Gallbladder Cancer Tissues Research Articles

Circular RNA circERBB2 promotes gallbladder cancer progression by regulating PA2G4-dependent rDNA transcription

BackgroundCircRNAs are found to affect initiation and progression of several cancer types. However, whether circRNAs are implicated in gallbladder cancer (GBC) progression remains obscure.MethodsWe perform RNA sequencing in 10 pairs of GBC and para-cancer tissues. CCK8 and clone formation assays are used to evaluate proliferation ability of GBC cells. qPCR and Western blot are used to determine expression of RNAs and proteins, respectively. CircRNA-protein interaction is confirmed by RNA pulldown, RNA immunoprecipitation, and fluorescence in situ hybridization.ResultsWe find that circRNA expression pattern is tremendously changed during GBC development. Among dozens of significantly changed circRNAs, a circRNA generated from the oncogene ERBB2, named as circERBB2, is one of the most significant changes. CircERBB2 promotes GBC proliferation, in vitro and in vivo. Other than being a miRNA sponge, circERBB2 accumulates in the nucleoli and regulates ribosomal DNA transcription, which is one of the rate-limiting steps of ribosome synthesis and cellular proliferation. CircERBB2 regulates nucleolar localization of PA2G4, thereby forming a circERBB2-PA2G4-TIFIA regulatory axis to modulate ribosomal DNA transcription and GBC proliferation. Increased expression of circERBB2 is associated with worse prognosis of GBC patients.ConclusionsOur findings demonstrate that circERBB2 serves as an important regulator of cancer cell proliferation and shows the potential to be a new therapeutic target of GBC.

Circular RNA FOXP1 promotes tumor progression and Warburg effect in gallbladder cancer by regulating PKLR expression

BackgroundCircular RNAs (circRNAs) have recently been identified as potential functional modulators of the cellular physiology processes. The study aims to uncover the potential clinical value and driving molecular mechanisms of circRNAs in gallbladder cancer (GBC).Patients and methodsWe performed RNA sequencing from four GBC and paired adjacent normal tissues to analyze the circRNA candidates. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the circFOXP1 expression from 40 patient tissue samples. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro and in vivo assays were performed to prove the functional significance of circFOXP1. Double luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed.ResultsBy performing RNA sequencing from GBC and paired adjacent normal tissues to analyze the circRNA candidates, we identified that circFOXP1 (hsa_circ_0008234) expression was significantly upregulated in GBC tissues and positively associated with lymph node metastasis, advanced TNM stage and poor prognosis in patients. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro assays demonstrated that circFOXP1 has pleiotropic effects, including promotion of cell proliferation, migration, invasion, and inhibition of cell apoptosis in GBC. In vivo, circFOXP1 promoted tumor growth. Mechanistically, double luciferase reporter, RNA immunoprecipitation (RIP) and biotin-labeled RNA pull-down assays clarified that circFOXP1 interacted with PTBP1 that could bind to the 3’UTR region and coding region (CDS) of enzyme pyruvate kinase, liver and RBC (PKLR) mRNA (UCUU binding bites) to protect PKLR mRNA from decay. Additionally, circFOXP1 acted as the sponge of miR-370 to regulate PKLR, resulting in promoting Warburg effect in GBC progression.ConclusionsThese results demonstrated that circFOXP1 serve as a prognostic biomarker and critical regulator in GBC progression and Warburg effect, suggesting a potential target for GBC treatment.

Cholesterol depletion sensitizes gallbladder cancer to cisplatin by impairing DNA damage response

