The Relationship Between TNF-?-VEGF-D Axis And Lymphangiogenesis Of Gallbladder Cancer And The Mechanism Of TNF-?-induced Upregulation Of VEGF-D

Background: Our previous studies found that,(1) the inflammatory cytokine tumor necrosis factor-alpha(TNF-?) can promote the lymphangiogenesis of gallbladder cancer(GBC),(2) vascular endothelial growth factor D(VEGF-D) is positively correlated with lymphangiogenesis and lymph node metastasis of GBC. However, whether VEGF-D is involved in TNF-?-induced lymphangiogenesis of GBC remains undetermined. Thus, this study aims to clarify the relationship between TNF-?-VEGF-D axis and lymphangiogenesis of GBC in nude mice, and the molecular mechanism of TNF-?-induced upregulation of VEGF-D by animal experiment, promoter activity analysis and signal pathway study.Methods:1. The cryopreserved cell lines NOZ/sh VEGF-D, NOZ/sh CTRL and NOZ were subcultured and used in the establishment of the orthotopic xenograft models of GBC in nude mice. 2 weeks after cell injection, the experimental groups were intraperitoneally injected with TNF-?(2 ?g/kg) one time every 3 days. The control groups were injected with equal volume of normal saline. After continuous injection for 3 weeks, nude mice were killed and the in situ carcinoma and metastatic lymph nodes were dissected and made into paraffin section. The lymphatic vessel density(LVD) of in situ carcinoma and lymph node metastasis(LNM) were detected by immunohistochemistry and HE staining, and the effect of TNF-?-VEGF-D axis on the ymphangiogenesis and lymph node metastasis of orthotopic xenograft models were analyzed.2. Recombinant DNA technology was used to build constructions which contain a series of 5'-deletion VEGF-D promoter fragments and the firefly luciferase reporter.The dual-luciferase reporter assay was sudsequently employed to analyze the activity of VEGF-D promoter.3. The programs TFbind and Promoter Scan were used to search for potential AP-1 and/or NF-?B transcription factor binding site motifs.4. The AP-1 and/or NF-?B binding sites on VEGF-D promoter and the effect of TNF-? on its activity were determined using site-directed mutagenesis, electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation assay(Ch IP).5. RNAi silencing technique and protein specific blocking agent inhibition test were performed for the detection of signaling pathway molecules on TNF-?- VEGF-D axis.6. The comparison of overall mean of measurement data use t test for statistical analysis(?= 0.05, P < 0.05 for a statistically significant difference).Results:1. The effect of TNF-?-VEGF-D axis on the lymphangiogenesis and lymph node metastasis(LNM) of GBC in nude mice:1) The results of LVD counting indicated that: After treatment with TNF-?, the LVD of GBC in nude mice of the NOZ group, NOZ/sh CTRL group and the NOZ/sh VEGF-D group(23.73±2.17?23.20±2.18?10.07±1.83) were significantly increased respectively compared to the TNF-? untreated groups(11.73±2.28?14.27±1.36? 5.67±1.25)(P<0.05). But the effect of TNF-?-induced lymphangiogenesis in the NOZ/sh VEGF-D group was significantly weaker than that of the NOZ group and NOZ/sh CTRL group(P<0.05). These data revealed that TNF-? can promote lymphangiogenesis of GBC in nude mice through VEGF-D.2) When treatment with TNF-?, the LNM rates of GBC in nude mice of the NOZ group, NOZ/sh CTRL group and the NOZ/sh VEGF-D group were significantly increased, respectively compared to the TNF-? untreated groups. But the effect of TNF-?-induced LNM in the NOZ/sh VEGF-D group was significantly weaker than that of the NOZ group and NOZ/sh CTRL group(P<0.05). These results suggested that TNF-? might promote LNM of GBC in nude mice through VEGF-D.2. The molecular mechanism of TNF-?