Gallbladder Carcinoma Cells Research Articles

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.

Gene silencing of heparanase results in suppression of invasion and migration of gallbladder carcinoma cells.

This study investigated the effect of transcriptional gene silencing of the heparanase gene on standard gallbladder carcinoma cells (GBC-SD). The miRNAs targeting the promoter region and coding region of the heparanase gene were designed and synthesized. We transfected four recombinant miRNA vectors into GBC-SD. We performed the wound healing assays and invasion assays. The result shows that the heparanase expression was significantly decreased by recombinant vectors in transfected GBC-SD cells (p<0.01), of which pmiR-Hpa-2 showed best interference effect (p<0.05). The penetrated and migrating cells numbers and adherence rate of GBC-SD cells were significantly decreased by pmiR-Hpa-2 (p<0.05).

Tumor suppressor LKB1 inhibits the progression of gallbladder carcinoma and predicts the prognosis of patients with this malignancy.

Gallbladder carcinoma(GBC) represents the most common fatal tumors of the biliary tract. The 3-year or 5-year survival rate for patients with this disease are 30 and 5%, respectively. Liver kinase B1 (LKB1), a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK) necessary for maintaining cell metabolism and energy homeostasis, has been found to be an important tumor suppressor gene in recent years, and its inactivation has also found to be closely associated with tumor growth, metastasis and cancer stem cell (CSC) proliferation. Nevertheless, the function of LKB1 in GBC remains unclear. In this study, we found that the expression of LKB1 in GBC tissues was decreased compared with that in non-cancerous tissues. LKB1 overexpression suppressed the proliferation, metastasis and expansion of GBC CSCs. Mechanically, LKB1 suppressed GBC cell progression via the JAK/signal transducer and activator of transcription 3 (STAT3) pathway. The use of the JAK2 inhibitor, AZD‑1480, attenuated the suppressive effects of LKB1 overexpression on the growth, metastasis and self-renewal ability of the GBC cells, which further demonstrated that JAK/STAT3 was involved in the LKB1-induced suppression of GBC cell growth, metastasis and self-renewal ability. More importantly, the decreased expression of LKB1 was a predictor of a poor prognosis of patients with GBC. On the whole, our data indicate that LKB1 inhibits GBC cell growth, metastasis and self-renewal ability by disrupting JAK/STAT3 signaling, and may thus prove to be a novel prognostic biomarker for patients with GBC.

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...

Effect of CD147 inhibitor tunicamycin and activator angiotensin II on gallbladder carcinoma cells

Objective To research the effect of CD147 inhibitor tunicamycin and activator angiotensin Ⅱ (AngⅡ) on proliferation, invasion, and expression of CD147 and matrix metalloproteinase (MMP)-9 in GBC-SD cells. Methods GBC-SD cells were treated with different concentrations of tunicamycin (5, 10, 20 μl/ml), AngⅡ (5, 10, 20 μl/ml) alone or tunicamycin plus AngⅡ (tunicamycin 10 μl/ml + AngⅡ 20 μl/ml). The rate of proliferation was detected by cell counting kit-8 (CCK-8). The expression of CD147 and MMP-9 was detected by Western blotting. The ability of invasion was examined by Transwell assay. Results (1) As compared with the control group, proliferation rate of GBC-SD in tunicamycin group was significantly decreased in a time- and concentration-dependent manner [(88.17±8.36)% vs. (100.00±0.00)%, P=0.005; (85.35±6.42)% vs. (100.00±0.00)%, P=0.003; (70.15±6.89)% vs. (100.00±0.00)%, P=0.000]. As compared with the control group, the CD147 value of K was significantly decreased (0.75±0.04 vs. 0.88±0.07, P=0.046; 0.71±0.04 vs. 0.88±0.07, P=0.009), and MMP-9 value of K was also decreased (0.84±0.04 vs. 1.10±0.08, P=0.004; 0.67±0.10 vs. 1.10±0.08, P=0.000) in a concentration-dependent manner in in tunicamycin group. The invasive ability in tunicamycin group was significantly decreased as compared with control group (43.00±10.98 vs. 67.65±8.65, P=0.034; 32.46±7.71 vs. 67.65±8.65, P=0.005). (2) The cell proliferation rate in AngⅡ group was gradually increased in a time- and concentration-dependent manner [(104.32±5.05)% vs. (100.00±0.00)%, P=0.056; (108.25±6.57)% vs. (100.00±0.00)%, P=0.019; (115.50±8.65)% vs. (100.00±0.00)%, P=0.003], the CD147 value of K increased in a concentration-dependent manner (1.02±0.08 vs. 0.88±0.07, P=0.039; 1.08±0.05 vs. 0.88±0.07, P=0.005), MMP-9 value of K increased (1.28±0.06 vs. 1.10±0.08, P=0.034; 1.57±0.07 vs. 1.10±0.08, P=0.001), and the invasion ability increased (98.00±12.56 vs. 67.65±8.65, P=0.013) as compared with the control group. There was significant difference in cell invasion, CD147 expression and MMP-9 expression between the combined group and the control group. Conclusion Tunicamycin can inhibit the proliferation of gallbladder cancer cells, reduce the expression of CD147 and MMP-9, and AngⅡcan accelerate the proliferation of gallbladder cancer cells, and increase the expression of CD147 and MMP-9. The combined use of tunicamycin and AngⅡ can counteract the interaction. Key words: Gallbladder carcinoma cell; Tunicamycin; Angiotensin Ⅱ; CD147; Matrix metalloproteinase-9

RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway.

BackgroundTumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear.MethodsThe RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover, VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction, VEGF-C protein levels were measured by immunoblotting and enzyme-linked immunosorbent assay, and VEGF-C promoter and NF-κB activities were quantified using a dual luciferase reporter assay. The association of NF-κB with the VEGF-C promoter was analysed by chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells. The expression of RIP1 protein, TNF-α protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-α protein and lymphatic vessel density was analysed.ResultsTNF-α dose- and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated VEGF-C expression at the protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α protein and the lymphatic vessel density in GBC tissues.ConclusionWe conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway.

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.

Oxymatrine induces apoptosis and inhibits invasion in Gallbladder carcinoma via PTEN/PI3K/AKT pathway.

Oxymatrine extracted from Sophora flavescens Ait as a natural polyphenolic phytochemical has been demonstrated to exhibit anti-tumor effects on various cancers, including Gallbladder carcinoma (GBC). However, its underlying mechanisms of function are largely unknown in GBC cells. The present study is conducted to investigate the anti-tumor effects and the underlying mechanisms of oxymatrine on GBC cells in vitro and in vivo. The results showed that oxymatrine inhibited cell viability, metastatic ability and induced cell apoptosis in dose-dependent manners. Furthermore, we found that the expression of p-AKT, MMP-2, MMP-9 and the ratio of Bcl-2/Bax were significantly down-regulated, while the expression of PTEN was up-regulated in GBC cells. In addition, pretreatment with a specific PI3K/AKT activator (IGF-1) significantly antagonized the oxymatrine-mediated inhibition of GBC-SD cells. Subsequently, our in vivo studies showed that administration of oxymatrine induced a significant dose-dependent decrease in tumor growth. In conclusion, these findings indicated that the inhibition of cells proliferation, migration, invasion and the induction of apoptosis in response to oxymatrine in GBC cells, may function through the suppression of PTEN/PI3K/AKT pathway, which was considered as the vital signaling pathway in regulating tumorigenesis. These results suggested that oxymatrine might be a novel effective candidate as chemotherapeutic agent against GBC.

Targeting of NT5E by miR-30b and miR-340 attenuates proliferation, invasion and migration of gallbladder carcinoma

MicroRNAs (miRNAs) have been closely associated with the proliferation, invasion and migration of various cancers, including gallbladder carcinoma (GBC). Previous studies have revealed dysregulation of miR-30b and miR-340 in many types of cancer. However, the role of miR-30b and miR-340 in the development and progression of GBC remains unclear. Moreover, epithelial-to-mesenchymal transition (EMT) has been gradually viewed as a significant contributor to tumor metastasis. In this study, the cell line GBC-SD was used and we explored that EMT promoted GBC cells invasion and migration and inhibited the expression level of miR-30b and miR-340 compared with the control. We showed that overexpression of miR-30b and miR-340 suppressed GBC cells proliferation, invasion and migration, as well as the expression of EMT-associated genes. In addition, we identified ecto-5′-nucleotidase (NT5E) as a common target of miR-30b and miR-340 using bioinformatics analysis and a luciferase assay. Further experiments found that exogenous expression of NT5E in GBC cells could partially reverse the inhibitory effect of miR-30b and miR-340 on cell proliferation, invasion and migration. Our findings suggest that NT5E-targeting miRNAs (miR-30b and miR-340) function as tumor suppressors and may represent promising therapeutic targets for GBC.

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.

