Gallbladder Cancer Cell Proliferation Research Articles

Parthenolide induces gallbladder cancer cell apoptosis via MAPK signalling.

Parthenolide (PTL) has a wide range of clinical applications owing to its anti-inflammatory and antitumor effects. To date, the antitumor effect of PTL on gallbladder cancer (GBC) remains largely unknown. Therefore, we aimed to investigate the biological effects of PTL on GBC. The cellular viability and proliferation of GBC-SD and NOZ cell lines after treatment with different concentrations of PTL were analyzed using the Cell Counting Kit-8 (CCK8)assay and colony formation assay. Apoptosis analysis was performed using flow cytometry. Hoechst staining was performed. RNA sequencing (RNA-seq) was performed to identify PTL-related genes and signalling pathways. Furthermore, we confirmed the involvement of these signalling pathways by qRT-PCR and western blotting. For the in-vivo experiments, a xenograft model was used to evaluate the effects of PTL on the proliferation of NOZ cells. PTL significantly inhibited GBC cell growth in vitro and induced apoptosis in the GBC-SD and NOZ cell lines in a dose-dependent manner. RNA sequencing data showed that the immune response and mitogen-activated protein kinase (MAPK) signalling pathways are closely associated with PTL-induced gallbladder cancer cell apoptosis. PTL upregulated BAX, cleaved PARP-1, cleaved caspase-3, cleaved caspase-9, P53 and decreased the expression of BCL-2, phosphorylated ERK, and phosphorylated MEK in vitro. Tumour volume and weight were also suppressed by PTL in vivo. Moreover, the effects of PTL on GBC cells might be mediated by the MAPK pathway. PTL significantly inhibits gallbladder cancer cell proliferation and induces apoptosis through the MAPK pathway, which is a potential molecular reagent for treating GBC. However, further exploration is needed to verify the antitumor effects of PTL and its intracellular signalling mechanism.

SIRT7: A potential prognostic marker and therapeutic target in gallbladder cancer

Gallbladder cancer (GBC) is a highly aggressive malignancy with limited treatment options and poor prognosis. In this study, we aimed to investigate the role of SIRT7, a member of the sirtuin family, in GBC and its potential as a prognostic marker and therapeutic target. Through immunohistochemistry analysis of GBC tissue samples, we observed elevated levels of SIRT7, which were correlated with worse clinicopathological parameters and shorter overall survival in GBC patients. Additionally, through cellular and animal experiments, we have discovered that interfering with SIRT7 can effectively suppress the proliferation, migration, and invasive capabilities of GBC cells. Conversely, overexpressing SIRT7 yields the opposite outcome. Furthermore, interference with SIRT7 triggers cell cycle arrest and enhances apoptosis in GBC cells. Mechanistically, we found that SIRT7 inhibition led to reduced activation of the NF-κB signaling pathway, suggesting its involvement in modulating GBC cell behavior. Our findings shed light on the oncogenic role of SIRT7 in GBC and highlight its potential as a promising prognostic marker and therapeutic target. Further research is warranted to explore the therapeutic implications of targeting SIRT7 in GBC treatment.

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway.

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial-mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A-NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.

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.

SNORA38B promotes proliferation, migration, invasion and epithelial-mesenchymal transition of gallbladder cancer cells <em>via</em> activating TGF-β/Smad2/3 signaling

Evidence has shown that small nucleolar RNAs (snoRNAs) participate in the tumorigenesis in multiple cancers, including gallbladder cancer (GBC). Our results showed that SNORA38B level was increased in GBC tissues compared to adjacent normal tissues. Thus, this research aimed to explore the role and molecular mechanisms of SNORA38B in GBC. SNORA38B level between normal and GBC tissues was evaluated by RT-qPCR. Cell proliferation, apoptosis, migration, and invasion were tested by EdU assay, TUNEL staining and transwell assay, respectively on human intrahepatic biliary epithelial cells (HIBEpiCs) and the GBC cell lines, NOZ and GBC-SD. Expression of proteins in GBC cells was evaluated by immunofluorescence and Western blot assays. We found that, relative to normal tissues, SNORA38B level was notably elevated in GBC tissues. SNORA38B overexpression obviously enhanced GBC cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), but weakened cell apoptosis. Conversely, SNORA38B downregulation strongly suppressed the proliferation and EMT of GBC cells and induced cell apoptosis and ferroptosis, whereas these phenomena were obviously reversed by TGF-β. Meanwhile, SNORA38B downregulation notably reduced the levels of phosphorylated-Smad2 and phosphorylated-Smad3 in GBC cells, whereas these levels were elevated by TGF-β. Collectively, downregulation of SNORA38B could inhibit GBC cell proliferation and EMT and induce ferroptosis via inactivating TGF-β1/Smad2/3 signaling. These findings showed that SNORA38B may be potential target for GBC treatment.

