expression. The results revealed that miR-1179 is a target of TMPO-AS1, which was confirmed via the dual-luciferase reporter assay and RNA pull-down analysis. Overexpression of miR-1179 inhibited the viability, proliferation, migration, invasion and EMT of GBC cell lines. Furthermore, E2F2 was verified as a direct target of miR-1179 by binding to its 3′-untranslated region. E2F2 expression was significantly upregulated in clinical GBC tissues and cell lines, and negatively correlated with miR-1179 expression. Notably, E2F2 knockdown partially hindered the effects of TMPO-AS1/miR-1179 on the proliferation and metastasis of GBC cell lines. Taken together, the results of the present study suggest that TMPO-AS1 potentially plays a tumor-promoting role in the occurrence and development of GBC, which may be achieved by regulating the miR-1179/E2F2 axis.

Long Non-Coding RNA Myosin Light Chain Kinase Antisense 1 Plays an Oncogenic Role in Gallbladder Carcinoma by Promoting Chemoresistance and Proliferation.

BackgroundLong non-coding RNAs (lncRNAs) have been reported to play critical roles in human tumours, including gallbladder carcinoma (GBC). However, their biological functions and molecular mechanisms in tumorigenesis and progression remain largely unknown.MethodsQuantitative polymerase chain reaction (qPCR) was used to verify the expression of lncRNA myosin light chain kinase antisense RNA 1 (MYLK-AS1) in 120 pairs of GBC tissues and paired adjacent non-tumour tissues, as well as in six different GBC cell lines (NOZ, EH-GB1, OCUG-1, GBC-SD, SGC-996 and QBC-939). Cell counting kit 8 was applied to explore cell proliferation and drug sensitivity assays. The target miRNAs (miR) of MYLK-AS1 and downstream target genes were predicted using Starbase 3.0 software and confirmed by double luciferase reporting test. The expression of proteins was assessed using Western blot assay.ResultsHere, we demonstrated that MYLK-AS1 was significantly upregulated and correlated with a poor prognosis and poor clinical characteristics in GBC. Furthermore, the forced expression of MYLK-AS1 significantly promoted GBC cell proliferation and resistance to gemcitabine in vitro. Mechanistically, MYLK-AS1 functioned as an efficient miR-217 sponge, thereby releasing the inhibition of enhancer of zeste 2 polycomb repressive complex 2 (EZH2) subunit expression. MYLK-AS1 promoted GBC cell proliferation and resistance to gemcitabine by upregulating EZH2 expression, and EZH2 was confirmed as a direct target of miR-217.DiscussionOur results confirmed that the chemoresistant driver MYLK-AS1 might be a promising candidate as a therapeutic target for the treatment of advanced GBC.

MiR-193a-3p Enhanced the Chemosensitivity to Trametinib in Gallbladder Carcinoma by Targeting KRAS and Downregulating ERK Signaling.

Objective: In this study, the authors identified miR-193a-3p as a tumor-suppressing microRNA, and its effects on the chemosensitivity to trametinib in gallbladder carcinoma (GBC) were evaluated. Materials and Methods: The levels of miR-193a-3p in clinical GBC tissues and GBC cells were determined by quantitative real-time polymerase chain reaction. The protein levels of KRAS, ERK, and phosphorylated ERK (p-ERK) were examined by Western blot. Dual-luciferase reporter assays were performed to confirm the interaction between miR-193a-3p and KRAS. The effect of miR-193a-3p knockdown or overexpression on the malignant behaviors and chemosensitivity of GBC was determined by 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide and flow cytometry assays in vitro and further examined in a xenograft model. Results: The levels of miR-193a-3p were significantly decreased in GBC cell lines, especially with KRAS mutations. In addition, miR-193a-3p overexpression retarded cell proliferation of GBC, but induced cell apoptosis. Moreover, miR-193a-3p overexpression significantly improved the chemosensitivity of GBC to trametinib both in in vitro assays and in vivo xenograft mouse model. Further mechanisms disclosed that KRAS was a target of miR-193a-3p and levels of p-ERK were increased by treatment with miR-193a-3p inhibitor in GBC. Conclusions: These data suggested that miR-193a-3p enhanced the chemosensitivity to trametinib in GBC with wild-type KRAS or KRAS mutations by directly targeting KRAS and finally downregulated ERK signaling.