ABSTRACT Gallbladder cancer (GBC) is the common malignancy of the bile tract system with extremely poor clinical outcomes, owing to its metastatic property and intrinsic resistance to the first-line drugs. Although it is well-established that cholesterol abnormity contributes to gallstone formation, a leading risk factor for GBC, the link of cholesterol homeostasis with GBC has not been investigated. The present study systematically examined the genes implicated in cholesterol homeostasis, and revealed altered gene expressions of de novo cholesterol biosynthesis and sterol sulfonation (SULT2B1), reduced bile acid synthesis (CYP7B1 and CYP39A1) and impaired sterol efflux (ABCA1, ABCG5, LCAT, and CETP) in GBC tissues. Suppression of cholesterol biosynthesis by lovastatin inhibited GBC cell proliferation possibly through attenuating the DNA repair process. Further investigation revealed lovastatin sensitized GBC cells to cisplatin-induced apoptosis and suppressed the activation of CHK1, CHK2, and H2AX during DNA damage response. By using chemically distinct statins, HMGCR depletion or supplementing mevalonate, the product of HMGCR, we showed the inhibitory effects on DNA repair process of lovastatin were due to the blockage of the mevalonate pathway. Subcutaneous xenograft mice model suggested lovastatin promoted the therapeutic efficacy of cisplatin, and significantly prolonged the survival times of tumor-bearing mice. Moreover, HMGCR ablation repressed tumor growth in vivo, which can be rescued partially by restored expression of HMGCR, suggesting the on-target effects of lovastatin. Therefore, our study provides the clinical relevance of cholesterol homeostasis with GBC progression, and highlights a novel intervention of combined use of lovastatin and cisplatin for GBC.

Identification of messenger and long noncoding RNAs associated with gallbladder cancer via gene expression profile analysis.

The present study aimed to investigate the long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in the progression of gallbladder cancer and explore the potential physiopathologic mechanisms of gallbladder cancer in terms of competing endogenous RNAs (ceRNAs). The original lncRNA and mRNA expression profile data (nine gallbladder cancer tissues samples and nine normal gallbladder samples) in GSE76633 was downloaded from the Gene Expression Omnibus database. Differentially expressed mRNAs and lncRNAs between gallbladder cancer tissue and normal control were selected and the pathways in which they are involved were analyzed using bioinformatics analyses. MicroRNAs (miRNAs) were also predicted based on the differentially expressed mRNAs. Finally, the co-expression relation between lncRNA and mRNA was analyzed and the ceRNA network was constructed by combining the lncRNA-miRNA, miRNA-mRNA, and lncRNA-mRNA pairs. Overall, 373 significantly differentially expressed mRNAs and 47 lncRNAs were identified between cancer and normal tissue samples. The upregulated genes were significantly enriched in the extracellular matrix (ECM)-receptor interaction pathway, while the downregulated genes were involved in the complement and coagulation cascades. Altogether, 128 co-expression relations between lncRNA and mRNA were obtained. In addition, 196 miRNA-mRNA regulatory relations and 145 miRNA-lncRNA relation pairs were predicted. Finally, the lncRNA-miRNA-gene ceRNA network was constructed by combining the three types of relation pairs, such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6. mRNAs and lncRNAs may be involved in gallbladder cancer progression via ECM-receptor interaction pathways and the complement and coagulation cascades. Moreover, ceRNAs such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6 can also be implicated in the pathogenesis of gallbladder cancer.

MBD1 promotes the malignant behavior of gallbladder cancer cells and induces chemotherapeutic resistance to gemcitabine

BackgroundMethyl-CpG binding domain protein 1 (MBD1), which couples DNA methylation to transcriptional repression, has been implicated in transcriptional regulation, heterochromatin formation, genomic stability, cell cycle progression and development. It has also been proven that MBD1 is involved in tumor development and progression. However, whether MBD1 is involved in tumorigenesis, especially in gallbladder cancer, is totally unknown.MethodsHuman GBC-SD and SGC996 cells were used to perform experiments. Invasion, wound healing and colony formation assays were performed to evaluate cell viability. A CCK-8 assay was performed to assess gallbladder cancer cell viability after gemcitabine treatment. Western blot analysis was used to evaluate changes in protein expression. Human gallbladder cancer tissues and adjacent nontumor tissues were subjected to immunohistochemical staining to detect protein expression.ResultsWe found that MBD1 expression was significantly upregulated in gallbladder cancer tissues compared with that in surrounding normal tissues according to immunohistochemical analysis of 84 surgically resected gallbladder cancer specimens. These data also indicated that higher MBD1 expression was correlated with lymph node metastasis and poor survival in gallbladder cancer patients. Overexpression and deletion in vitro validated MBD1 as a potent oncogene promoting malignant behaviors in gallbladder cancer cells, including invasion, proliferation and migration, as well as epithelial–mesenchymal transition. Studies have demonstrated that epithelial–mesenchymal transition is common in gallbladder cancer, and it is well known that drug resistance and epithelial–mesenchymal transition are very closely correlated. Herein, our data show that targeting MBD1 restored gallbladder cancer cell sensitivity to gemcitabine chemotherapy.ConclusionsTaken together, the results of our study revealed a novel function of MBD1 in gallbladder cancer tumor development and progression through participation in the gallbladder cancer epithelial–mesenchymal transition program, which is involved in resistance to gemcitabine chemotherapy. Thus, MBD1 may be a potential therapeutic target for gallbladder cancer.

