NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Ponicidin inhibited gallbladder cancer proliferation and metastasis by decreasing MAGEB2 expression through FOXO4.

BACKGROUND: Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC. METHODS: CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin. RESULTS: Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety. CONCLUSION: Ponicidin may be a promising agent for the treatment of GBC effectively and safely.

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

BACKGROUNDS: Long 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: Our 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.

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 ß-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.

MYBL2 is a Potential Prognostic Marker that Promotes Cell Proliferation in Gallbladder Cancer.

BACKGROUND: Gallbladder cancer (GBC) is an aggressive and highly lethal biliary tract malignancy, with extremely poor prognosis. In the present study, we analyzed the potential involvement of MYBL2, a member of the Myb transcription factor family, in the carcinogenesis of human GBC. METHODS: MYBL2 expression levels were measured in GBC and cholecystitis tissue specimens using quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) assays. The effects of MYBL2 on cell proliferation and DNA synthesis were evaluated using Cell Counting Kit-8 assay (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) retention assay, flow cytometry analysis, western blot, and a xenograft model of GBC cells in nude mice. RESULTS: MYBL2 expression was increased in GBC tissues and associated with histological differentiation, tumour invasion, clinical stage and unfavourable overall survival in GBC patients. The downregulation of MYBL2 expression resulted in the inhibition of GBC cell proliferation, and DNA replication in vitro, and the growth of xenografted tumours in nude mice. Conversely, MYBL2 overexpression resulted in the opposite effects. CONCLUSIONS: MYBL2 overexpression promotes GBC cell proliferation through the regulation of the cell cycle at the S and G2/M phase transitions. Thus, MYBL2 could serve as a potential prognostic and therapeutic biomarker in GBC patients.

MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway.

Gallbladder cancer (GBC) is a leading cause of cancer-related deaths worldwide, and its prognosis remains poor, with a 5-year survival rate of ~5%. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of the metastasis-associated miRNA miR-29c-5p in GBC.We validated that expression of miR-29c-5p was significantly downregulated in GBC and was closely associated with lymph node metastasis, overall survival and disease-free survival in 40 GBC patients who were followed clinically. Ectopic overexpression of miR-29c-5p dramatically repressed proliferation, metastasis, and colony formation and induced apoptosis in vitro, and it suppressed tumorigenicity in vivo through the MAPK pathway. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) was identified as a critical effector target of miR-29c-5p. Enforced expression of miR-29c-5p significantly inhibited the expression of CPEB4, and restoration of CPEB4 expression reversed the inhibitory effects of miR-29c-5p on GBC cell proliferation and metastasis. Transforming growth factor-ß (TGF-ß) upregulated CPEB4 by downregulating miR-29c-5p, leading to MAPK pathway activation. In conclusion, the TGF-ß/miR-29c-5p/CPEB4 axis has a pivotal role in the pathogenesis and poor prognosis of GBC, suggesting that miR-29c-5p is a tumor-suppressive miRNA that may serve as potential prognostic biomarker or therapeutic target for GBC.

Correction: Xiang, S., et al. Schisandrin B Induces Apoptosis and Cell Cycle Arrest of Gallbladder Cancer Cells. Molecules 2014, 19, 13235-13250.

We would like to change the Affiliation addresses on Page 13235 of paper [1], [...].

miR-101 targeting ZFX suppresses tumor proliferation and metastasis by regulating the MAPK/Erk and Smad pathways in gallbladder carcinoma.

Gallbladder cancer (GBC), the most common malignancy of the bile duct, is highly aggressive and has an extremely poor prognosis, which is a result of early metastasis. As it is regulated being at multiple levels, the metastatic cascade in GBC is complex. Recent evidence suggests that microRNAs (miRNAs) are involved in cancer metastasis and are promising therapeutic targets. In this study, miR-101 was significantly downregulated in tumor tissues, particularly in metastatic tissues. In GBC patients, low miR-101 expression was correlated with tumor size, tumor invasion, lymph node metastasis, TNM stage, and poor survival. Moreover, miR-101 was an independent prognostic marker for GBC. Additionally, miR-101 inhibited GBC cell proliferation, migration, invasion, and TGF-ß-induced epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the gene encoding the zinc finger protein X-linked (ZFX) was identified as a direct target of miR-101. More importantly, miR-101 significantly reduced activation of the MAPK/Erk and Smad signaling pathways, resulting in inhibition of TGF-ß-mediated induction of EMT. Altogether, our findings demonstrate a novel mechanism by which miR-101 attenuates the EMT and metastasis in GBC cells and suggest that miR-101 can serve as a potential biomarker and therapeutic target for GBC management.