FGF19 and FGFR4 promotes the progression of gallbladder carcinoma in an autocrine pathway dependent on GPBAR1-cAMP-EGR1 axis.

Treatment options for gallbladder carcinoma (GBC) are limited and GBC prognosis remains poor. There is no well-accepted targeted therapy to date, so effective biomarkers of GBC are urgently needed. Here we investigated the expression and correlations of fibroblast growth factor receptors (FGFR1-4) and 18 fibroblast growth factors (FGFs) in two independent patient cohorts and evaluated their prognostic significance. Consequently, we demonstrated that both FGF19 and FGFR4 were unfavorable prognostic biomarkers, and their co-expression was a more sensitive predictor. By analyzing the correlations between all 18 FGFs and FGFR4, we showed that FGF19 expression was significantly associated with FGFR4 and promoted GBC progression via stimulating FGFR4. With experiments using GBC cells, GPBAR1-/- mice models, and human subjects, we demonstrated that elevated bile acids (BAs) could increase the transcription and expression of FGF19 and FGFR4 by activating GPBAR1-cAMP-EGR1 pathway. FGF19 secreted from GBC cells promoted GBC progression by stimulating FGFR4 and downstream ERK in an autocrine manner with bile as a potential carrier. Patients with GBC had significantly higher FGF19 in serum and bile, compared to patients with cholelithiasis. BLU9931 inhibited FGFR4 and attenuated its oncogenic effects in GBC cell line. In conclusion, upregulation of BAs elevated co-expression of FGF19 and FGFR4 by activating GPBAR1-cAMP-EGR1 pathway. Co-expression of FGF19 and FGFR4 was a sensitive and unfavorable prognostic marker. GBC cells secreted FGF19 and facilitated progression by activating FGFR4 with bile as a potential carrier in an autocrine pathway.

Expression and inhibitory effects of p53-upregulated modulator of apoptosis in gallbladder carcinoma.

The p53-upregulated modulator of apoptosis (PUMA) has been reported to be involved in various types of cancer. However, its potential biological role in gallbladder carcinoma (GBC) has not been fully elucidated. The present study aimed to determine the expression levels of PUMA and its biological effects on GBC. The mRNA and protein expression levels of PUMA in GBC tissues and cell lines were measured using reverse transcription-quantitative PCR and western blotting, respectively. The effects of PUMA overexpression on cell viability, proliferation and invasive ability were determined in vitro using the MTT, colony formation and Transwell invasion assays, respectively. The apoptotic rates were detected using the Annexin V-FITC apoptosis detection kit. Furthermore, follow-up of patients with GBC was performed to identify the association between PUMA expression levels and GBC prognosis. The results of the present study demonstrated that the expression levels of PUMA were significantly lower in the GBC tissues and cell lines compared with those in adjacent normal gallbladder tissues and normal gallbladder cells, respectively. Further experiments indicated that overexpression of PUMA inhibited the viability, proliferation and invasive ability of GBC cells compared with those in the control-transfected GBC cells. In addition, overexpression of PUMA significantly promoted apoptosis in GBC cells. Furthermore, overexpression of PUMA inhibited epithelial-mesenchymal transition, and promoted Bax upregulation and Bcl-2 downregulation compared with those in the control group. Low PUMA expression levels were associated with a short overall survival time in patients with GBC. In conclusions, PUMA may act as a tumor suppressor in GBC and may serve as a potential novel treatment target for human GBC.

