Yiyu Qin

Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer

BackgroundProtein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown.MethodsA computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays.ResultsWe demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a.ConclusionTogether, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.

Long non‐coding RNA‐LET is a positive prognostic factor and exhibits tumor‐suppressive activity in gallbladder cancer

The identification of cancer‐associated long non‐coding RNAs (lncRNAs) and the investigation of their molecular and biological functions are vital for understanding the molecular biology and progression of cancer. The lncRNA‐LET, a newly identified lncRNA, was demonstrated to be down‐regulated in hepatocellular cancer. However, little is known about its role in gallbladder cancer. In the present study, an obvious down‐regulation of lncRNA‐LET was observed in gallbladder cancer compared to their adjacent normal tissues. Meanwhile, patients with low expression of lncRNA‐LET have significantly poorer prognosis than those with high expression. We confirmed that hypoxia decreased lncRNA‐LET levels in gallbladder cancer cells. Moreover, lncRNA‐LET overexpression was further validated to inhibit the invasion of gallbladder cancer cells under hypoxic or normoxic conditions in vitro. We demonstrated that lncRNA‐LET overexpression conferred a proliferative advantage to tumor cells under hypoxic conditions. The ectopic expression of lncRNA‐LET led to the promotion of cell cycle arrest at G0/G1 phase and to the induction of apoptosis under hypoxic conditions. Ectopic expression of LncRNA‐LET also suppressed gallbladder tumor growth in vivo. Our findings indicate that lncRNA‐LET may represent a prognostic marker and a potential therapeutic target for gallbladder cancer.

Long Noncoding RNA GCASPC, a Target of miR-17-3p, Negatively Regulates Pyruvate Carboxylase-Dependent Cell Proliferation in Gallbladder Cancer

Long noncoding RNAs (lncRNA) are being implicated in the development of many cancers. Here, we report the discovery of a critical role for the lncRNA GCASPC in determining the progression of gallbladder cancer. Differentially expressed lncRNAs and mRNAs between gallbladder cancer specimens and paired adjacent nontumor tissues from five patients were identified and validated by an expression microarray analysis. Quantitative real-time PCR was used to measure GCASPC levels in tissues from 42 gallbladder cancer patients, and levels of GCASPC were confirmed further in a separate cohort of 89 gallbladder cancer patients. GCASPC was overexpressed or silenced in several gallbladder cancer cell lines where molecular and biological analyses were performed. GCASPC levels were significantly lower in gallbladder cancer than adjacent nontumor tissues and were associated with tumor size, American Joint Committee on Cancer tumor stage, and patient outcomes. GCASPC overexpression suppressed cell proliferation in vitro and in vivo, whereas GCASPC silencing had opposite effects. By RNA pull-down and mass spectrometry, we identified pyruvate carboxylase as an RNA-binding protein that associated with GCASPC. Because GCASPC is a target of miR-17-3p, we confirmed that both miR-17-3p and GCASPC downregulated pyruvate carboxylase level and activity by limiting protein stability. Taken together, our results defined a novel mechanism of lncRNA-regulated cell proliferation in gallbladder cancer, illuminating a new basis for understanding its pathogenicity. Cancer Res; 76(18); 5361-71. ©2016 AACR.

The microRNA miR-33a suppresses IL-6-induced tumor progression by binding Twist in gallbladder cancer

Cytokine is a key molecular link between chronic inflammation and gallbladder cancer (GBC) progression. The potential mechanism of cytokine-associated modulation of microRNAs (miRNAs) expression in GBC progression is not fully understood. In this study, we investigated the biological effects and prognostic significance of interleukin-6 (IL-6) -induced miRNAs in the development of GBC. We identify that inflammatory cytokine, IL-6 promotes proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GBC both in vitro and in vivo. Among all the changed miRNAs in miRNA profiling, miR-33a expression was significantly decreased in IL-6 treated GBC cell lines, as well as in GBC tissues compared with case-matched normal tissues and cholecystitis tissues. In turn, miR-33a suppresses IL-6−induced tumor metastasis by directly binding Twist which was identified as an EMT marker. High expression of miR-33a suppressed xenograft tumor growth and dissemination in nude mice. The downregulation of miR-33a was closely associated with advanced clinical stage, lymph node metastasis, and poor clinical outcomes in patients with GBC. miR-33a acts as a tumor suppressor miRNA in GBC progression and may be considered for the development of potential therapeutics against GBC.

Expression of interleukin-6 is associated with epithelial-mesenchymal transition and survival rates in gallbladder cancer