Upregulation of eukaryotic translation initiation factor 4E associates with a poor prognosis in gallbladder cancer and promotes cell proliferation in vitro and in vivo.

Eukaryotic translation initiation factor 4 (eIF4E) has been demonstrated to promote tumorigenesis in different types of cancer; however, whether eIF4E is involved in the development of GBC is unclear. The present study aimed to explore the biological function of eIF4E in gallbladder cancer (GBC) and identified that the expression level of eIF4E was significantly increased in GBC tissues compared with that in normal gallbladder tissues. The overall survival (OS) was also shorter in the group of patients with GBC with increased eIF4E expression. Increased eIF4E was correlated with advanced stage and higher histologic grade. Knockdown of eIF4E significantly inhibited cell proliferation, colony formation and cell cycle-associated protein expression levels in 2 GBC cell lines. The weight of the tumors in the eIF4E knockdown group was remarkably decreased compared with the control group. It also was revealed that knockdown of eIF4E is associated with upregulating cyclin-dependent kinase inhibitor 1B and down-regulating the expression levels of cyclin E1 and cyclin D1 in vitro and in vivo. These data demonstrated that eIF4E is a novel prognostic marker in GBC and may serve a critical role in the regulation of cell proliferation.

Genetic Changes of P53 and Kras in Gallbladder Carcinoma in Kumaon Region of Uttarakhand.

Gallbladder carcinoma is a highly lethal but relatively rare neoplasm of the digestive tract. p53 mutations are one of the most frequent genetic alterations in human cancers and are thought to play a role in pathogenesis of several malignancies. Kras oncogene is responsible for high frequency recognized as an early event in pancreatic and colonic carcinogenesis. We investigated the genetic change of p53, Kras and histopathological changes in gallbladder cancer tissue samples. P53 mutation was seen in the axons 5, 6, 7 and 8 of p53 gene and Kras codon 12 mutations in 25 operative specimens of gallbladder carcinoma. The hispathology observations and polymerase chain reaction-based techniques of these patients were used for point mutations study in p53 and in codon 12 of Kras gene. Mutations of p53 analyzed from exons 5 to 8 using the method of PCR-SSCP were 44%, PCR-RFLP was carried out, and incidence of mutation in codon 12 of Kras was 48% in the adenocarcinoma patients. There was a significant correlation between presence of gall stone, histopathological type, cellular differentiation, grade, lymphovascular invasion, perineural invasion, lymph node invasion, involvement of cystic duct end margin, liver invasion, omental tissue invasion, and T.N.M. staging. Curative surgical resection remains the only effective approach for treatment of GBC. Studies with large number of cases with latest application, next-generation sequencing, DNA microarray, transcriptome analysis, and real-time PCR would help in exploring more targets and better classification of these cancers at genetic level.