Long non-coding RNA SSTR5-AS1 facilitates gemcitabine resistance via stabilizing NONO in gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most aggressive carcinoma of the biliary tract, effective chemotherapy was critical for the patients with unresectable GBC. However, chemotherapy resistance is still problematic for clinicians. Here, we identified a specific long non-coding RNA, SSTR5-AS1, in GBC patient that facilitates gemcitabine resistance. SSTR5-AS1 is significantly increased in GBC samples and cell lines, especially in gemcitabine-resistant cell lines, and higher SSTR5-AS1 expression was correlated with poorer overall survival rate in GBC patients. Our data revealed that upregulated SSTR5-AS1 facilitates gemcitabine resistance via inhibiting apoptosis. Knockdown of SSTR5-AS1 sensitized drug resistant GBC cells to gemcitabine in vitro and strongly inhibited xenografts formed by drug resistant GBC cells in vivo. Moreover, we found via streptavidin pull down assay that NONO specifically binds to sense sequence of SSTR5-AS1 and prevented proteasome mediated NONO degradation, which resulted in increased NONO protein level without affecting the transcription of NONO. NONO functions as the downstream effector of SSTR5-AS1 and is required for SSTR5-AS1 mediated gemcitabine resistance. Collectively, our data provided novel insights into lncRNA-mediated chemotherapy resistance and suggested a novel therapeutic target to improve chemotherapy strategies for unresectable GBC patients.

LncRNA-HGBC stabilized by HuR promotes gallbladder cancer progression by regulating miR-502-3p/SET/AKT axis

BackgroundsLong non-coding RNAs (lncRNAs) are essential factors that regulate tumor development and metastasis via diverse molecular mechanisms in a broad type of cancers. However, the pathological roles of lncRNAs in gallbladder carcinoma (GBC) remain largely unknown. Here we discovered a novel lncRNA termed lncRNA Highly expressed in GBC (lncRNA-HGBC) which was upregulated in GBC tissue and aimed to investigate its role and regulatory mechanism in the development and progression of GBC.MethodsThe expression level of lncRNA-HGBC in GBC tissue and different cell lines was determined by quantitative real-time PCR. The full length of lncRNA-HGBC was obtained by 5′ and 3′ rapid amplification of the cDNA ends (RACE). Cellular localization of lncRNA-HGBC was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. In vitro and in vivo assays were preformed to explore the biological effects of lncRNA-HGBC in GBC cells. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay were used to identify lncRNA-HGBC-interacting proteins. Dual luciferase reporter assays, AGO2-RIP, and MS2-RIP assays were performed to verify the interaction between lncRNA-HGBC and miR-502-3p.ResultsWe found that lncRNA-HGBC was upregulated in GBC and its upregulation could predict poor survival. Overexpression or knockdown of lncRNA-HGBC in GBC cell lines resulted in increased or decreased, respectively, cell proliferation and invasion in vitro and in xenografted tumors. LncRNA-HGBC specifically bound to RNA binding protein Hu Antigen R (HuR) that in turn stabilized lncRNA-HGBC. LncRNA-HGBC functioned as a competitive endogenous RNA to bind to miR-502-3p that inhibits target gene SET. Overexpression, knockdown or mutation of lncRNA-HGBC altered the inhibitory effects of miR-502-3p on SET expression and downstream activation of AKT. Clinically, lncRNA-HGBC expression was negatively correlated with miR-502-3p, but positively correlated with SET and HuR in GBC tissue.ConclusionsOur study demonstrates that lncRNA-HGBC promotes GBC metastasis via activation of the miR-502-3p-SET-AKT cascade, pointing to lncRNA-HGBC as a new prognostic predictor and a therapeutic target.

MiR-335 promotes cell proliferation by inhibiting MEF2D and sensitizes cells to 5-Fu treatment in gallbladder carcinoma.