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells.

Oleanolic acid (OA), a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma.

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway.

BACKGROUND: Gallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC. METHODS: SPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice. RESULTS: SPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC. CONCLUSIONS: SPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.

Baicalein inhibits progression of gallbladder cancer cells by downregulating ZFX.

Baicalein, a widely used Chinese herbal medicine, has multiple pharmacological activities. However, the precise mechanisms of the anti-proliferation and anti-metastatic effects of baicalein on gallbladder cancer (GBC) remain poorly understood. Therefore, the aim of this study was to assess the anti-proliferation and anti-metastatic effects of baicalein and the related mechanism(s) on GBC. In the present study, we found that treatment with baicalein induced a significant inhibitory effect on proliferation and promoted apoptosis in GBC-SD and SGC996 cells, two widely used gallbladder cancer cell lines. Additionally, treatment with baicalein inhibited the metastasis of GBC cells. Moreover, we demonstrated for the first time that baicalein inhibited GBC cell growth and metastasis via down-regulation of the expression level of Zinc finger protein X-linked (ZFX). In conclusion, our studies suggest that baicalein may be a potential phytochemical flavonoid for therapeutics of GBC and ZFX may serve as a molecular marker or predictive target for GBC.

Ursolic acid induces cell cycle arrest and apoptosis of gallbladder carcinoma cells.

BACKGROUND: Ursolic acid (UA), a plant extract used in traditional Chinese medicine, exhibits potential anticancer effects in various human cancer cell lines in vitro. In the present study, we evaluated the anti-tumoral properties of UA against gallbladder carcinoma and investigated the potential mechanisms responsible for its effects on proliferation, cell cycle arrest and apoptosis in vitro. METHODS: The anti-tumor activity of UA against GBC-SD and SGC-996 cells was assessed using MTT and colony formation assays. An annexin V/PI double-staining assay was used to detect cell apoptosis. Cell cycle changes were detected using flow cytometry. Rhodamine 123 staining was used to assess the mitochondrial membrane potential (ΔΨm) and validate UA's ability to induce apoptosis in both cell lines. The effectiveness of UA in gallbladder cancer was further verified in vivo by establishing a xenograft GBC model in nude mice. Finally, the expression levels of cell cycle- and apoptosis-related proteins were analyzed by western blotting. RESULTS: Our results suggest that UA can significantly inhibit the growth of gallbladder cancer cells. MTT and colony formation assays indicated dose-dependent decreases in cell proliferation. S-phase arrest was observed in both cell lines after treatment with UA. Annexin V/PI staining suggested that UA induced both early and late phases of apoptosis. UA also decreased ΔΨm and altered the expression of molecules regulating the cell cycle and apoptosis. In vivo study showed intraperitoneally injection of UA can significantly inhibited the growth of xenograft tumor in nude mice and the inhibition efficiency is dose related. Activation of caspase-3,-9 and PARP indicated that mitochondrial pathways may be involved in UA-induced apoptosis. CONCLUSIONS: Taken together, these results suggest that UA exhibits significant anti-tumor effects by suppressing cell proliferation, promoting apoptosis and inducing 7cell cycle arrest both in vitro and in vivo. It may be a potential agent for treating gallbladder cancer.

Cordycepin induces S phase arrest and apoptosis in human gallbladder cancer cells.