Effect of CD147 antagonist peptide and doxycycline on gallbladder carcinoma cells

Objective To research the effect of GBC-SD on proliferation, apoptosis, expression of CD147 and activity of matrix metalloproteinase (MMP)-9 in gallbladder carcinoma cells, with single and combination therapy of CD147 antagonist peptide and doxycycline. Methods Gallbladder carcinoma cells were treated with antagonist peptide (AP-9) (50, 100, 200, 400 μg/ml) or DOX (5, 10, 20, 40 μg/ml) alone and combination therapy (AP-9 200 μg/ml + DOX 20 μg/ml) for 12, 24 and 48 h, then the inhibition rate of proliferation was detected by cell counting kit-8 (CCK-8), expression of CD147 was detected by Western blotting, the activity of MMP-9 was detected by gelatin zymography, and the early apoptosis rate was detected by flow cytometry with Annexin V/propidium iodide (PI) staining. Results After treatment with several concentrations of AP-9, the apoptosis rate [(2.13±0.10)% and (2.92±0.19)%] was higher (P=0.002, P=0.001) than control group [(1.58±0.08)%]; the activity of MMP-9 [(320.08±33.90), (249.59±33.63), (259.63±34.08) and (172.31±35.20)] was reduced (P=0.013, 0.002, 0.003 and 0.001 respectively) as compared with that in control group; and cell proliferation was inhibited as compared with that in control group (P=0.001). After treatment with several concentrations of doxycycline, the apoptosis rate [(3.25±0.12)% and (3.63±0.24)%] was higher (P=0.001) than in the control group [(1.58±0.08)%]; the activity of MMP-9 [(203.52±18.25), (175.22±23.30), (147.86±22.83) and (109.99±29.22)] was reduced (P=0.001) as compared with control group [(446.08±42.42)]; cell proliferation was inhibited as compared with control group (P=0.001); the expression level of CD147 [(0.97±0.12), (0.80±0.10), (0.69±0.11) and (0.51±0.12)] was reduced (P=0.001) as compared with cotrol group [(2.04±0.15)]. As compared with treatment alone, cell proliferation [(38.78±0.39)%, (43.76±0.71)% and (52.00±0.91)%] was increased (P=0.001, P=0.003), the activity of MMP-9 [(110.19±24.74)] was reduced (P=0.001, P=0.024), and the apoptosis rate [(3.73±0.29)%] was elevated after combination therapy (P=0.001, P=0.048). Conclusion DOX can down-regulate the expression of CD147. AP-9 and DOX alone or in combination can inhibit the proliferation of gallbladder cancer cells, inhibit the activity of MMP-9, and promote apoptosis, and the inhibitory effect was enhanced after combination therapy. Key words: Gallbladder carcinoma; CD147; Extracellular matrix metalloproteinase-9; Antagonist peptide; Doxycycline

Heparanase overexpression down-regulates syndecan-1 expression in a gallbladder carcinoma cell line.

ObjectiveTo discuss the relevance of heparanase and syndecan-1 and regulation of the heparanase-syndecan1 axis in the invasiveness of gallbladder carcinoma cells.Methods1. Generation of a gallbladder cancer cell line overexpressing a heparanase (GBD-SD) transgene. 2. Western blot analysis of syndecan-1 levels of GBD-SD and control gallbladder carcinoma (GBC-SD) cells. 3. RT-PCR analysis of syndecan-1 mRNA levels of GBD-SD and GBC-SD. 4. Evaluation of invasion and migration of GBD-SD and GBC-SD cells.Results1. Heparanase expression in GBD-SD cells was significantly increased. 2. The syndecan-1 mRNA level of GBD-SD cells was significantly lower compared with that of GBC-SD cells. 3. The syndecan-1 DNA copy number in GBD-SD cells was significantly lower compared with that of GBC-SD. 4. The invasiveness and migration of GBD-SD cells were significantly higher compared with GBC-SD cells.Conclusions1. The expression of heparanase negatively correlated with that of syndecan-1 in a gallbladder carcinoma cell line. 2. The expression of heparanase and syndecan-1 in gallbladder carcinomas negatively correlated, similar to other tumours. 3. The heparanase/syndecan1 axis in gallbladder carcinoma plays an important role in the invasion and metastasis, thus providing a new therapeutic target. 4. Further research is required to identify the detailed mechanisms.

Combination of celecoxib and PD184161 exerts synergistic inhibitory effects on gallbladder cancer cell proliferation.

Cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) may serve as potential targets in various types of cancer; however, the roles of these proteins in gallbladder carcinoma (GBC) have not been reported previously. In the present study, the expression levels of COX-2 and phospho (p)-ERK1/2 in GBC were examined and the biological activities of celecoxib and PD184161 (specific inhibitors of COX-2 and p-ERK1/2, respectively) on the proliferation, cell cycle and apoptosis of the GBC-SD and NOZ human GBC cell lines were evaluated by a series of in vitro and in vivo studies. COX-2 and p-ERK1/2 protein expression levels were found to be significantly elevated in GBC tissues as well as in GBC-SD and NOZ cells. Treatments with celecoxib and PD184161 significantly inhibited GBC-SD and NOZ cell growth in a concentration-dependent manner, and their combination produced a synergistic inhibitory effect. In addition, celecoxib and PD184161 significantly inhibited tumor growth in xenograft nude mice. Celecoxib treatment led to G1 arrest via the upregulation of p21 and p27 expression in GBC-SD and NOZ cells, whereas PD184161 did not affect cell cycle distribution. The combination of celecoxib and PD184161 was able to promote cell apoptosis by triggering a collapse of mitochondrial membrane potential and activating caspase-3-mediated apoptosis. In conclusion, COX-2 and p-ERK1/2 protein may serve as potential targets for GBC chemotherapy, and the combination of celecoxib and PD184161 could significantly inhibit GBC cell growth, induce cell G1 arrest and trigger cell apoptosis of GBC cells.

Upregulation of long non-coding RNA HOXA-AS2 promotes proliferation and induces epithelial-mesenchymal transition in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most common malignancy of the bile duct and patients with GBC have extremely poor prognoses. Increasing evidence indicates that long non-coding RNAs (lncRNAs) regulate diverse cellular processes, including cell growth, differentiation, apoptosis, and cancer progression. However, the function of lncRNAs in the progression of GBC remains largely unknown. Here, we reported that HOXA cluster antisense RNA2 (HOXA-AS2) was upregulated in GBC. In vitro experiments revealed that HOXA-AS2 knockdown significantly inhibited GBC cells proliferation by causing G1 arrest and promoting apoptosis, whereas HOXA-AS2 overexpression promoted cell growth. Further functional assays indicated that HOXA-AS2 overexpression significantly promoted GBC cell migration and invasion by promoting EMT. Taken together, our study demonstrates that HOXA-AS2 could act as a functional oncogene in GBC, as well as a potential therapeutic target to inhibit GBC metastasis.

Influence of Yes-associated protein-1 on proliferation and metastasis of gallbladder carcinoma

Objective To investigate the expressions of Yes-associated protein-1 (YAP1) in gallbladder mucosal epithelium of normal persons, in patients with simple/calculous cholecystitis, and in patients with gallbladder carcinoma; and to study the mechanism of YAP1 in gallbladder carcinoma development. Methods Immunohistochemistry was used to detect the expression and distribution of YAP1 protein in 50 persons with normal gallbladder, 101 patients with simple cholecystitis/calculous cholecystitis and 100 patients with gallbladder carcinoma. RT-PCR and western-blot were used to detect the mRNA and protein levels of YAP1 in normal and malignant gallbladder mucosal epithelium cells. siRNA was used to shut down the expression of YAP1 in SGC996 cells. MTT was used to test cell vitality. Flow cytometry was used to measure cell cycle. Results Immunohistochemistry revealed the expression rates of YAP1 in the gallbladder carcinoma group, the cholecystitis/gallstone group and the control group to be 87.0% (87/100), 56.4% (57/101) and 5.0% (1/20), respectively (P<0.01). The YAP1 protein levels were higher in gallbladder carcinoma tissues and cells when compared to normal tissues and cells. RT-PCR showed the mRNA levels of gallbladder carcinoma cells to be 12.5±1.2 times of normal gallbladder mucosal epithelial cells (P<0.05). After using siRNA to shut down the YAP1 expression, EMT associated proteins were down-regulated, cell vitality was decreased, and cell cycle was arrested in the S-phase. Conclusions YAP1 is closely related to cell proliferation and metastasis of gallbladder carcinoma. It may promote tumor progression through epithelial-mesenchymal transition. Key words: Primary gallbladder carcinoma; Yes-associated protein 1; Epithelial-mesenchymal transition; Tumor progress

Long non-coding RNA TUG1 promotes cell proliferation and metastasis by negatively regulating miR-300 in gallbladder carcinoma.

