Downregulation of Notch-regulated Ankyrin Repeat Protein Exerts Antitumor Activities against Growth of Thyroid Cancer.

BACKGROUND: The Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer. METHODS: Thirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2012. Immunohistochemistry was used to detect the level of NRARP in cancer tissues. Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student's t-test, one-way analysis of variance (ANOVA), or Kaplan-Meier were used to analyze the differences between two group or three groups. RESULTS: NRARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05). Lenti-NRARP-shRNA-induced G1 arrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression. CONCLUSION: Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

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.

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

BACKGROUND: Protein 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. METHODS: A 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. RESULTS: We 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. CONCLUSION: Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.

[The role of tissue factor pathway inhibitor-2 gene in gallbladder cancer].

OBJECTIVE: To examine the expression of tissue factor pathway inhibitor-2 (TFPI-2) in gallbladder cancer (GBC) and to investigate the anti-cancer activities of TFPI-2 against the growth of GBC. METHODS: TFPI-2 expression in gallbladder normal tissues, gallbladder polyp (GBP) tissues and GBC tissues were examined by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemical staining. Adenovirus carrying human TFPI-2 gene (Ad5-TFPI-2) were constructed and its anti-cancer effects were investigated in xenograft tumors. Xenograft tumors were constructed by injection of GBC-SD and SGC-996 cells into the flank of nude mice and the volume of xenograft tumors was measured every 3 days until the sacrifice of mice. The apoptosis index of xenograft tumors was examined by TUNEL assay. The status of Bax, Bcl-2 and caspase-3 was examined by Western blot assay. RESULTS: TFPI-2 expression was profoundly lower in GBC tissues (87.0%) when compared to normal tissues (23.3%) and GBP tissues (52.2%; χ(2) = 21.104, P = 0.000). Ad-TFPI-2 significantly inhibited the growth of xenograft tumors in nude mice. Ad-TFPI-2 inhibited GBC-SD cell growth through the induction of apoptosis. The means of total apoptotic cells per field were much higher in Ad5-TFPI-2 group than those in PBS and Ad5-GFP groups. Ad5-TFPI-2 elevated the expression of Bax and cleaved caspase-3, while it decreased the expression of Bcl-2. CONCLUSIONS: TFPI-2 gene and protein was down-regulated in GBC and the down-regulation of TFPI-2 may play a role in the tumorigenesis of GBC. Adenovirus-mediated TFPI-2 can inhibit GBC growth through the induction of apoptosis.

[Somatostatin enhanced anti-tumor effect of doxorubicin on gallbladder cancer cells through the regulation of intracellular drug concentration].

OBJECTIVE: To investigate the possible mechanisms by which Somatostatin (SST) enhances the anti-tumor effect of doxorubicin (DOX) on gallbladder cancer cells. METHODS: GBC-SD cells were grouped into 4 groups: SST-treated group, DOX-treated group, SST+DOX co-treated group and control group. The concentrations of SST and DOX were 75 µg/ml and 5 µg/ml based on our previous studies. In control group, cells were cultivated with phosphate buffered saline (PBS). In experimental groups, cells were cultivated with medium and the corresponding drugs. After drug treatment, cell viability was examined by MTT assay at 6, 12, 24 and 36 h respectively. Meanwhile, intracellular concentrations of doxorubicin in each group was determined by microspectrofluorimetry; Real-time polymerase chain reaction (RT-PCR) was used to determine the expressions of MDR1 mRNA in the cells at different time points and the expressions of P-gp protein, a product of MDR1 mRNA, were determined by Western blot analysis. RESULTS: SST did not exhibit significant inhibitory effect on the proliferation of GBC-SD cells as compared to that of control group (P>0.05). SST+DOX co-treatment group and DOX showed significantly inhibitory effect on the growth of GBC-SD cells at Hour 12 post-treatment. However no statistical difference was found between SST+DOX and DOX groups. Interestingly, at Hour 24 post-treatment, SST+DOX group showed more robust inhibitory effect on GBC-SD cells as compared to DOX alone group. Moreover, SST could significantly down-regulate the expressions of MDR1 mRNA and P-gp protein. SST could increase intracellular DOX concentration. And the difference of intracellular DOX concentration between SST+DOX group and DOX group at Hour 24 was statistically significant. CONCLUSIONS: In our experiment, SST decreases the expression of MDR1 mRNA and P-gp protein so as to reduce the efflux of DOX and elevate DOX concentrations in GBC-SD cells. This eventually leads to enhanced cytotoxic effects of DOX on GBC-SD cells.