MUC16 C terminal fragment activates YAP1 through Src signaling to promote gallbladder cancer growth

AbstractThe Hippo pathway is crucial to organ size control and its dysregulation contributes to tumorigenesis. The aberrant activation of YAP1 was identified in gallbladder cancer (GBC). However, the underlying mechanism and role in GBC remains unclear. The C terminal fragment of Mucin16, also known as carbohydrate antigen 125 (CA125) encoded product, MUC16c, plays extensive roles in tumor initiation and development. Our study showed that MUC16c binding with 14‐3‐3ε disrupted the interaction of 14‐3‐3ε and phosphorylated yes‐associated protein 1 (YAP1), which led to the activation of YAP1 in GBC. Furthermore, MUC16c decreased the phosphorylation of YAP1 at serine 397 (ser397) by inhibiting LATS1, which upregulated YAP1 protein stability. Interestingly, there was a potential Src kinase site in the MUC16c fragment. The MUC16c_del15Y polypeptides with the deletion of the Src kinase site promoted the interaction of YAP1 with 14‐3‐3ε and downregulated the YAP1 protein levels. Consistently, SU6656, a Src kinase inhibitor also blocked the activation of YAP1 by MUC16c. The MUC16c_del15Y polypeptides decreased GBC cell proliferation in vitro and the growth of xenograft tumors in vivo. Our study revealed the underlying mechanism of the activation of MUC16c on YAP1 mediated by Src signaling and the antitumor effect of MUC16c_del15Y, providing a potential target for GBC therapy.

YTHDF2 promotes gallbladder cancer progression and gemcitabine resistance via m6A-dependent DAPK3 degradation.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic RNA and involved in the carcinogenesis of various malignancies. However, the functions and mechanisms of m6A in gallbladder cancer (GBC) remain unclear. In this study, we investigated the role and underlying mechanism of the RNA-binding protein YT521-B homology domain-containing family protein 2 (YTHDF2), an m6A reader, in GBC. Herein, we detected that YTHDF2 was remarkably upregulated in GBC tissues compared to normal gallbladder tissues. Functionally, YTHDF2 overexpression promoted the proliferation, tumor growth, migration, and invasion of GBC cells while inhibiting the apoptosis in vitro and in vivo. Conversely, YTHDF2 knockdown induced opposite results. Mechanistically, we further investigated the underlying mechanism by integrating RNA immunoprecipitation sequencing (RIP-seq), m6A-modified RIP-seq, and RNA sequencing, which revealed that death-associated protein kinase 3 (DAPK3) is a direct target of YTHDF2. YTHDF2 binds to the 3'-UTR of DAPK3 mRNA and facilitates its degradation in an m6A-dependent manner. DAPK3 inhibition restores the tumor-suppressive phenotype induced by YTHDF2 deficiency. Moreover, the YTHDF2/DAPK3 axis induces the resistance of GBC cells to gemcitabine. In conclusion, we reveal the oncogenic role of YTHDF2 in GBC, demonstrating that YTHDF2 increases the mRNA degradation of the tumor suppressor DAPK3 in an m6A-dependent way, which promotes GBC progression and desensitizes GBC cells to gemcitabine. Our findings provide novel insights into potential therapeutic strategies for GBC.

ZFP64 Promotes Gallbladder Cancer Progression through Recruiting HDAC1 to Activate NOTCH1 Signaling Pathway.