P-129 - Expression of female sex hormone receptors, connective tissue growth factor, and HER2 in gallbladder cancer and adjacent normal tissue

P-129 - Expression of female sex hormone receptors, connective tissue growth factor, and HER2 in gallbladder cancer and adjacent normal tissue

Consistency in biomarkers expression between matched tissue microarray cores from primary gallblader and ovarian cancers

Introduction. Tissue microarray (TMA) technique has been widely used, especially in immunohistochemical assays of new prognostic and predictive markers. The main objections raised by its opponents are the small amount of sampled material and the associated risk of inadequate assessment of analysed expression, resulting from the potential heterogeneity of tumour tissue. Material and methods. This study evaluated the compatibility of biomarker expression in two independent tissue cores, 1.5 mm in diameter, obtained by TMA technique from patients with gallbladder cancer (ERb, cytoPgR, HER2, CTGF) and ovarian cancer (PTEN, BCL2, PIK3CA, IGF1R). Comparison of the expression of individual biomarkers between cores was performed using the intraclass correlation coefficient (ICC), assuming a kappa Results. Evaluation of biomarker expression in the primary tumour was performed in 60 patients with gallbladder cancer and in 64 patients with high-grade serous ovarian cancer. Additionally, in patients with follicular cancer, the expression of the tested markers was assessed in the epithelium free from neoplastic malignancy. In both tumours, a good or sufficient level of homogeneity was observed in the expression of the analysed biomarkers between tissue cores. The correlation coefficient for the expression of individual markers in gallbladder cancer and adhering healthy tissue was: 0.68 (95% CI: 0.53–0.79)/0.62 (95% CI: 0.39–0.78) for ERb, 0.44 (95% CI: 0.23–0.61)/0.77 (95% CI: 0.61–0.87) for cytoPgR, 0.77 (95% CI: 0.65–0.85)/0.66 (95% CI: 0.44–0.80) for HER2, and 0.68 (95% CI: 0.53–0.79)/0.62 (95% CI: 0.39–0.78) for CTGF. In patients with ovarian cancer, the correlation coefficient within the primary tumour was 0.82 (95% CI: 0.71–0.89) for PTEN, 0.84 (95% CI: 0.75–0.90) for BCL2, 0.71 (95% CI: 0.56–0.81) for PIK3CA, and 0.77 (95% CI: 0.65–0.85) for IGF1R. Conclusions. Tissue microarray technique allows reliable assessment of the expression of tissue biomarkers within the primary tumour of gallbladder cancer and ovarian cancer.

Long non-coding RNA GBCDRlnc1 induces chemoresistance of gallbladder cancer cells by activating autophagy

BackgroundGallbladder cancer is the most common biliary tract malignancy and not sensitive to chemotherapy. Autophagy is an important factor prolonging the survival of cancer cells under chemotherapeutic stress. We aimed to investigate the role of long non-coding RNAs (lncRNAs) in autophagy and chemoresistance of gallbladder cancer cells.MethodsWe established doxorubicin (Dox)-resistant gallbladder cancer cells and used microarray analysis to compare the expression profiles of lncRNAs in Dox-resistant gallbladder cancer cells and their parental cells. Knockdown or exogenous expression of lncRNA combined with in vitro and in vivo assays were performed to prove the functional significance of lncRNA. The effects of lncRNA on autophagy were assessed by stubRFP-sensGFP-LC3 and western blot. We used RNA pull-down and mass spectrometry analysis to identify the target proteins of lncRNA.ResultsThe drug-resistant property of gallbladder cancer cells is related to their enhanced autophagic activity. And we found a lncRNA ENST00000425894 termed gallbladder cancer drug resistance-associated lncRNA1 (GBCDRlnc1) that serves as a critical regulator in gallbladder cancer chemoresistance. Furthermore, we discovered that GBCDRlnc1 is upregulated in gallbladder cancer tissues. Knockdown of GBCDRlnc1, via inhibiting autophagy at initial stage, enhanced the sensitivity of Dox-resistant gallbladder cancer cells to Dox in vitro and in vivo. Mechanically, we identified that GBCDRlnc1 interacts with phosphoglycerate kinase 1 and inhibits its ubiquitination in Dox-resistant gallbladder cancer cells, which leads to the down-regulation of autophagy initiator ATG5-ATG12 conjugate.ConclusionsOur findings established that the chemoresistant driver GBCDRlnc1 might be a candidate therapeutic target for the treatment of advanced gallbladder cancer.

Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

BackgroundThe long non-coding RNA PVT1 (lncRNA PVT1) has been reported to act as an oncogenic regulator of several cancers. However, its expression and function in gallbladder cancer (GBC) remain largely unknown.MethodsIn situ hybridization (ISH) and quantitative real-time PCR (qPCR) were performed to detect the expression of PVT1 and miR-143 in GBC tissues and cell lines. Immunohistochemistry (IHC) assays were performed to assess the expression of the hexokinase 2 (HK2) protein. The relationships among PVT1, miR-143 and HK2 were evaluated using dual-luciferase reporter, RNA immunoprecipitation (RIP) and biotin pull-down assays. The biological functions of PVT1, miR-143 and HK2 in GBC cells were explored with cell counting kit 8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), colony formation, transwell, wound healing and glucose metabolism assays in vitro. For in vivo experiments, a xenograft model was used to investigate the effects of PVT1 and HK2 on GBC.ResultsPVT1 was upregulated in GBC tissues and cells and was positively associated with malignancies and worse overall survival. PVT1 knockdown inhibited cell proliferation, migration, and invasion in vitro and restrained tumor growth in vivo. Further studies demonstrated that PVT1 positively regulated HK2 expression via its competing endogenous RNA (ceRNA) activity on miR-143. Additionally, HK2 expression and function were positively correlated with PVT1. Furthermore, we observed that the PVT1/miR-143/HK2 axis promoted cell proliferation and metastasis by regulating aerobic glucose metabolism in GBC cells.ConclusionsThe results of our study reveal a potential ceRNA regulatory pathway in which PVT1 modulates HK2 expression by competitively binding to endogenous miR-143 in GBC cells, which may provide new insights into novel molecular therapeutic targets for GBC.

SLC25A22 promotes proliferation and metastasis by activating MAPK/ERK pathway in gallbladder cancer

BackgroundSLC25A22, a member of mitochondrial carrier system (MCS) family encoding a mitochondrial glutamate transporter, has been reported to have vital roles in promoting proliferation and migration in cancer. Gallbladder cancer (GBC) is the most common biliary tract malignancy and has a poor prognosis. We aimed to determine the expression and function of SLC25A22 in GBC.MethodsImmunohistochemistry (IHC) staining analysis and quantitative real-time PCR (qRT-PCR) were conducted to determine the expression of SLC25A22 in GBC tissues. Human NOZ and GBC-SD cells were used to perform the experiments. The protein expression was detected by western-blot analysis. Cell viability was evaluated via CCK-8 assay and colony formation assay. Cell migration and invasion in vitro were investigated by wound healing and transwell assay. Annexin V/PI staining assay for apoptosis were measured by flow cytometry. The effect of SLC25A22 in vivo was conducted with subcutaneous xenograft.ResultsWe indicated that the expression of SLC25A22 was significantly upregulated in GBC tumor tissues as well as cell lines. Downregulation of SLC25A22 inhibited GBC cell growth and proliferation in vitro and in vivo and also had an effect on metastasis of GBC cells through the EMT processes. In addition, inhibition of SLC25A22 promoted mitochondrial apoptosis via downregulating BCL-2 and upregulating cleaved PARP, Cytochrome-c, and BAX mediated by MAPK/ERK pathway.ConclusionsOur study identified that SLC25A22 promoted development of GBC activating MAPK/ERK pathway. SLC25A22 has a potential to be used as a target for cancer diagnosis of GBC and related therapies.

Tamoxifen Sensitizes Gallbladder Cancer to Cisplatin Resistance by Impaired Glucose Metabolism via Activation of AMPK Signaling