The mechanisms of somatostatin induced enhanced chemosensitivity of gallbladder cancer cell line to doxorubicin: cell cycle modulation plus target enzyme up-regulation.

BACKGROUND: Gallbladder carcinoma is known to be an aggressive malignancy and nonsensitive to routine chemotherapy. Its prognosis is quite poor. We have illustrated that somatostatin (SST) can enhance chemosensitivity of gallbladder cancer to Doxorubicin (DOX) in our precious studies. Here, we explored the possible mechanisms by which SST used to enhance the cytotoxicity of DOX on gallbladder carcinoma cell line. METHODS: Human gallbladder cancer cells line (GBC-SD cell line) were divided into four groups: control group, SST group, DOX group, SST+DOX co-treated-group. Cell cycle was detected by flow cytometry (FCM). Cell apoptosis index was detected by using Annexin V/Propidium Iodide Binding on FCM. The expressions of certain key cell cycle-related factors, including retinoblastoma protein (Rb) and E2F-1 protein were investigated by western blotting. ICBP90 protein, which could be a new downstream effector of E2F-1, was also detected by western blotting. The expression of Topo IIα protein, target enzyme of DOX, was assessed in synchronized GBC-SD cells by western blotting. RESULTS: After 24h treatment with SST alone, cell cycle was arrested at S phase in GBC-SD cells line, followed by indistinctive increment of apoptosis index. After 24h treatment with SST and DOX, apoptosis index significantly increased than that of DOX alone (P<0.05). Compared with control group, the expressions of Rb and E2F-1 protein were significantly up-regulated at 24h after treatment with SST. Similarly, the expressions of ICBP90 and Topo IIα protein were also enhanced at 24h after treatment with SST. CONCLUSION: These results suggested that SST could induce cell cycle block in S phase in GBC-SD cells line, the most sensitive phase of the cell cycle for DOX, through up-regulating Rb, E2F-1 and ICBP90 protein expression. Furthermore, ICBP90 induced the enhanced expression of Topo IIα protein which is the target enzyme of DOX and enhanced its cytotoxic effect on GBC-SD cells. We concluded that the mechanisms of SST enhanced chemosensitivity of GBC-SD cell line to DOX might be cell cycle arrest plus up-regulated target enzyme.

[Mechanism research in somatostatin reverting the chemosensitivity of GBC-SD cell line].

OBJECTIVE: To investigate the mechanism of increasing chemosensitivity of gallbladder carcinoma stimulated by somatostatin. METHODS: GBC-SD cells were divided into four groups: SST-alone-treated group, Doxorubicin (DOX)-alone-treated group and co-treated group (co-treatment of SST and DOX). In the control group, the cells were cultivated by medium only. In SST-alone-treated group, the cells were cultivated by medium with SST in the concentration of 75 microg/ml. In DOX-alone-treated group, the cells were cultivated by medium with DOX in the gradient concentrations of 5, 10, 20 microg/ml. In the co-treated group, cells were first cultivated by medium with 75 microg/ml SST for 24 h, followed by the addition of DOX in the gradient concentrations mentioned above. Cell viability curve was measured by MTT assay at 24, 48, 72 and 96 h, respectively. Meanwhile, the alterations of protein expressions of ICBP90 and Topo IIalpha after treatment of SST were examined by Western blot. RESULTS: The treatment of SST alone on GBC-SD cells did not exert significantly inhibitory effect compared to the control group (P > 0.05). However, 24 h after the treatment of SST, the protein expressions of ICBP90 and Topo IIalpha were both up-regulated (P < 0.05). CONCLUSION: Up-regulated the expression of ICBP90 by somatostatin maybe the cause of overexpression of Topo IIalpha, which leads to the enhanced lethal effect of DOX.