The lack of meaningful and effective early-stage markers remains the major challenge in the diagnosis of gallbladder cancer (GBC) and a huge barrier to timely treatment. Zinc finger protein 64 (ZFP64), a member of the zinc finger protein family, is considered to be a promising predictor in multiple tumors, but its potential effect in GBC still remains unclear. Here, we identified that ZFP64 was a vital regulatory protein in GBC. We found that ZFP64 expressed higher in GBC gallbladder carcinoma tissues than in normal tissues and was positively correlated with poor prognosis. Furthermore, ZFP64 was responsible for the migration, invasion, proliferation, anti-apoptosis, and epithelial mesenchymal transition (EMT) of GBC cells in vitro and in vivo. Mechanistically, through Co-IP assay, we confirmed that ZFP64 recruits HDAC1 localized to the promoter region of NUMB for deacetylation and therefore inhibits NUMB expression. The downregulation of NUMB enhanced the activation of the Notch1 signaling pathway, which is indispensable for the GBC-promotion effect of ZFP64 on GBC. In conclusion, ZFP64 regulated GBC progression and metastasis through upregulating the Notch1 signaling pathway, and thus ZFP64 is expected to become a new focus for a GBC prognostic marker and targeted therapy.

Isoliquiritigenin induces HMOX1 and GPX4-mediated ferroptosis in gallbladder cancer cells.

Gallbladder cancer (GBC) is the most common malignant tumor of biliary tract. Isoliquiritigenin (ISL) is a natural compound with chalcone structure extracted from the roots of licorice and other plants. Relevant studies have shown that ISL has a strong anti-tumor ability in various types of tumors. However, the research of ISL against GBC has not been reported, which needs to be further investigated. The effects of ISL against GBC cells in vitro and in vivo were characterized by cytotoxicity test, RNA-sequencing, quantitative real-time polymerase chain reaction, reactive oxygen species (ROS) detection, lipid peroxidation detection, ferrous ion detection, glutathione disulphide/glutathione (GSSG/GSH) detection, lentivirus transfection, nude mice tumorigenesis experiment and immunohistochemistry. ISL significantly inhibited the proliferation of GBC cells in vitro . The results of transcriptome sequencing and bioinformatics analysis showed that ferroptosis was the main pathway of ISL inhibiting the proliferation of GBC, and HMOX1 and GPX4 were the key molecules of ISL-induced ferroptosis. Knockdown of HMOX1 or overexpression of GPX4 can reduce the sensitivity of GBC cells to ISL-induced ferroptosis and significantly restore the viability of GBC cells. Moreover, ISL significantly reversed the iron content, ROS level, lipid peroxidation level and GSSG/GSH ratio of GBC cells. Finally, ISL significantly inhibited the growth of GBC in vivo and regulated the ferroptosis of GBC by mediating HMOX1 and GPX4 . ISL induced ferroptosis in GBC mainly by activating p62-Keap1-Nrf2-HMOX1 signaling pathway and down-regulating GPX4 in vitro and in vivo . This evidence may provide a new direction for the treatment of GBC.

MAML3 Contributes to Induction of Malignant Phenotype of Gallbladder Cancer Through Morphogenesis Signalling Under Hypoxia.

Hedgehog (HH) signalling is a potential therapeutic target for gallbladder cancer (GBC), and Mastermind-like 3 (MAML3) is involved in the transcription of Smoothened (SMO), which is a key protein of HH signalling during hypoxia in the cancer microenvironment. MAML3 is a NOTCH signalling activator, and HH and NOTCH are involved in morphogenesis signalling. However, the association between MAML3-NOTCH and HH signalling and its role in regulating GBC cells remain unknown. This study aimed to determine whether NOTCH signalling affects tumour aggressiveness in GBC under hypoxic conditions and if MAML3 could be a new comprehensive therapeutic target that regulates morphogenesis signalling, HH, and NOTCH in GBC. We used three cell lines (NOZ, TYGBK1, and TGBC2TKB) and 58 resected specimens. These samples were subjected to cell proliferation, RNA interference, invasion, western blot, and immunohistochemical analyses. MAML3 expression was higher under hypoxic conditions than under normoxic conditions and was involved in the activation of HH and NOTCH signalling. It contributed to the proliferation, migration, and invasion of GBC cells through the NOTCH signalling pathway and enhanced gemcitabine sensitivity. Immunohistochemical analysis showed that MAML3 expression was related to lymphatic invasion, lymph node metastasis, stage category, and a poor prognosis. MAML3 contributes to the induction of the malignant phenotype of GBC under hypoxia through the HH and NOTCH signalling pathways and may be a comprehensive therapeutic target of morphogenesis signalling in GBC.

