FoxM1 overexpression promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma

AIM: To investigate the expression of forkhead box protein M1(Fox M1) in the process of epithelial mesenchymal transition in hepatocellular carcinoma(HCC) and its role in metastasis.METHODS: Fox M1 and E-cadherin expression in HCC tissue microarray specimens was evaluated by immunohistochemical staining,and statistical methods were applied to analyze the correlation between FoxM 1 and epithelial-mesenchymal transition(EMT).KaplanMeier analysis of the correlation between the Fox M1 expression level and recurrence or overall survival of HCC patients was performed.The expression of FoxM 1,E-cadherin and snail homologue 1(SNAI1) in HCC cell lines was evaluated by real-time reverse transcriptionpolymerase chain reaction and Western blot.Hepatocyte growth factor(HGF) was used to induce EMT and stimulate cell migration in HCC cells.The expression of Fox M1 and SNAI1 was regulated by transfection with plasmids pc DNA3.1 and si RNAs in vitro.The occurrence of EMT was evaluated by Transwell assay,morphologic analysis and detection of the expression of EMT markers(E-cadherin and vimentin).Luciferase and chromatin immunoprecipitation assays were used to evaluate whether SNAI1 is a direct transcriptional target of FoxM 1.RESULTS: FoxM 1 expression was increased significantly in HCC compared with para-carcinoma(10.7 ± 0.9 vs 8.2 ± 0.7,P < 0.05) and normal hepatic(10.7 ± 0.9 vs 2.7 ± 0.4,P < 0.05) tissues.Overexpression of Fox M1 was correlated with HCC tumor size,tumor number,macrovascular invasion and higher TNM stage,but was negatively correlated with E-cadherin expression in microarray specimens and in cell lines.Fox M1 overexpression was correlated significantly with HCC metastasis and EMT.In vitro,we found that FoxM 1 plays a key role in HGF-induced EMT,and overexpression of Fox M1 could suppress E-cadherin expression and induce EMT changes,which were associated with increased HCC cell invasiveness.Next,we confirmed that FOXM1 directly binds to and activates the SNAI1 promoter,and we identified SNAI1 as a direct transcriptional target of FOXM1.Moreover,inhibiting the expression of SNAI1 significantly inhibited FoxM 1-mediated EMT.CONCLUSION: Fox M1 overexpression promotes EMT and metastasis of HCC,and SNAI1 plays a critical role in FoxM 1-mediated EMT.

Prognostic implications of estrogen receptor 1 and vascular endothelial growth factor A expression in primary gallbladder carcinoma

AIM: To investigate the prognostic significance of estrogen receptor 1(ER1) and vascular endothelial growth factor A(VEGF-A) expression in primary gallbladder carcinoma(GBC) to identify new prognostic markers for this malignancy.METHODS: Using immunohistochemistry, we investigated ER1 and VEGF-A expression in 78 GBC and 78 cholelithiasis(CS) tissues. The results were correlated with clinicopathological features. Univariate and multivariate analyses were performed to evaluate the relationship between ER1 and VEGF-A expression and patients' prognosis. Further Kaplan-Meier survival analysis was also performed. RESULTS: ER1 and VEGF-A expression was significantly higher in GBC compared with CS(47/78 vs 28/78, P < 0.05; 51/78 vs 33/78, P < 0.05). ER1 expression was correlated with gender(P < 0.05) and VEGF-A expression was correlated with tumor differentiation in GBC patients(P < 0.05). In univariate analysis, age and tumor node metastasis(TNM) stage were factors associated with GBC prognosis(P < 0.05). Although there was no statistical difference between the expression of ER1 or VEGF-A and overall survival, the high expression of ER1 combined with VEGF-A predicted a poor prognosis for GBC patients(16.30 ± 1.87 vs 24.97 ± 2.09, log-rank P < 0.05). In multivariate analysis, combined expression of ER1 and VEGF-A and TNM stage were independent prognostic factors for GBC patients(P < 0.05).CONCLUSION: Combined expression of ER1 and VEGF-A is a potential prognostic marker for GBC patients. Clinical detection of ER1 and VEGF-A in surgically resected GBC tissues would provide animportant reference for decision-making of postoperative treatment programs.

Down-regulation of FoxM1 inhibits viability and invasion of gallbladder carcinoma cells, partially dependent on inducement of cellular senescence

AIM:To investigate the effect of knockdown of forkhead box M1(foxM1)on the proliferation and invasion capacities of human gallbladder carcinoma(Gbc)-sd cells.METHODS:four foxM1 shRNAs were transfected into Gbc-sd cells with lipofectamine 2000 to select the appropriate shRNA for down-regulation of foxM1.A recombinant lentivirus for shfoxM1(lv-shfoxM1),which expresses FoxM1-specific shRNA,and a negative control carrying green fluorescent protein,which expresses a scrambled RNA,were constructed.After transfection with the recombinant adenovirus and screened with puromycin,RT-PcR and Western blot were utilized to evaluate the inhibition efficiency.cell viability was evaluated by MTT assay,and cell migration and invasion were assessed using the Transwell system.cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated(for migration assay)or coated(for invasion assay)with growth factor-reduced Matrigel.To verify the involvement of foxM1 in the senescence of tumor cells,staining of senescenceβ-galactosidase(sAβ-gal),the widely used biomarker of cellular senescence,was also performed.RESULTS:After successful transfection of four foxM1small interfering RNAs(shRNAs)with lipofectamine2000,the shf1822 was selected as the most appropriate shRNA according to its obvious inhibitory effect.The recombinant adenovirus was then constructed with the shf1822 and successfully transfected into the Gbcsd cells,resulting in the significant inhibition of foxM1expression at both the mRNA and protein levels,compared with the negative control(P<0.05).After transfection,down-regulation of foxM1 significantly inhibited cell viability according to the MTT assay(P<0.05).In addition,Transwell migration and invasion assays also suggested the suppression of invasion ability of the transfected cells.sAβ-gal staining showed that downregulation of foxM1 could induce more senescent Gbc cells(P<0.05),suggesting the possible involvement of the senescence process of the foxM1-deficient cells in Gbc.CONCLUSION:foxM1 is functionally involved in viability of Gbc cells,partially dependent on the inducement of cellular senescence,and is a potential target for Gbc therapy.