Gallbladder carcinoma is a malignant tumor in the bile duct with poor prognosis. Although aberrant expression of miR-335 has been reported in the tumor tissues of gallbladder carcinoma, the biological role of miR-335 was still largely unknown. This study was intended to explore the role of miR-335 in the progression of gallbladder carcinoma. The gallbladder carcinoma cell lines GBC-SD and SGC-996 were used in our study. MiR-335 mimic, miR-335 inhibitor, and si-myocyte enhancer factor 2D (MEF2D) were transfected into gallbladder carcinoma cells, respectively. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay analysis was used to determine cell viability. The colony formation was also analyzed. Cell cycle progression was determined using flow cytometer. To verify the target gene of miR-335, the luciferase assay was used. MiR-335 overexpression inhibited cell viability and colony formation of GBC-SD and SGC-996 cells. The percentage of cells in first gap phase (G1)/resting phase (G0) was significantly increased, and the expression of cell division cycle 2 (cdc2) and cell division cycle 25 (cdc25) was decreased after miR-335 was overexpressed, indicating its role in inducing the cell cycle arrest of GBC-SD and SGC-996 cells. MEF2D was up-regulated in gallbladder cancer and associated with tumor size and clinical stage. Down-regulation of MEF2D inhibited cell viability and colony formation, induced cell cycle arrest in G1/G0 phase, and decreased the expression of cdc2 and cdc25 in GBC-SD and SGC-996 cells. Bioinformatics analysis by TargetScan and luciferase assay verified that MEF2D could be targeted by miR-335. Importantly, the effects of miR-335 inhibitor on cell growth were rescued by small interfering RNA of MEF2D (siMEF2D) in GBC-SD and SGC-996 cells. Besides, miR-335 overexpression increased cell sensitivity to 5-Fluoracil (Fu) treatment and decreased the expression levels of ATP-binding cassette transporter B1 (ABCB1) and ATP-binding cassette G2 (ABCG2) in GBC-SD and SGC-996 cells. MiR-335 participates in the progression of gallbladder carcinoma by targeting MEF2D. MiR-335 may be a potential therapeutic target for gallbladder carcinoma.

Modulation on gallbladder carcinoma by TGF-β1 via IGFBP-2.

Gallbladder carcinoma (GC) occupies more than 90% of all cancers in biliary tract with an increasing incidence. Most patients with GC are already at terminal stage at the time of primary diagnosis, causing unfavorable prognosis and high mortality. Transformation growth factor-beta (TGF-β) is up-regulated in GC. However, the mechanism by how TGF-β is involved in GC remains unclear. The aim of this study was to investigate the effect and mechanism of TGF-β in GC using GC cell line NOZ cells.In vitro cultured NOZ cell was randomly assigned into control, si-NC and TGF-β1 siRNA groups and were transfected with siRNA negative control (NC) or TGF-β1 siRNA followed by analysis of TGF-β1 expression by Real-time PCR, cell proliferation by MTT assay, cell apoptosis and cell invasion, as well as expression of proteins in epithelial-mesenchymal transition (EMT), p38, Smad2/3 and Smad4 phosphorylation by Western blot, Insulin-like growth factor-binding protein-2 (IGFBP-2) level by ELISA. After transfecting TGF-β1 siRNA into NOZ cells, TGF-β1 expression was suppressed and cell proliferation and invasion were inhibited, together with enhanced Caspase-3 activity. Meanwhile, E-cadherin expression was increased, with decreased Vimentin, IGFBP-2, p38, Smad2/3 and Smad4 phosphorylation (P< 0.05 comparing to control group). In conclusion, inhibition of TGF-β1 expression facilitates GC cell apoptosis, inhibits GC cell proliferation, invasion and EMT occurrence.

MiR-1-5p is down-regulated in gallbladder carcinoma and suppresses cell proliferation, migration and invasion by targeting Notch2

BackgroundNumerous studies have demonstrated that aberrant microRNAs (miRNAs) are involved in tumorigenesis and tumor progression. Nevertheless, the precise role of miR-1-5p in gallbladder carcinoma cell growth and metastasis remains not fully revealed. Material and methodsThe levels of miR-1-5p were detected in gallbladder carcinoma tissues and cell lines using qRT-PCR method. A series of functional assays, including cell proliferation, colony formation, wound healing and Transwell invasion were conducted using miR-1-5p or miR-1-5p inhibitor transfected cells. ResultsMiR-1-5p was remarkably down-regulated in gallbladder carcinoma tissues and cell lines compared to normal. In addition, over-expression of miR-1-5p markedly suppressed the growth, migration and invasion of gallbladder carcinoma cell. Conversely, down-expression of miR-1-5p facilitated gallbladder carcinoma cell proliferation and aggressiveness. Mechanistic investigations demonstrated that neurogenic locus notch homolog protein 2 (Notch2) was the directly target of miR-1-5p and Notch2 mediated the inhibitory effect of miR-1-5p in gallbladder carcinoma cell growth and aggressiveness. ConclusionOur findings demonstrated that miR-1-5p acted as a suppressive miRNA and played vital roles in the growth, migration and invasion of gallbladder carcinoma cell through targeting Notch2.