Taurodeoxycholic acid-YAP1 upregulates OTX1 in promoting gallbladder cancer malignancy through IFITM3-dependent AKT activation.

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.

TRF-3013b inhibits gallbladder cancer proliferation by targeting TPRG1L

BackgroundtRNA-derived fragments (tRFs) are newly discovered noncoding RNAs and regulate tumor progression via diverse molecular mechanisms. However, the expression and biofunction of tRFs in gallbladder cancer (GBC) have not been reported yet.MethodsThe expression of tRFs in GBC was detected by tRF and tiRNA sequencing in GBC tissues and adjacent tissues. The biological function of tRFs was investigated by cell proliferation assay, clonal formation assay, cell cycle assay, and xenotransplantation model in GBC cell lines. The molecular mechanism was discovered and verified by transcriptome sequencing, fluorescence in situ hybridization (FISH), target gene site prediction, and RNA binding protein immunoprecipitation (RIP).ResultstRF-3013b was significantly downregulated in GBC compared with para-cancer tissues. Decreased expression of tRF-3013b in GBC patients was correlated with poor overall survival. Dicer regulated the production of tRF-3013b, and its expression was positively correlated with tRF-3013b in GBC tissues. Functional experiments demonstrated that tRF-3013b inhibited GBC cell proliferation and induced cell-cycle arrest. Mechanically, tRF-3013b exerted RNA silencing effect on TPRG1L by binding to AGO3, and then inhibited NF-κB. TPRG1L overexpression could rescue the effects of tRF-3013b on GBC cell proliferation.ConclusionsThis study indicated that Dicer-induced tRF-3013b inhibited GBC proliferation by targeting TPRG1L and repressed NF-κB, pointing to tRF-3013b as a novel potential therapeutic target of GBC.

Knockdown of IGF2BP3 inhibits the tumorigenesis of gallbladder cancer and modifies tumor microenvironment.

Gallbladder cancer (GBC) ranks seventh among the gastrointestinal cancers. Messenger RNAs (mRNAs) could regulate the progression of GBC. For the purpose of exploring the targets for GBC treatment, RNA sequencing was used to identify the differential expressed mRNAs between GBC and adjacent tissues. Next, CCK8 assay was used to assess the cell viability, and cell proliferation was investigated by colony formation assay. Flow cytometry was performed to evaluate the cell apoptosis. Protein and mRNA expression were analyzed by western blot and RT-qPCR, respectively. Transwell was performed to evaluate the cell metastasis. GBC-derived exosomes were isolated with ultracentrifugation. To evaluate the function of exosomes in GBC, in vivo model of GBC was constructed. The data revealed IGF2BP3 was identified to be upregulated in GBC, and IGF2BP3 silencing was able to decrease GBC cell proliferation by promoting the apoptosis. The migration and invasion of GBC cells were reduced by IGF2BP3 knockdown. Silencing of IGF2BP3 obviously suppressed the level of p-STAT3 in GBC cells. Meanwhile, GBC cell-derived exosomes notably promoted macrophage M2 polarization via carrying IGF2BP3, and then the polarized macrophages promoted the malignant behavior of GBC cells. Furthermore, exosomes markedly promoted the tumor growth of GBC via promoting macrophage M2 polarization. In summary, knockdown of IGF2BP3 suppressed the malignant behavior of GBC cells. Additionally, knockdown of IGF2BP3 modified tumor microenvironment during the progression of GBC. Thus, these findings might provide a new theoretical basis for exploring a strategies against GBC.

