Background Gallbladder cancer is the most common biliary tract malignancy and not sensitive to chemotherapy. Autophagy is an important factor prolonging the survival of cancer cells under chemotherapeutic stress. We aimed to investigate the role of long non-coding RNAs (lncRNAs) in autophagy and chemoresistance of gallbladder cancer cells. Methods We established doxorubicin (Dox)-resistant gallbladder cancer cells and used microarray analysis to compare the expression profiles of lncRNAs in Dox-resistant gallbladder cancer cells and their parental cells. Knockdown or exogenous expression of lncRNA combined with in vitro and in vivo assays were performed to prove the functional significance of lncRNA. The effects of lncRNA on autophagy were assessed by stubRFP-sensGFP-LC3 and western blot. We used RNA pull-down and mass spectrometry analysis to identify the target proteins of lncRNA. Results The drug-resistant property of gallbladder cancer cells is related to their enhanced autophagic activity. And we found a lncRNA ENST00000425894 termed gallbladder cancer drug resistance-associated lncRNA1 (GBCDRlnc1) that serves as a critical regulator in gallbladder cancer chemoresistance. Furthermore, we discovered that GBCDRlnc1 is upregulated in gallbladder cancer tissues. Knockdown of GBCDRlnc1, via inhibiting autophagy at initial stage, enhanced the sensitivity of Dox-resistant gallbladder cancer cells to Dox in vitro and in vivo. Mechanically, we identified that GBCDRlnc1 interacts with phosphoglycerate kinase 1 and inhibits its ubiquitination in Dox-resistant gallbladder cancer cells, which leads to the down-regulation of autophagy initiator ATG5-ATG12 conjugate. Conclusions Our findings established that the chemoresistant driver GBCDRlnc1 might be a candidate therapeutic target for the treatment of advanced gallbladder cancer. Electronic supplementary material The online version of this article (10.1186/s12943-019-1016-0) contains supplementary material, which is available to authorized users.
Background Circular RNAs (circRNAs) have recently been identified as potential functional modulators of the cellular physiology processes. The study aims to uncover the potential clinical value and driving molecular mechanisms of circRNAs in gallbladder cancer (GBC). Patients and methods We performed RNA sequencing from four GBC and paired adjacent normal tissues to analyze the circRNA candidates. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the circFOXP1 expression from 40 patient tissue samples. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro and in vivo assays were performed to prove the functional significance of circFOXP1. Double luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed. Results By performing RNA sequencing from GBC and paired adjacent normal tissues to analyze the circRNA candidates, we identified that circFOXP1 (hsa_circ_0008234) expression was significantly upregulated in GBC tissues and positively associated with lymph node metastasis, advanced TNM stage and poor prognosis in patients. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro assays demonstrated that circFOXP1 has pleiotropic effects, including promotion of cell proliferation, migration, invasion, and inhibition of cell apoptosis in GBC. In vivo, circFOXP1 promoted tumor growth. Mechanistically, double luciferase reporter, RNA immunoprecipitation (RIP) and biotin-labeled RNA pull-down assays clarified that circFOXP1 interacted with PTBP1 that could bind to the 3’UTR region and coding region (CDS) of enzyme pyruvate kinase, liver and RBC (PKLR) mRNA (UCUU binding bites) to protect PKLR mRNA from decay. Additionally, circFOXP1 acted as the sponge of miR-370 to regulate PKLR, resulting in promoting Warburg effect in GBC progression. Conclusions These results demonstrated that circFOXP1 serve as a prognostic biomarker and critical regulator in GBC progression and Warburg effect, suggesting a potential target for GBC treatment.
Long non-coding RNA LINC00152 had been reported as an oncogene in gastric and hepatocellular cancer. In this study, we show that LINC00152 is overexpressed in gallbladder cancer (GBC) tissue samples and cell lines. The high LINC00152 levels correlated negatively with the overall survival time in GBC patients. Functionally, LINC00152 dramatically promoted cell migration, invasion and epithelial–mesenchymal transition (EMT) progression in vitro. In vivo, LINC00152 overexpression significantly promoted tumour peritoneal spreading and metastasis. Mechanistic analyses indicated that LINC00152 functions as a molecular sponge for miR-138, which directly suppresses the expression of hypoxia inducible factor-1α (HIF-1α). We revealed that miR-138 is a suppressor of GBC cell metastasis and EMT progression, and a similar phenomenon was observed in HIF-1α knockdown NOZ cells. Through binding to miR-138, LINC00152 has an oncogenic effect on GBC. Overall, our study suggested that the LINC00152/miR-138/HIF-1α pathway potentiates the progression of GBC, and LINC00152 may be a novel therapeutic target.
