miR-138 inhibits the growth of tumor in vivo.

<p>(A) Twelve nude mice were used to establish the gallbladder carcinoma xenograft models. Representative image of tumors formed at the fifth week after injection. Experiments were repeated at least three times. (B) Growth curve was drawn by measuring tumor volumes at the indicated times. **<i>P</i><0.01.</p

Bag-1 is a direct target of miR-138.

<p>(A) A schematic representation showing the putative target site of Bag-1 and mutated target site for miR-138 with the seed region and base substitutions underlined. (B and C) Luciferase reporter assay in HEK293T cells with co-transfection of Bag-1-UTR-WT or Bag-1-UTR-MUT at indicated times. (D and E) The mRNA and protein expression levels of Bag-1 in OCUG-1 and NOZ cells transfected with miR-138 mimic or the control. β-Actin was used as an internal quantitative control. Error bars represent the SD from three independent trials. **<i>P</i><0.01.</p

Effects of overexpression of Bag-1 on cell proliferation and apoptosis.

<p>(A) Expression of Bag-1 protein in OCUG-1 and NOZ cells stably expressing miR-138 transfected with pcDNA3.1 vector as a control or Bag-1 vector was detected using Western blot at 48 h after transfection. (B and C) Cell proliferation assay was measured in pcDNA3.1 vector or Bag-1-transfected OCUG-1 and NOZ cells stably expressing miR-138. (D) Apoptosis assay was performed after transfection of pcDNA3.1 vector or Bag-1 in gallbladder carcinoma cells. Error bars represented the SD from three independent trials. **<i>P</i><0.01.</p

Effect of miR-138 on gallbladder carcinoma cell proliferation and apoptosis.

<p>(A) The qRT-PCR analysis confirmed that the expression of miR-138 was clearly increased in cells transduced with miR-138 compared with the control vector. (B and C) Effect of miR-138 on cell proliferation was measured using MTT assay in OCUG-1 and NOZ cells transduced with miR-138 or the control vector. (D) Flow cytometric analysis of the effect of miR-138 on apoptosis of OCUG-1 and NOZ cells. Error bars represented the SD from three independent trials. **<i>P</i><0.01.</p

Silencing the expression of Bag-1 could inhibit the proliferation of gallbladder carcinoma cells.

<p>(A) Immunoblots were performed to analyze Bag-1, BCL-2, Bax, and cleavage of caspase-3 expression in Bag-1 siRNA-transfected OCUG-1 and NOZ cells. (B and C) MTT assay was used to measure cell proliferative capacity in gallbladder carcinoma cells treated with the control siRNA or Bag-1 siRNA. (D) Gallbladder carcinoma cells transduced with miR-138 compared with the control vector were subjected to Western blot analysis with the indicated antibodies. Error bars represented the SD from three independent trials. *<i>P</i><0.05; **<i>P</i><0.01.</p

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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

FOXL1 overexpression induced mitochondrial dysfunction and caspase-mediated apoptosis.

<p>(<b>A</b>) The loss of mitochondrial membrane potential (ΔΨm) is indicated by a decrease in the red/green fluorescence intensity ratio. X axis indicates green fluorescence intensity; Y axis indicates red fluorescence intensity. The results shown are representative of three experiments. (<b>B</b>) The proportion of red fluorescence-positive cells decreased while the proportion of green fluorescence-positive cells increased after transfection with pcDNA-FOXL1 (P<0.05). (<b>C</b>) Western blot analysis showed the level of cytosolic cytochrome c markedly increased, while the level of mitochondrial cytochrome c markedly decreased. (<b>D</b>) The levels of cleaved caspase-9, 3 and PARP and bax were elevated, while the level of bcl-2 was decreased. *indicates significant difference.</p

FOXL1 overexpression promoted apoptosis in vitro and in vivo.

<p>(<b>A</b>) Apoptosis of NOZ cells in vitro was examined by Annexin V/PI double staining. Cells that stain negative for both Annexin V and PI are viable cells (lower left). Cells that stain positive for Annexin V and negative for PI are undergoing early apoptosis (lower right). Cells that stain positive for both Annexin V and PI are either in the end stage of apoptosis, or undergoing necrosis (upper right). The results shown are representative of three experiments. (<b>B</b>) The number of apoptotic NOZ cells (including early apoptosis, late apoptosis and necrosis) was significantly increased after transfection with pcDNA-FOXL1 (P<0.05). (<b>C</b>) Apoptosis of NOZ cells in vivo was determined using TUNEL assay. Apoptotic cells are visualized as intensely green fluorescent cells. The number of apoptotic cells was recorded under high-power fields (400×). Localization in nuclei was visualized by counterstaining with DAPI (blue). (<b>D</b>) FOXL1 overexpression significantly promoted apoptosis of NOZ cells in vivo (P<0.05). *indicates significant difference.</p

Overexpression of NOTCH-regulated Ankyrin Repeat Protein is associated with papillary thyroid carcinoma progression

<div><p>Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation.</p></div

Meta-analysis of the safety of liver resection and radiofrequency ablation.

<p>LR: liver resection. RFA: radiofrequency ablation.</p