LncRNA MNX1-AS1 sustains inactivation of Hippo pathway through a positive feedback loop with USP16/IGF2BP3 axis in gallbladder cancer

The long non-coding RNAs (lncRNAs) have been implicated in multiple human cancers, which may offer great potential as putative targets for cancer diagnosis and treatment. However, the roles of most lncRNAs in gallbladder cancer (GBC) remain poorly understood. The objective of this research involves investigating the clinical implications and underlying mechanism of lncRNA motor neuron and pancreas homeobo×1 antisense RNA 1 (MNX1-AS1) in GBC. This study shows that MNX1-AS1 expression is elevated in the tissues of GBC patients, and is strongly associated with reduced patient survival. Functionally, MNX1-AS1 significantly stimulates the proliferation and metastasis of GBC cells in vitro and in vivo. Mechanistically, MNX1-AS1 is transcriptionally activated by TEA domain family member 4 (TEAD4), and suppresses insulin-like growing factor 2 mRNA-binding protein 3 (IGF2BP3) degradation by recruiting ubiquitin specific peptidase 16 (USP16). Furthermore, MNX1-AS1/IGF2BP3 axis inhibits the Hippo signaling pathway and subsequently activates TEAD4, thereby forming a positive feedback loop. According to our results, MNX1-AS1 facilitates tumorigenesis, progression and metastasis of GBC through a MNX1-AS1/IGF2BP3/Hippo pathway positive feedback loop, which could be both diagnostically and therapeutically helpful in GBC.

Inhibition of AMPK/PFKFB3 mediated glycolysis synergizes with penfluridol to suppress gallbladder cancer growth

BackgroundPenfluridol (PF) is an FDA-approved antipsychotic drug that has recently been shown to have anticancer activity. However, the anticancer effects and underlying mechanisms of PF are not well-established in gallbladder cancer (GBC).MethodsThe anticancer efficacy of PF on GBC was investigated via a series of cell functions experiments, including cell viability, colony formation, apoptosis assays, and so on. The corresponding signaling changes after PF treatment were explored by western blotting. Then, nude mice were utilized to study and test the anticancer activity of PF in vivo. Besides, glucose consumption and lactic production assays were used to detect the glycolysis alteration.ResultsIn this study, we discovered that PF greatly inhibited the proliferation and invasion ability of GBC cells (GBCs). The glucose consumption and lactic generation ability of GBCs were dramatically elevated following PF treatment. Additionally, we discovered that inhibiting glycolysis could improve PF's anticancer efficacy. Further studies established that the activation of the AMPK/PFKFB3 signaling pathway medicated glycolysis after PF treatment. We proved mechanistically that inhibition of AMPK/PFKFB3 singling pathway mediated glycolysis was a potential synergetic strategy to improve the anticancer efficacy of PF on GBC.ConclusionsBy inhibiting AMPK, the anticancer effects of PF on GBCs were amplified. As a result, our investigations shed new light on the possibility of repurposing PF as an anticancer drug for GBC, and AMPK inhibition in combination with PF may represent a novel therapeutic strategy for GBC.Graphical abstractEbwk-zRxpETjsimLmTXTMCVideo abstract

Circulating miR-141 as a potential biomarker for diagnosis, prognosis and therapeutic targets in gallbladder cancer