BackgroundLong non-coding RNA (lncRNA) H19 has been reported to involve in many kinds of human cancers and functions as an oncogene. Our previous study found that H19 was over-expressed in gallbladder cancer (GBC) and was shown to promote tumor development in GBC. However, the competing endogenous RNA (ceRNA) regulatory network involving H19 in GBC progression has not been fully elucidated. We aim to detect the role of H19 as a ceRNA in GBC.Methods and ResultsIn this study, the expression of H19 and miR-342-3p were analyzed in 35 GBC tissues and matched normal tissues by using quantitative polymerase chain reaction (qRT-PCR). We demonstrated H19 was overexpressed and negatively correlated with miR-342-3p in GBC. By dual-luciferase reporter assays, RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays, we verified that H19 was identified as a direct target of miR-342-3p. QRT-PCR and Western-blotting assays demonstrated that H19 silencing down-regulated, whereas over-expression enhanced the expression of miR-342-3p targeting FOXM1 through competitively ‘sponging’ miR-342-3p. Furthermore, transwell invasion assays and cell cycle assays indicated that H19 knockdown inhibited both cells invasion and proliferation, but this effects was attenuated by co-transfection of siRNA-H19 and miR-342-3p inhibitor in GBC cells. In vivo, tumor volumes were decreased significantly in H19 silenced group compared to the control group, but was attenuated by co-transfection of shRNA-H19 and miR-342-3p inhibitor, which were stablely constructed through lenti-virus vector.ConclusionOur results suggest a potential ceRNA regulatory network involving H19 regulates FOXM1 expression by competitively binding endogenous miR-342-3p in GBC. This mechanism may contribute to a better understanding of GBC pathogenesis and provides potential therapeutic strategy for GBC.
Gallbladder carcinoma (GBC) is an aggressive neoplasm, and the treatment options for advanced GBC are limited. Recently, long non‐coding RNAs (lncRNAs) have emerged as new gene regulators and prognostic markers in several cancers. In this study, we found that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) expression was up‐regulated in GBC tissues (P < 0.05). Luciferase reporter assays and RNA pull down assays showed that MALAT1 is a target of miR‐363‐3p. Real‐time quantitative PCR and Western blot analysis indicated that MALAT1 regulated Myeloid cell leukaemia‐1 (MCL‐1) expression as a competing endogenous RNA (ceRNA) for miR‐363‐3p in GBC cells. Furthermore, MALAT1 silencing decreased GBC cell proliferation and the S phase cell population and induced apoptosis in vitro. In vivo, tumour volumes were significantly decreased in the MALAT1 silencing group compared with those in the control group. These data demonstrated that the MALAT1/miR‐363‐3p/MCL‐1 regulatory pathway controls the progression of GBC. Inhibition of MALAT1 expression may be to a novel therapeutic strategy for gallbladder cancer.
Gallbladder cancer (GBC) is a highly malignant cancer with poor prognosis. Although long noncoding RNA (lncRNA) H19 has been reported to play vital role in many human cancers, whether it is involved in GBC proliferation is still unknown. This study was designed to explore the effect of H19 in GBC cell proliferation. The expression of H19 and AKT2 were significantly elevated in GBC tissues, and the level of miR-194-5p is markedly decreased. Moreover, the RNA levels of H19 and AKT2 were positively correlated, and H19 elevation was significantly associated with tumor size. Cell proliferation decreased significantly after knockdown of H19 in GBC-SD and NOZ cells and after knockdown of AKT2 in NOZ cells. Results from cell cycle studies indicated that the S phase were significantly decreased after knockdown of H19 in NOZ cells but significantly elevated after overexpression of H19 in GBC-SD cells. Furthermore, knockdown of H19 upregulated miR-194-5p levels, yet significantly decreased miR-194-5p targeting AKT2 gene expression in NOZ cells. Inhibitor against miR-194-5p reversed these effects. In addition, overexpression of H19 in GBC-SD cells downregulated miR-194-5p and markedly increased AKT2 expression, and miR-194-5p mimic reversed these effects. Eventually, GBC cells were arrested in G0/G1-phase after H19 knockdown, inhibition of miR-194-5p markedly promoted cells into S-phase and co-transfection of siH19, and miR-194-5p inhibitor exerted mutually counter-regulated effects on cell cycle. These results suggested that H19/miR-194-5p/AKT2 axis regulatory network might modulate cell proliferation in GBC.
Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (Malat1) functions as an oncogene in many types of human cancer. In this study, we show that Malat1 is overexpressed in gallbladder cancer (GBC) tissue and cells. The high Malat1 levels correlated positively with tumor size and lymphatic metastasis, and correlated negatively with overall survival. We also show that Malat1 functions as a competing endogenous RNA (ceRNA) for miR-206. Because miR-206 directly suppresses expression of ANXA2 and KRAS, which are thought to promote GBC progression, Malat1 binding of miR-206 in GBC tissue and cells has an oncogenic effect. Conversely, Malat1 knockdown inhibits proliferation and invasion by GBC cells while increasing apoptosis. In vivo, silencing Malat1 decreases tumor volume. These results suggest Malat1 could potentially serve as a therapeutic target and prognostic marker for GBC.
LncRNA-ROR has been reported to be involved in many kinds of human cancers. However, whether LncRNA-ROR is involved in gallbladder cancer progression remains largely unknown. The objective of this study is to investigate the role of LncRNA-ROR in gallbladder cancer. We found that LncRNA-ROR expression level was upregulated in gallbladder cancer tissues (P < 0.05) and was significantly associated with tumor sizes (P < 0.05) and lymph node metastasis (P < 0.05). High expression of LncRNA-ROR was significantly associated with poor prognosis in gallbladder cancer patients (P < 0.05). Moreover, knockdown of LncRNA-ROR inhibited cell proliferation, migration, and invasion. The epithelial-mesenchymal transition (EMT) phenotype induced by TGF-β1 was reversed after LncRNA-ROR knocking down in SGC-996 and Noz cells. LncRNA-ROR plays an important role in the development of gallbladder cancer and mediates the EMT in gallbladder cancer. LncRNA-ROR might act as a marker of prognosis and therapeutic target for gallbladder cancer.
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