Purpose: Gallbladder cancer (GBC) is the leading malignancy of biliary system showing refractory chemoresistance to current first-line drugs. Growing epidemiological evidences have established that the incidence of GBC exhibits significant gender predominance with females 2-3 fold higher than males, suggesting estrogen/estrogen receptors (ERs) signaling might be a critical driver of tumorigenesis in gallbladder. This study aims to evaluate the antitumor activity of tamoxifen (TAM), a major agent of hormonal therapy for breast cancer, in preclinical GBC model. Experimental Design: The antitumor activity of TAM alone or with cisplatin was examined using cell viability, apoptosis and in vivo models. Results: Increased ERɑ expression was found in GBC tissues and predicted poor clinical outcomes. TAM was showed effective against a variety of GBC cell lines. Mechanical investigations revealed that TAM enabled potent reactive oxygen species (ROS) production by reduced nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression and its target genes, leading to the activation of Adenosine Monophosphate Activated Protein Kinase (AMPK), which subsequently induced impaired glycolysis and survival advantages. Moreover, the inhibitory effect of TAM for GBC cells was remarkably attenuated by N-acetyl-L-cysteine (NAC) and AMPK inhibitor, suggesting ROS/AMPK axis was required for TAM tumor-suppressive function. Notably, TAM was demonstrated to sensitize GBC cells to cisplatin (CDDP) both in vitro and in vivo. In agreement with these findings, elimination of estrogens by ovariectomy in nude mice prevented CDDP resistance. Conclusions: These results provide basis for TAM-mediated chemosensitivity for GBC and shed novel light on the potential application of endocrine therapy for patients with GBC. Funding Statement: This work was supported by National Science Foundation of China (81472240，81272748 and 81072011)，Science and Technology Commission of Shanghai Municipality (16411952700, 10411955400, and 09411960800), Program for Outstanding Academic Leader of Shanghai (2016, JW) Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The collection and analysis of patient samples were approved by the Ethical Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine, and written informed consent was obtained from all patients. All methods and experiments were carried out in accordance with the approved guidelines and regulations. Animal maintenance and experimental procedures were strictly performed following the guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University and approved by IACUC committee of Shanghai Jiao Tong University

Effects of claudin‑1 downregulation on the physiological processes of gallbladder cancer SGC996 cells

Gallbladder cancer has a high recurrence and mortality rate, with limited treatment options. Therefore, elucidating the underlying molecular mechanisms of this disease would be beneficial to achieve an earlier diagnosis and potentially identify novel treatment targets. Claudin-1 is a tight junction protein associated with the development and prognosis of several types of cancer, and our preliminary studies have demonstrated that claudin-1 expression is elevated in gallbladder cancer tissues. Therefore, the aim of the present study was to investigate the effects of downregulating claudin-1 on the physiological processes of gallbladder cancer cells. The gallbladder cancer SGC996 cell line was transfected with claudin-1-RNA interference lentivirus (LV-CLDN1-RNAi) to downregulate claudin-1 expression, and the downstream effects on cell proliferation, the cell cycle, apoptosis and cell invasion were investigated. Following transfection with LV-CLDN1-RNAi, the results of an MTT assay revealed that downregulating claudin-1 did not affect the proliferation of the SGC996 cells. However, flow cytometry analysis demonstrated that the number of cells arrested in the G1 phase increased significantly, whereas the amount of cells arrested in the S phase was significantly reduced. Annexin V-APC single-color staining demonstrated that downregulating claudin-1 expression increased the ratio of cell apoptosis, which was confirmed by the results of western blot analysis, in which levels of the pro-apoptotic B-cell lymphoma 2 (Bcl-2)-associated X protein and anti-apoptotic Bcl-2 protein were increased and decreased, respectively. Finally, a Transwell assay indicated that claudin-1 downregulation inhibited cell invasion. Overall, the results from the present study indicated that downregulating claudin-1 expression promoted the apoptosis of gallbladder cancer cells and inhibited cell invasion, indicating that claudin-1 may be involved in the recurrence and metastasis of gallbladder cancer. These insights provide theoretical and experimental foundations for considering claudin-1 as a novel target for the treatment of gallbladder cancer.

CCL18 secreted from M2 macrophages promotes migration and invasion via the PI3K/Akt pathway in gallbladder cancer.