Gallbladder cancer is the most common malignant tumor of the biliary tract, and this condition has a rather dismal prognosis, with an extremely low five-year survival rate. To improve the outcome of unresectable and recurrent gallbladder cancer, it is necessary to develop new effective treatments and drugs. The purpose of the present study was to evaluate the effects of cordycepin on human gallbladder cells and uncover the molecular mechanisms responsible for these effects. The Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that cordycepin affected the viability and proliferation of human gallbladder cancer cells in a dose- and time-dependent manner. Flow cytometric analysis showed that cordycepin induced S phase arrest in human gallbladder cancer cell lines(NOZ and GBC-SD cells). Cordycepin-induced apoptosis was observed using an Annexin V/propidium iodide (PI) double-staining assay, and the mitochondrial membrane potential (ΔΨm) decreased in a dose-dependent manner. Additionally, western blot analysis revealed the upregulation of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP and Bax and the downregulation of Bcl-2, cyclin A and Cdk-2 in cordycepin-treated cells. Moreover, cordycepin inhibited tumor growth in nude mice bearing NOZ tumors. Our results indicate that this drug may represent an effective treatment for gallbladder carcinoma.

Clinical and prognostic significance of preoperative plasma hyperfibrinogenemia in gallbladder cancer patients following surgical resection: a retrospective and in vitro study.

BACKGROUND: Coagulation and fibrinolysis activation is frequently observed in cancer patients, and the tumors in these cases are thought to be associated with a higher risk of invasion, metastasis, and worse long-term outcome. The objective of this study was to elucidate the prognostic significance of blood coagulation tests and various clinicopathological characteristics in patients with gallbladder cancer (GBC) after surgical resection. METHODS: We retrospectively reviewed the medical records of 115 patients with histologically confirmed GBC who underwent surgical resection in our department. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), international normalized ratio (INR), fibrinogen levels, and platelet counts were measured pretreatment at the time of diagnosis. The predictive value of fibrinogen levels for tumor staging was evaluated using a receiver operating characteristic (ROC) curve analysis. Correlations between the preoperative hyperfibrinogenemia and clinicopathological characteristics were analyzed, and univariate and multivariate survival analyses were performed to identify the factors associated with overall survival (OS). Cancer cell migration and invasion in vitro were examined to investigate the function of fibrinogen in GBC cell migration. RESULTS: The plasma levels for all coagulation tests, with the exception of INR, were significantly different between the GBC patients and control patients (p < 0.001). Hyperfibrinogenemia (>402 mg/dL) was associated with poorly differentiated tumors, advanced tumor invasion, lymphatic metastasis, and advanced tumor stage (p < 0.001), and had a statistically significant adverse effect on survival (p = 0.001). In the multivariate analysis, hyperfibrinogenemia (p = 0.031) was independently associated with worse OS, tumor stage (p = 0.016), margin status (p < 0.001), and lymphatic metastasis (p = 0.035). Moreover, cell migration and invasion in vitro were significantly enhanced by fibrinogen. CONCLUSIONS: Preoperative plasma fibrinogen levels was associated with tumor progression and may be an independent marker of poor prognosis in GBC patients. Furthermore, fibrinogen may contribute to cell migration by inducing epithelial-mesenchymal transition.

Schisandrin B induces apoptosis and cell cycle arrest of gallbladder cancer cells.

Gallbladder cancer, with high aggressivity and extremely poor prognosis, is the most common malignancy of the bile duct. The main objective of the paper was to investigate the effects of schisandrin B (Sch B) on gallbladder cancer cells and identify the mechanisms underlying its potential anticancer effects. We showed that Sch B inhibited the viability and proliferation of human gallbladder cancer cells in a dose-, time -dependent manner through MTT and colony formation assays, and decrease mitochondrial membrane potential (ΔΨm) at a dose-dependent manner through flow cytometry. Flow cytometry assays also revealed G0/G1 phase arrest and apoptosis in GBC-SD and NOZ cells. Western blot analysis of Sch B-treated cells revealed the upregulation of Bax, cleaved caspase-9, cleaved caspase-3, cleaved PARP and downregulation of Bcl-2, NF-κB, cyclin D1 and CDK-4. Moreover, this drug also inhibited the tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data demonstrated that Sch B induced apoptosis in gallbladder cancer cells by regulating apoptosis-related protein expression, and suggests that Sch B may be a promising drug for the treatment of gallbladder cancer.