| Background and aim TNF-α is a key inflammatory cytokines responded to chronic inflammatory of gallbladder, which is one of the important risk factor of gallbladder carcinoma(GBC). Studies have shown that that TNF-α may be associated with the progress of the gallbladder, but the mechanism is unclear. In fibroblasts or rheumatoid synoviocytes, it has been reported TNF-α stimulated the m RNA and protein of VEGF-C, which is an important lymphangiogenic factor of gallbladder carcinoma.This study aims to investigate whether TNF-α promotes the expression of VEGF-C and lymphangiogenesis of gallbladder carcinoma and the probable mechanism of it.Methods The correlation between the TNF-α and VEGF-C, TNF-α and the lymphatic vessel density(LVD) of clinical GBC specimens were analysed. The transcription and protein level of VEGF-C in NOZ cell line were detected by real-time PCR and Enzyme linked immunosorbent assay(ELISA). The mixed-cultured system consisting of NOZ cells Human Dermal Lymphatic Endothelial Cells(HDLECs) was constructed and treated with TNF-α,then the tube formation was observed. The orhtotopic xenograft models of GBC in nude mice were established, and the LVD of GBC tumors were detected by immunohistochemistry. A series of luciferase reporter assay of VEGF-C promoter were constructed, and the binding site of NF-κB on the promoter was identified using site-directed mutagenesis, electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation assay(Ch IP).Results 1. The concentration of TNF-α in the gallbladder bile was higher that of the control group(609.0 ± 43.43pg/ml Vs 193.3 ± 13.47pg/ml, p < 0.0001); A linear correlation was found between the concentration ofTNF-α and that of VEGF-C in the bile of GBC patients(p < 0.01). A significant correlation was also found between TNF-α level of the GBC bile and LVD of the GBC tissue(p < 0.01). 2. TNF-α could dose and time-dependently enhanced the transcription and protein expression of VEGF-C in NOZ cells. 3. In the Lymphatic tubu formation assay, when HDLECs was mixed-cultured with NOZ cell line, the tube number of the group with TNF-α(10.89±1.10) was higher than that of the group without TNF-α(6.82±0.62, p < 0.05), the tube length of the group with TNF-α(10.26±1.034mm) was higher than that of the group without TNF-α(6.14±0.74 mm, p < 0.05), the tube area of the group with TNF-α(0.86±0.04mm2) was also higher than that of the group without TNF-α(0.65 ± 0.06mm2, p < 0.05).When VEGF-C was knocked down by si RNA, the tube number of the group with TNF-α(4.11±0.35) was higher than that of the group without TNF-α(3.44±0.29, p < 0.05), but the increased rate of the group of VEGF-C/si RNA(39.27%±5.60%) was lower than that of the control group(71.14%±5.15%, p < 0.01). However, there was no significant difference between the tube length of the group with TNF-α(4.34±0.24mm) and that of the group without TNF-α(3.48±0.43 mm, p > 0.05); there was no significant difference between the tube area of the group with TNF-α(0.39±0.02mm2) and that of the group without TNF-α(0.33±0.04mm2, p > 0.05). 4. In the orhtotopic xenograft models of GBC in nude mice, the LVD of the group with TNF-α(14.33±1.17) was higher than that of the group without TNF-α(10.33±1.17, p < 0.05). when VEGF-C was knocked down by si RNA, the LVD of the group with TNF-α(7.44±0.44) was also higher than that of group without TNF-α(5.89±0.29, p < 0.05), but the increased rate of the group of VEGF-C/si RNA(26.37%±4.85%) was lower than that of the control group(44.19%±3.85%, p < 0.05). 5. TNF-α could activate NF-κB, which improved the activation of VEGF-C promoter and the expression of VEGF-C via combining with the NF-κB binding site(-315 to-306 nt,GGGGAGCTCC) on the promoter.Conclusion TNF-α can promote the expression of VEGF-C and the lymphangiogenesis of gallbladder carcinoma via NF-κB combining with the binding site of VEGF-C promoter and up-regulating the expression of VEGF-C... |
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