-induced upregulation of VEGF-D.1) Recombinant plasmids containing a series of 5'-deletion fragments of VEGF-D promoter were successfully constructed and identified correctly by sequencing. Dual luciferase test showed that the recombinant plasmid PGL4-988 compared to PGL4-717, PGL4-444 compared to PGL4-325, PGL4-154 compared to PGL4-57, exhibited a stronger fluorescence activity, and the difference was statistically significant(P<0.05). These results suggested that there maybe exist some control sites for regulation of VEGF-D promoter activity in the-988 to-717 nt,-444 to-325 nt and-154 to-57 nt regions upstream of the transcriptional start site ATG of VEGF-D gene.2) Two AP-1 binding sites but no NF-?B site in the VEGF-D promoter(-444 to-325 nt region upstream the transcription start site ATG) were identified: GTGTGTCAT(-401 to-393 nt) and CTGAGATAC(-345 to-337 nt) by using TFbind and Promoter Scan programs. Neither of the other two regions contained AP-1/ NF-?B binding sites.3) Site-directed mutagenesis, electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation assay(Ch IP) were performed to confirm that TNF-? can promote AP-1 binding to these two AP-1 binding sites.4) The effect of AP-1 binding sites' mutation on the TNF-?-induced promoter activity of VEGF-D: The fluorescent activity of “PGL4-AP1 mut1+TNF-?” group(2.77±0.23) and “PGL4-AP1 mut2+TNF-?” group(3.14±0.33) were significantly reduced, respectively compared to that of “PGL4-444+TNF-?” group(3.98±0.15)(P<0.05). These data indicated that TNF-? could regulate the promoter activity of VEGF-D through the two AP-1 binding sites.5) The Effect of AP-1 silencing on the TNF-?-induced promoter activity and protein expression of VEGF-D: 1) The fluorescent activity of PGL4-444 in the “si AP-1+TNF-?” group was significantly reduced compared to that of TNF-? group(2.69±0.34 vs 5.88±0.15, P<0.05). The VEGF-D expression of “si AP-1+TNF-?” group was significantly reduced compared to that of TNF-?-treated group(0.59±0.13 vs 1.39±0.10, P<0.05). The data revealed that TNF-? can enhance VEGF-D promoter activity and upregulate VEGF-D protein expression by AP-16) The Effect of MAPKs inhibitors on the TNF-?-induced promoter activity of VEGF-D: The fluorescent activity of PGL4-444 of the PD98059+TNF-?-treated group was significantly reduced compared to that of TNF-?-treated group(1.74±0.23 vs 5.88±0.15, P<0.05). The activity of PGL4-444 of the SP600125+TNF-?-treated group(5.80±0.31) and the SB203580+TNF-?-treated group(6.49±0.54) were not obviously changed, respectively compared to that of TNF-?-treated group.(P<0.05) These data indicated that TNF-? can enhance VEGF-D promoter activity through the ERK1/2 pathway.7) The Effect of MAPKs inhibitors on the TNF-?-induced protein expression of VEGF-D: The VEGF-D expression of the PD98059+TNF-?-treated group was significantly reduced compared to that of TNF-?-treated group(0.14±0.03 vs 0.33±0.02, P<0.05). The VEGF-D expression of the SP600125+TNF-?-treated group(0.34±0.05) and the SB203580+TNF-?-treated group(0.32±0.03) were not obviously changed, respectively compared to that of TNF-?-treated group.(P>0.05) These data indicated that TNF-? can upregulate VEGF-D protein expression through the ERK1/2 pathway.Conclusions:1. TNF-? can promote lymphangiogenesis of the orthotopic transplanted GBC in nude mice through VEGF-D.2.-444 to-325 nt region of VEGF-D promoter contains two active AP-1 binding sites and TNF-? can enhance the combination of AP-1 and these two AP-1 binding sites. NF-?B binding site was not detected.3.TNF-? enhances the activity of VEGF-D promoter and upregulates the expression of VEGF-D protein through the ERK1/2- AP-1 pathway.