NCT02442414;Pre-results.

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

Gallbladder cancer (GBC) is the most common malignant tumour of the biliary track system. Angiogenesis plays a pivotal role in the development and progression of malignant tumours. miR-143-3p acts as a tumour suppressor in various cancers. Their role in GBC is however less well defined. Here we show that the expression levels of miR-143-3p were decreased in human GBC tissues compared with the non-tumour adjacent tissue (NAT) counterparts and were closely associated with overall survival. We discovered that miR-143-3p was a novel inhibitor of tumour growth and angiogenesis in vivo and in vitro. Our antibody array, ELISA and PLGF rescue analyses indicated that PLGF played an essential role in the antiangiogenic effect of miR-143-3p. Furthermore, we used miRNA target-prediction software and dual-luciferase assays to confirm that integrin α6 (ITGA6) acted as a direct target of miR-143-3p. Our ELISA and western blot analyses confirmed that the expression of PLGF was decreased via the ITGA6/PI3K/AKT pathway. In conclusion, miR-143-3p suppresses tumour angiogenesis and growth of GBC through the ITGA6/PI3K/AKT/PLGF pathways and may be a novel molecular therapeutic target for GBC.

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.

NOP2/Sun domain family, member 2 (NSUN2) is a nuclear RNA methyl‐transferase catalyzing 5‐methylcytosine formation. Evidence shows that NSUN2 is correlated with cell unlimited proliferation. However, its functional role in gallbladder carcinoma (GBC), which is the most common biliary tract malignancy and has a poor prognosis, remains to be determined. Here we found that NSUN2 was highly expressed in GBC tissues as well as cell lines. NSUN2 silencing repressed GBC cell proliferation and tumorigenesis both in vitro and in vivo. Conversely, upregulation of NSUN2 enhanced GBC cell growth and colony formation. We further discovered that RPL6 was a closely interacting partner with NSUN2. Silencing RPL6 resulted in insufficient NSUN2 translational level and accumulative NSUN2 transcriptional level. Exogenous expression of NSUN2 partially rescued the effect of RPL6 in gallbladder cancer progression. Taken together, our data provided novel mechanic insights into the function of NSUN2 in GBC, thus pointing to NSUN2 as a potential and effective therapeutic approach to GBC treatment.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary tract system. Epithelial–mesenchymal transition (EMT) plays a vital role in the process of tumor metastasis. Mesenchymal‐like cells can serve as a source of cancer stem cells, which can confer the EMT phenotype. Placental growth factor (PLGF) belongs to the vascular endothelial growth factor family and plays a vital role in cancer. However, the underlying molecular mechanisms about the influence of PLGF on EMT in GBC remain unknown. Here we show that PLGF expression levels were higher in GBC tissues than in normal adjacent tissues and were associated with poor prognosis in GBC patients. Exogenous PLGF enhanced the migration, invasion, and tumorsphere formation of GBC cells. Conversely, knockdown of PLGF decreased the aggressive phenotype of GBC cells. Mechanistically, exogenous PLGF upregulated microRNA‐19a (miR‐19a) expression through the activation of c‐MYC. Moreover, Spearman's correlation analysis showed a positive pairwise correlation among PLGF, c‐MYC, and miR‐19a expression in GBC tissues. Taken together, these results suggest that PLGF promotes EMT and tumorsphere formation through inducing miR‐19a expression by upregulating c‐MYC. Thus, PLGF could be a promising molecular therapeutic target for GBC.

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Metformin is the most commonly used drug for type 2 diabetes and has potential benefit in treating and preventing cancer. Previous studies indicated that membrane proteins can affect the antineoplastic effects of metformin and may be crucial in the field of cancer research. However, the antineoplastic effects of metformin and its mechanism in gallbladder cancer (GBC) remain largely unknown. In this study, the effects of metformin on GBC cell proliferation and viability were evaluated using the Cell Counting Kit‐8 (CCK‐8) assay and an apoptosis assay. Western blotting was performed to investigate related signaling pathways. Of note, inhibition, knockdown and upregulation of the membrane protein Chloride intracellular channel 1 (CLIC1) can affect GBC resistance in the presence of metformin. Our data demonstrated that metformin apparently inhibits the proliferation and viability of GBC cells. Metformin promoted cell apoptosis and increased the number of early apoptotic cells. We found that metformin can exert growth‐suppressive effects on these cell lines via inhibition of p‐Akt activity and the Bcl‐2 family. Notably, either dysfunction or downregulation of CLIC1 can partially decrease the antineoplastic effects of metformin while upregulation of CLIC1 can increase drug sensitivity. Our findings provide experimental evidence for using metformin as an antitumor treatment for gallbladder carcinoma.