The present study aimed to investigate the expression of interleukin‑6 (IL‑6) in gallbladder cancer (GBC) tissues and its correlation with survival rate. The association between IL‑6 and epithelial‑mesenchymal transition (EMT)‑associated markers was also examined. Using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, the protein and mRNA expression levels of IL‑6, Twist, E‑cadherin and Vimentin in 20 GBC tissues were analyzed. The IL‑6, Twist and Vimentin proteins were overexpressed in 40, 20 and 70% of the human GBC samples, respectively. The protein expression of E‑cadherin was higher in only 5% of the GBC samples. These differences were significant (P<0.05). Western blot analysis also revealed overexpression of IL‑6, Twist and Vimentin and underexpression of E‑cadherin in the GBC samples with poor differentiation, local invasion and a higher tumor‑node‑metastasis (TNM) stage (P<0.05). Higher mRNA expression levels of IL‑6, Twist and Vimentin and a reduced expression level of E‑cadherin were also demonstrated in the GBC tissues (P<0.05). The degree of differentiation, local invasion, lymph node metastasis and clinical stage were significantly associated with the mRNA expression levels of IL‑6, Twist and E‑cadherin. The increased expression levels of IL‑6 and Twist and the reduced expression of E‑cadherin correlated with shorter median survival rates (P<0.05). Line regression results revealed correlation among the mRNA expression levels of IL‑6, Twist, E‑cadherin and Vimentin. To the best of our knowledge, the present study is the first to demonstrate that IL‑6 is associated with EMT‑associated markers, tumor differentiation, local invasion, TNM stage and survival rates in GBC.

Forkhead Box L1 Is Frequently Downregulated in Gallbladder Cancer and Inhibits Cell Growth through Apoptosis Induction by Mitochondrial Dysfunction

Background Forkhead box L1 (FOXL1), considered as a novel candidate tumor suppressor, suppresses proliferation and invasion in certain cancers. However, the regulation and function of FOXL1 in gallbladder cancer (GBC) remains unclear. Methods FOXL1 expression at mRNA and protein levels in GBC tissues and cell lines were examined by RT-PCR, immunohistochemistry and western blot assay. FOXL1 expression in GBC cell lines was up-regulated by transfection with pcDNA-FOXL1. The effects of FOXL1 overexpression on cell proliferation, apoptosis, migration and invasion were evaluated in vitro or in vivo. In addition, the status of mediators involved in migration, invasion and apoptosis was examined using western blot after transfection with pcDNA-FOXL1. Results FOXL1 was frequently downregulated in GBC tissues and cell lines. Its higher expression is associated with better prognosis, while its lower expression is correlated with advanced TNM stage and poor differentiation. FOXL1 overexpression in NOZ cells significantly suppresses cell proliferation, migration and invasion in vitro and tumorigenicity in nude mice. FOXL1 overexpression disrupted mitochondrial transmembrane potential and triggered mitochondria-mediated apoptosis in NOZ cells. In addition, FOXL1 overexpression suppressed ZEB1 expression and induced E-cadherin expression in NOZ cells. Conclusion Our findings suggested that dysregulated FOXL1 is involved in tumorigenesis and progression of GBC and may serve as a predictor of clinical outcome or even a therapeutic target for patients with GBC.

Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivo

BackgroundGallbladder carcinoma (GBC) is highly lethal, and effective treatment will require synergistic anti-tumor management. The study is aimed at investigating the oncolytic value of myxoma virus (MYXV) infection against GBC and optimizing MYXV oncolytic efficiency.MethodsWe examined the permissiveness of GBC cell lines to MYXV infection and compared the effects of MYXV on cell viability among GBC and control permissive glioma cells in vitro and in vivo after MYXV + rapamycin (Rap) treatment, which is known to enhance cell permissiveness to MYXV by upregulating p-Akt levels. We also assessed MYXV + hyaluronan (HA) therapy efficiency by examinating Akt activation status, MMP-9 expression, cell viability, and collagen distribution. We further compared hydraulic conductivity, tumor area, and survival of tumor-bearing mice between the MYXV + Rap and MYXV + HA therapeutic regimens.ResultsMYXV + Rap treatment could considerably increase the oncolytic ability of MYXV against GBC cell lines in vitro but not against GBC xenografts in vivo. We found higher levels of collagen IV in GBC tumors than in glioma tumors. Diffusion analysis demonstrated that collagen IV could physically hinder MYXV intratumoral distribution. HA–CD44 interplay was found to activate the Akt signaling pathway, which increases oncolytic rates. HA was also found to enhance the MMP-9 secretion, which contributes to collagen IV degradation.ConclusionsUnlike MYXV + Rap, MYXV + HA therapy significantly enhanced the anti-tumor effects of MYXV in vivo and prolonged survival of GBC tumor-bearing mice. HA may optimize the oncolytic effects of MYXV on GBC via the HA–CD44 interaction which can promote viral infection and diffusion.

The inhibitory effects of deleted in liver cancer 1 gene on gallbladder cancer growth through induction of cell cycle arrest and apoptosis

The biological function of tumor suppressor deleted in liver cancer 1 (DLC1) has been investigated in several types of human cancer, but its role in gallbladder cancer (GBC) is yet to be determined. In this research, we conducted in vitro and in vivo analysis to evaluate the inhibitory activities of DLC1 gene against GBC growth.

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor-2 inhibits gallbladder carcinoma growth in vitro and in vivo.

Tissue factor pathway inhibitor‐2 (TFPI‐2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI‐2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus‐mediated gene transfer of TFPI‐2 (Ad5‐TFPI‐2) was constructed to restore the expression of TFPI‐2 in GBC cell lines (GBC‐SD, SGC‐996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI‐2 was significantly downregulated in GBC tissue specimens. Ad5‐TFPI‐2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5‐TFPI‐2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase‐3 and ‐9 and suppressing Bcl‐2 activity. These data indicated that TFPI‐2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC. (Cancer Sci 2012; 103: 723–730)

Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway

Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA–epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.