MiR-139-5p is associated with poor prognosis and regulates glycolysis by repressing PKM2 in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most highly aggressive cancer of biliary tract, but effective therapeutics are lacking. Emerging evidence has unveiled that miR-139-5p is aberrantly downregulated in cancers, including GBC. However, the functions and mechanisms of miR-139-5p in GBC remain unclear. MiR-139-5p-overexpression was established in GBC cell lines, after which cell proliferation, migration, invasion, colony formation, and glucose metabolism were assayed in vitro. Subsequently, bioinformatics prediction and dual-luciferase reporter were performed to confirm that pyruvate kinase M2 (PKM2) was a direct target of miRNA-139-5p. Xenograft mouse models were applied to investigate the role of miR-139-5p in GBC tumourigenicity in vivo. In situ hybridization and immunohistochemical assays were performed to determine the relationships among miR-139-5p, PKM2 expression and clinical malignancies in GBC samples. We found that miR-139-5p was substantially downregulated in GBC tissues. Low expression of miR-139-5p was significantly associated with poor clinical outcomes. GBC cell proliferation, migration, and invasion could be inhibited by overexpression of miR-139-5p either in vitro or in vivo. In addition, miR-139-5p overexpression could directly inhibit PKM2 expression and lead to suppression of glucose consumption, lactate production, and cellular ATP levels. Moreover, PKM2 was frequently upregulated in GBC and correlated with poor prognosis. Mechanistically, miRNA-139-5p inhibited cell proliferation, migration, and glycolysis in GBC, at least in part, by repressing PKM2. These results demonstrated a novel role for miR-139-5p/PKM2 in GBC progression and provided potential prognostic predictors for GBC patients.

MiRNA-145 Induces Apoptosis in a Gallbladder Carcinoma Cell Line by Targeting DFF45.

BakcgroundWe measured expression of miRNA-145 in gallbladder carcinoma and its influence on propagation, invasion, and apoptosis of gallbladder carcinoma cells in vitro.MethodsmiRNA-145 expression was compared between normal gallbladder epithelial cells and GBS-SD (gallbladder series) cells using miRNA chip technology. Propagation, apoptosis, and invasion properties of each cell group were tested using MTT, a clone-formation assay, flow cytometry, Western blot, and Transwell assays.ResultsExpression of miRNA-145 was observed to be down-regulated and GBC-SD cell clones transiently transfected with hsa-miRNA-145 were substantially reduced compared with controls (p<0.01). We observed that GBC-SD cells transfected with hsa-miRNA-145 and double-positive (Annexin V and PI) for apoptosis were more numerous than controls. Moreover, GBC-SD cells over-expressing miRNA-145 had significantly greater expression of apoptosis-related protein, caspase-3. A Transwell assay confirmed that GBC-SD cells over-expressing miRNA-145 that migrated to the lower chamber were fewer compared with controls. Post-transcriptional regulation of gene expression was measured using dualluciferase reporter assays and data show that miRNA-145 facilitates the inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45.ConclusionThus, we speculate that miRNA-145 facilitates inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45; however, miRNA-145 does not directly affect the GBC-SD cell cycle.

Genomic ERBB2/ERBB3 mutations promote PD-L1-mediated immune escape in gallbladder cancer: a whole-exome sequencing analysis

ObjectivesPatients with gallbladder carcinoma (GBC) lack effective treatment methods largely due to the inadequacy of both molecular characterisation and potential therapeutic targets. We previously uncovered a spectrum of genomic alterations...