The presence of M2 macrophages within primary tumors has been correlated with a poor prognosis for many types of cancer. However, little is known about the role of M2 macrophages in gallbladder cancer (GBC). The number of M2 macrophages in 78 GBC and 16 normal gallbladder tissue samples was assessed by immunohistochemistry. The THP-1 monocyte cell line was differentiated into M2 macrophages and co-cultured with GBC-derived cell lines. The effect of M2 macrophages on promoting GBC cell migration and invasion was analyzed using migration, invasion and scratch wound healing assays. Western blotting and real-time PCR were used to assess the expression of epithelial-mesenchymal transition (EMT) markers and the activation status of the PI3K/Akt signaling pathway in GBC cells co-cultured with THP-1-derived macrophages. The average number of M2 macrophages was found to be significantly higher in GBC tissues than in normal gallbladder tissues. We also found that GBC patients with higher M2 macrophage counts exhibited poorer overall survival rates. Co-culture with M2 macrophages significantly promoted the migration, invasion and EMT of GBC cells. Moreover, we found that CCL18 secreted from M2 macrophages had the same effect on GBC cells as M2 macrophages. Blocking the function of CCL18 with a neutralizing antibody reversed this effect. Finally, we found that M2 macrophages could activate PI3K/Akt signaling in GBC cells, thereby leading to migration, invasion and EMT of these cells. Our findings contribute to our understanding of the role of chronic inflammation in GBC development and progression, and may offer potential therapeutic targets for GBC.

Long noncoding RNA MEG3 regulates LATS2 by promoting the ubiquitination of EZH2 and inhibits proliferation and invasion in gallbladder cancer

Gallbladder cancer (GBC) is the most common type of biliary tract cancer worldwide. Long noncoding RNAs (lncRNAs) play essential roles in physiological and pathological development. LncRNA MEG3, a tumor suppressor, has been reported to play important roles in some cancers, but the role of MEG3 in GBC remains largely unknown. The purpose of the present study was to explore the role of MEG3 in proliferation and invasion and the potential molecular mechanism in GBC. We found that MEG3 was downregulated in GBC tissues and cells, and low expression of MEG3 was correlated with poor prognostic outcomes in patients. Overexpression of MEG3 inhibited GBC cell proliferation and invasion, induced cell apoptosis and decreased tumorigenicity in nude mice. Moreover, we found that MEG3 was associated with EZH2 and attenuated EZH2 by promoting its ubiquitination. Furthermore, MEG3 executed its functions via EZH2 to regulate the downstream target gene LATS2. Taken together, these findings suggest that MEG3 is an effective target for GBC therapy and may facilitate the development of lncRNA-directed diagnostics and therapeutics against GBC.

Downregulation of PIM1 regulates glycolysis and suppresses tumor progression in gallbladder cancer.

BackgroundPIM1, a serine/threonine kinase, plays an essential role in tumorigenesis of multiple types of tumors. However, the expression pattern and functions of PIM1 in gallbladder cancer (GBC) remain largely unknown.Materials and methodsImmunohistochemistry, quantitative real-time PCR, and western blot analysis were performed to measure the expression of PIM1. Tissue microarray analysis was used to confirm the relationship between PIM1 expression and clinical outcomes of GBC patients. Finally, in vivo and in vitro functional studies were performed to detect the inhibition of PIM1 by RNAi or specific inhibitor in GBC cells.ResultsWe observed that PIM1 was dramatically overexpressed in GBC tissues, and its expression levels were positively related with clinical malignancies and a poor prognosis. Inhibition of PIM1 via RNAi or enzyme-specific inhibitor could suppress GBC cell proliferation, migration, and invasion both in vitro and vivo. Additionally, flow cytometry assays and cell cycle assays indicated that PIM1 inhibition promoted cell apoptosis and induced cell cycle arrest. Remarkably, inhibition of PIM1 could drive a metabolic shift from aerobic glycolysis to oxidative phosphorylation. We found that inhibition of PIM1 mechanistically reduced glucose consumption by regulating key molecules in aerobic glycolysis.ConclusionPIM1 may serve as an oncogene in GBC and be involved in the regulation of glycolysis. PIM1 is a promising therapeutic target for the treatment of human GBC.