MicroRNA-141(miR-141) has been reported to play vital roles in the regulation of carcinogenesis and cancer progression. However, the biological function of miR-141 in GBC has received less attention. The aim of this study was to estimate the potential value of the expression level of miR-141 as a diagnostic and prognostic blood-based biomarker in gallbladder cancer (GBC) patients. Meanwhile, to explore its biological role in GBC cells. RT-PCR was employed to confirm the expression of miR-141 in ten paired tissue samples (10 GBC tissues and 10 adjacent normal gallbladder tissues), GBC cell lines and peripheral blood specimens from 98 GBC patients and 60 healthy controls. MTT assay was used to evaluate the GBC cells proliferation and flow cytometry was used to detect the cell apoptosis. Receiver operating characteristic curve analysis and the area under the curve (AUC) were used to evaluate the value of miR-141 plasma levels for GBC diagnosis. Finally, clinicopathological and survival data of all GBC patients were collected and analyzed. Here, we confirmed that the expression of miR-141 were upregulated in primary gallbladder cancer cells and tissues compared with human gallbladder epithelial cells and adjacent normal tissues (P < 0.0001). Meanwhile, we found that downregulated expression of miR-141 by miR-141 inhibitor could induce apoptosis and inhibit proliferation of GBC cells. Additionally, elevated plasma miR-141 expression was also detected in the peripheral blood of GBC patients compared with healthy controls (P < 0.0001). The AUC value of miR-141 for GBC diagnosis was 0.894 (95% CI 0.843–0.945), which was more valuable than those including carcinoembryonic antigen (CEA) (0.713, 95% CI 0.633–0.793), carbohydrate antigen 125 (CA125) (0.837, 95% CI 0.776–0.899) and carbohydrate antigen 19–9 (CA19-9) (0.869, 95% CI 0.813–0.924). The high expression level of miR-141 in plasma was significantly associated with tumor invasion (P = 0.008), lymph node metastasis (P < 0.0001) and advanced pathologic tumor/node/metastasis (pTNM) stage (P = 0.009). More importantly, high plasma miR-141 expression was an independent prognostic factor for predicting poorer long-term survival in GBC patients. Elevated expression of circulating miR-141 in peripheral blood might be a potential novel biomarker for diagnosis and prognosis of GBC patients. Downregulated expression of miR-141 could inhibit proliferation and induce apoptosis of GBC cells, that provide a potential therapeutic target for GBC.

Fangchinoline induces gallbladder cancer cell apoptosis by suppressing PI3K/Akt/XIAP axis.

Gallbladder cancer (GBC) is the most common biliary tract malignancy with a dismal prognosis. The development of new drugs may help to improve prognosis. This study found that fangchinoline, a bisbenzylisoquinoline alkaloids, inhibited the proliferation and clone formation of GBC cells in a dose-dependent manner. Moreover, Hoechst staining, TUNEL assays, and flow cytometry demonstrated that fangchinoline effectively induced apoptosis in GBC cells. Further studies found that an anti-apoptotic pathway, the PI3K/Akt/XIAP axis, was significantly inhibited in GBC cells after treating with fangchinoline. Finally, we confirmed that fangchinoline restrained xenograft tumor growth in vivo. Our findings indicate that fangchinoline can be considered a potential drug for GBC treatment.

Long-term exposure to genistein inhibits the proliferation of gallbladder cancer by downregulating the MCM complex

Soy isoflavones are natural tyrosine kinase inhibitors closely associated with decreased morbidity and mortality of various tumors. The activation of tyrosine kinases such as ERBB2 is the mechanism by which cholecystitis transforms into gallbladder cancer (GBC), therefore, it is important to investigate the relationship between long-term exposure to soy isoflavones and the occurrence and progression of GBC. This case-control study (n = 85 pairs) found that the high level of plasma soy isoflavone—genistein (GEN) was associated with a lower risk of gallbladder cancer (≥326.00 ng/mL compared to ≤19.30 ng/mL, crude odds ratio 0.15, 95% CI 0.04–0.59; P for trend = 0.016), and that the level of GEN exposure negatively correlated with Ki67 expression in GBC tissue (n = 85). Consistent with these results, the proliferation of GBC cells was inhibited in the long-term exposure models of GEN in vitro and in vivo. The long-term exposure to GEN reduced the tyrosine kinase activity of ERBB2 and impaired the function of the PTK6-AKT-GSK3β axis, leading to downregulation of the MCM complex in GBC cells. In summary, long-term exposure to GEN associated with soy products intake might play a certain role in preventing GBC and even inhibiting the proliferation of GBC cells.

HMGA2 promotes the migration and invasion of gallbladder cancer cells and HMGA2 knockdown inhibits angiogenesis via targeting VEGFA.