MicroRNA-30a-5p inhibits gallbladder cancer cell proliferation, migration and metastasis by targeting E2F7

Gallbladder carcinoma (GBC), the most common malignant tumour of the bile duct, is highly aggressive and has a poor prognosis. MicroRNA-30a-5p (miR-30a-5p) is an important tumour suppressor that participates in many aspects of carcinogenesis and cancer development. However, the role of miR-30a-5p in GBC development remains to be determined, as do the mechanisms underlying its effects in GBC. Using samples collected from 42 subjects with gallbladder carcinoma (GBC), we showed decreased miR-30a-5p expression in the primary lesions vs. non-tumour adjacent tissues (NATs). Decreased miR-30a-5p was associated with shorter disease-free survival (DFS) and overall survival (OS). Inhibiting miR-30a-5p expression in 2 representative GBC cell lines (GBC-SD and NOZ) increased cell proliferation, migration, invasiveness, as well as β-catenin nuclear translocation, vice versa. In nude mice, NOZ cells transfected with miR-30a-5p mimics grew slower (vs. miR-NC) upon subcutaneous inoculation, and had lower rate of hepatic metastasis upon spleen inoculation. Dual luciferase assay confirmed that E2F transcription factor 7 (E2F7) was a direct target of miR-30a-5p and antagonized the effects induced by miR-30a-5p downregulation in GBC cells. MiR-30a-5p attenuates the EMT and metastasis in GBC cells by targeting E2F7, suggesting miR-30a-5p is a tumour suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for GBC.

STYK1 promotes cancer cell proliferation and malignant transformation by activating PI3K-AKT pathway in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract with extremely poor prognosis. The malignant transformation of GBC is associated with cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). However, the molecular mechanisms underlying GBC progression are poorly understood. We found that serine threonine tyrosine kinase 1 (STYK1) was elevated in GBC and was negatively correlated with clinical outcomes and prognosis. Overexpression of STYK1 in GBC cell lines gave rise to increased cell proliferation, colony formation, migration and invasion, thus committing cells to undergoing EMT. In contrast, silence of STYK1 led to opposite effects on cell transformation. Consistent with STYK1 gene knockdown, AKT specific inhibitor MK2206 abrogated tumor promoting action induced by STYK1, suggesting that PI3K/AKT pathway is essential for the oncogenic role of STYK1 in GBC. STYK1 shRNA in GBC cells inhibited development of xenografted tumors compared with control cells. Collectively, our findings suggest that STYK1 is a critical regulator of tumor growth and metastasis, and may serve as a potential target for GBC therapy.

Effects of matrix metalloproteinase inhibitor doxycycline and CD147 antagonist peptide-9 on gallbladder carcinoma cell lines.

Gallbladder carcinoma is the most common and aggressive malignancy of the biliary tree and highly expresses CD147, which is closely related to disease prognosis in a variety of human cancers. Doxycycline exhibited anti-tumor properties in many cancer cells. CD147 antagonist peptide-9 is a polypeptide and can specifically bind to CD147. The effect of these two drugs on gallbladder cancer cells has not been studied. The aim of this study is to investigate the effect of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells and the possible mechanism of inhibition on cancer cell of doxycycline. To investigate the effects of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells (GBC-SD and SGC-996), cell proliferation, CD147 expression, and early-stage apoptosis rate were measured after treated with doxycycline. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were measured after treated with different concentrations of doxycycline, antagonist peptide-9, and their combination. The results demonstrated that doxycycline inhibited cell proliferation, reduced CD147 expression level, and induced an early-stage apoptosis response in GBC-SD and SGC-996 cells. The matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were inhibited by antagonist peptide-9 and doxycycline, and the inhibitory effects were enhanced by combined drugs in gallbladder carcinoma cell lines. Taken together, doxycycline showed inhibitory effects on gallbladder carcinoma cell lines and reduced the expression of CD147, and this may be the mechanism by which doxycycline inhibits cancer cells. This study provides new information and tries to implement the design of adjuvant therapy method for gallbladder carcinoma.