Metformin suppresses hypoxia‑induced migration via the HIF‑1α/VEGF pathway in gallbladder cancer in�vitro and in�vivo

Hypoxia plays a crucial role in cancer development and progression. Overexpression of hypoxia-inducible factor-1α (HIF‑1α) has been demonstrated in a hypoxic microenvironment in various tumor types. Metformin has been identified as an antitumor drug in various tumor types. However, its role in cellular migration in a hypoxic microenvironment, and the associated regulatory mechanism, have yet to be fully elucidated. The present study aimed to investigate the clinical significance of HIF‑1α, and its biological role, in gallbladder cancer (GBC). Furthermore, the role of metformin in cellular migration, and its underlying mechanism in GBC, were also identified. Real‑time quantitative polymerase chain reaction analysis and immunohistochemistry experiments revealed that HIF‑1α was significantly upregulated in GBC tissues. HIF‑1α overexpression was closely associated with lymph node metastasis and tumor‑lymph node‑metastasis (TNM) stages. HIF‑1α was able to promote cell migration in a hypoxic microenvironment by overexpressing vascular endothelial growth factor (VEGF) in GBC‑SD cells, an effect which was partly reversed by small‑interfering RNA HIF‑1α (siHIF‑1α) and 2‑methoxyestradiol. Further experiments demonstrated that metformin inhibited hypoxia‑induced migration via HIF‑1α/VEGF invitro. In addition, metformin suppressed GBC growth and downregulated the expression of HIF‑1α and VEGF in a GBC‑SD cell xenograft model. Taken together, these results suggest that HIF‑1α may contribute to tumor migration via the overexpression of VEGF in GBC, while metformin is able to inhibit tumor migration by targeting the HIF‑1α/VEGF pathway.

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer.

Kindlin-2 promotes gallbladder cancer metastasis and invasion by inducing epithelial-mesenchymal transition

Objective: To investigate the effects of Kindlin-2 on malignant phenotypes of human gallbladder cancer cells and discuss the mechanisms. Methods: The expression level of Kindlin-2 in 30 cases of gallbladder cancer tissues and adjacent non-tumoral tissues collected from the First Affiliated Hospital of Zhengzhou University between September 2012 and May 2013 was assessed by real-time PCR and immunohistochemistry.Lentivirus-mediated Kindlin-2 overexpression was used in gallbladder cancer cell lines GBC-SD and SGC-996.Transwell assay and adhesion assay were investigated to explore the functional role of Kindlin-2 on gallbladder cancer cells.Western Blot was used to test the protein change of epithelial-mesenchymal transition(EMT) characteristics. The t-test was used to analyzed results. Results: The RNA and protein levels of Kindlin-2 in gallbladder cancer tissues were higher than in the non-tumoral tissues (t=4.372, P=0.001; t=7.477, P=0.000). The expression level of Kindlin-2 in gallbladder cancer tissues was correlated with Nevin stage(χ(2)=5.932, P=0.035). Compared with control groups, the cell-matrix adhesion ability of GBC-SD and SGC-996 with Kindlin-2 overexpression was obviously promoted(1.66±0.03 vs. 1.07±0.22, t=2.710, P=0.041; 2.66±0.24 vs. 1.03±0.02, t=6.610, P=0.020). The number of GBC-SD and SGC-996 cells with Kindlin-2 overexpression passing through the Transwell chamber matrix increased significantly compared with the control groups(116.1±13.9 vs. 54.7±8.4, t=3.781, P=0.019; 136.3±7.5 vs. 64.3±6.4, t=7.302, P=0.002). The wound healing rate of GBC-SD with Kindlin-2 overexpression at 12-hour and 24-hour was higher than that of the group ((42.9±2.2)% vs. (29.7±1.7)%, t=4.690, P=0.009; (65.0±2.4)% vs.(40.4±2.0)%, t=7.945, P=0.001). The wound healing rate of SGC-996 with Kindlin-2 overexpression at 12-hour and 24-hour was also higher than that of the group ((32.9±1.3)% vs. (24.1±1.5)%, t=4.518, P=0.011; (51.3±1.1)% vs. (39.2±1.1)%, t=8.001, P=0.001). The characteristics of EMT were induced in gallbladder cancer cells with Kindlin-2 overexpression, including the up-regulation of N-cadherin, Vemintin and the down-regulation of E-cadherin. Conclusion: The expression of Kindlin-2 is up-regulated in gallbladder cancer tissues and Kindlin-2 promoted the malignant phenotypes of gallbladder cancer cells partially by epithelial-mesenchymal transition.