The high mobility group AT-hook 2 (HMGA2) protein has been found to be upregulated in the majority of tumor types and is associated with a poor prognosis. Previous studies have suggested the oncogenic role of HMGA2 in gallbladder cancer (GBC). The present study aimed to investigate the effects of HMGA2 on the invasion, migration and angiogenesis of GBC cells. To achieve this aim, HMGA2 was overexpressed or silenced in the GBC cell line, EH-GB1, and then the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) abilities of EH-GB1 cells were investigated using Cell Counting Kit-8, wound healing, Transwell and western blotting assays. In addition, the expression levels of VEGFA were determined in EH-GB1 cells using western blotting and reverse transcription-quantitative PCR following HMGA2 overexpression or silencing. Furthermore, HMGA2-silenced EH-GB1 cells were transfected with VEGFA overexpression plasmids to evaluate the tube formation ability of HUVECs using tube formation assay. The results demonstrated that HMGA2 silencing inhibited GBC cell proliferation, migration, invasion and EMT, as evidenced by the downregulated expression of Ki67, proliferating cell nuclear antigen, MMP2, MMP9, N-cadherin, snail family transcriptional repressor 2 and zinc finger E-box-binding homeobox 1, and attenuated cell migration and invasion. However, the opposite results were obtained following HMGA2 overexpression. Moreover, HMGA2 knockdown and overexpression downregulated and upregulated VEGFA expression, respectively. In addition, the tube formation ability of HUVECs and the expression levels of CD31, VEGFR1 and VEGFR2 were downregulated following HMGA2 silencing. However, these effects were partially rescued by simultaneous VEGFA overexpression. In conclusion, the findings of the present study revealed that HMGA2 may promote GBC cell migration, invasion, EMT and angiogenesis. Therefore, inhibiting HMGA2 expression could be considered as a possible therapeutic approach for GBC.

MicroRNA-195-5p inhibits malignant progression of gallbladder cancer by regulating Wnt3a.

We aimed at investigating whether microRNA-195-5p inhibits the malignant proliferation of gallbladder cancer (GBC) via regulating Wnt3a; meanwhile, its relationship with the clinicopathological parameters and prognosis of patients with GBC was also explored. In this study, the tumor tissues and adjacent tissues of 47 GBC patients were tested for microRNA-195-5p expression level by real-time quantitative polymerase chain reaction (qPCR); the relationship between microRNA-195-5p expression and clinical indicators of GBC patients was further analyzed. Control group (NC mimic or NC inhibitor), microRNA-195-5p overexpression group (microRNA-195-5p mimic), and microRNA-195-5p knockdown group (microRNA-195-5p inhibitor) were set in GBC cell lines, respectively. In GBC cell lines GBC-SD and NOZ, cell counting kit-8 (CCK-8), plate cloning experiments and flow cytometry were carried out to assess microRNA-195-5p's effect on proliferation and apoptosis of GBC cells. Further, the interaction between microRNA-195-5p and its downstream gene Wnt3a was explored through Luciferase reporting assay. Our data showed that microRNA-195-5p expression in tumor tissues of GBC patients was remarkably lower than that in adjacent ones. In comparison to patients in highly expressed microRNA-195-5p group, those in lowly expressed microRNA-195-5p had more advanced pathological stage and larger tumor size. Overexpression of microRNA-195-5p markedly attenuated the proliferation capacity of GBC cells as compared to the NC mimic group; in contrast, knockdown of microRNA-195-5p enhanced GBC cell proliferation function of GBC cells in comparison to NC inhibitor group. At the same time, the opposite tendency in cell apoptosis was observed in the above four groups. In GBC tissue specimens, Wnt3a showed an increased expression, which was negatively correlated with microRNA-195-5p. Meanwhile, Luciferase assay verified a binding relationship between microRNA-195-5p and Wnt3a. In addition, we found that over-expressing Wnt3a counteracted the influence of upregulation of microRNA-195-5p on proliferation and apoptosis of GBC cells and thus modulate the malignant progress of GBC cells. In summary, the above studies suggest that low expression of microRNA-195-5p is remarkably relevant to pathological stage and tumor size of patients with GBC. In addition, microRNA-195-5p may suppress the malignant progression of GBC through down-regulating Wnt3a.