As we all know, long non-coding RNAs (lncRNAs) have been reported to play vital roles in various human cancers. In this study, we aimed to explore the role of lncRNA TUG1 in gallbladder carcinoma (GBC) development. Total RNA was extracted from the tissues of thirty GBC patients, four GBC cell lines. We detected the expression levels of TUG1 using quantitative real-time PCR. We performed CCK8, colony formation, transwell invasion and apoptosis assays to study the effects of TUG1 on GBC cell proliferation and invasion. Western blot assay was performed to assess to the expression level of epithelial-mesenchymal transition (EMT) markers in transforming growth factor-β1 (TGF-β1) treated and TUG1 knockdown GBC cell. Lastly, dual-luciferase reporter assay and quantitative real-time PCR were performed to verify the potential target microRNAs (miRNAs) of TUG1. TUG1 expression was significantly overexpressed in GBC tissues. Functionally, this study demonstrated that knockdown of TUG1 significantly inhibited GBC cell proliferation, metastasis. Mechanically, we found that TUG1 is upregulated by TGF-β1, and knockdown of TUG1 inhibited GBC cell EMT. Furthermore, we identified that miR-300, which has been reported as a suppressor in other types of cancer, is negatively regulated by TUG1. LncRNA TUG1 promotes GBC cell proliferation, metastasis and EMT progression by functioning as a miRNA sponge to abrogate the endogenous effect of miR-300.

Stathmin decreases cholangiocarcinoma cell line sensitivity to staurosporine-triggered apoptosis via the induction of ERK and Akt signaling.

Cholangiocarcinoma is a rare, but highly fatal malignancy. However, the intrinsic mechanism involved in its tumorigenesis remains obscure. An urgent need remains for a promising target for cholangiocarcinoma biological therapies. Based on comparative proteomical technologies, we found 253 and 231 different spots in gallbladder tumor cell lines and cholangiocarcinoma cell lines, respectively, relative to non-malignant cells. Using Mass Spectrometry (MS) and database searching, we chose seven differentially expressed proteins. High Stathmin expression was found in both cholangiocarcinoma and gallbladder carcinoma cells. Stathmin expression was validated using immunohistochemistry and western blot in cholangiocarcinoma tissue samples and peritumoral tissue. It was further revealed that high Stathmin expression was associated with the repression of staurosporine-induced apoptosis in the cholangiocarcinoma cell. Moreover, we found that Stathmin promoted cancer cell proliferation and inhibited its apoptosis through protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) signaling. Integrin, β1 appears to serve as a partner of Stathmin induction of ERK and Akt signaling by inhibiting apoptosis in the cholangiocarcinoma cell. Understanding the regulation of anti-apoptosis effect by Stathmin might provide new insight into how to overcome therapeutic resistance in cholangiocarcinoma.

A Microfluidic Chip for Cell Patterning Utilizing Paired Microwells and Protein Patterns

Cell patterning has been widely used in research on fundamental cell biology and in applications such as tissue engineering, neuron network formation, cell based biosensor and drug screening. Although various methods have been developed, cell patterning in an enclosed microfluidic device at single cell level remains challenging. This paper describes a microfluidic device with microwells and protein patterns paired together in a single microchannel for an easy cell patterning. Cells captured in the microwells were positioned directly onto the protein patterns within 5 min and the patterning performance was successfully demonstrated using HeLa cells and human gallbladder carcinoma cells (SGC-996). Cells survived for 6 days in the microchannel. Cell attachment, migration, proliferation and cell colony formation were observed. Our device is free of topographic constraint for the patterned cells and no complex chemical modification to the substrate is needed, offering a simple, fast, and easy-to-operate way of patterning cells at single cell level in an enclosed microfluidic channel.

Long non-coding RNA SPRY4-IT1 promotes gallbladder carcinoma progression.

Gallbladder carcinoma (GBC) is the most common malignancy of the bile duct and patients with GBC have extremely poor prognoses. Long non-coding RNAs (lncRNAs) are found to be dysregulated in a variety of cancers, including GBC. SPRY4-IT1 has been recently revealed as oncogenic regulator in many cancers. However, whether SPRY4-IT1 is involved in GBC progression remains largely unknown. To investigate the role of SPRY4-IT1 in GBC, we evaluated the expression SPRY4-IT1 in GBC tissues and cell lines, and investigated the effect of SPRY4-IT1 knockdown on cell proliferation, migration and invasion of GBC in vitro. Our result showed that SPRY4-IT1 was upregulated in GBC tissues. Further experiments revealed that SPRY4-IT1 knockdown significantly inhibited GBC cell proliferation. Furthermore, inhibitory effects of SPRY4-IT1 on cell migration and invasion were partly associated with EMT process. In conclusion, these data suggest that SPRY4-IT1 could be an oncogene for GBC, and may be served as a candidate target for new therapies in human GBC.