| Part one: Gene expression of vascular endothelial growth factor-C and -D and their receptors in esophageal squamous carcinoma tissuesObjective To investigate the gene expression of vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor-D (VEGF-D) and their receptors vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial growth factor receptor-3 (VEGFR-3) in esophageal squamous carcinoma. Methods The mRNA levels of VEGF-C, VEGF-D, VEGFR2 and VEGFR3 gene in 24 normal esophageal specimens and 60 esophageal squamous cell carcinomas (ESCCs) were determined by real-time PCR. Results The mRNA levels of VEGF-C, VEGF-D, VEGFR2 and VEGFR3 were significantly upregulated by 3.9-, 1.3-, 2.2- and 8.6-fold, respectively in human ESCC tissues compared with normal esophageal tissues. Conclusions Upregulation of VEGF-C, VEGF-D and their receptors maybe involved in esophageal tumor development and progression.Part two: Protein expression of vascular endothelial growth factor-C correlates with clinicopathological parameters and a poor prognosis of esophageal squamous cell carcinomasObjective: To specifically investigate the clinicopathological role of expression of VEGF-C as well as the correlation with clinical outcomes in ESCCs. Methods Seventy-three patients with ESCC resected in our institute were included in this study. Formalin-fixed paraffin-embedded specimens were stained for VEGF-C and the correlation between the staining, its clinicopathological parameters and its prognostic power were analyzed statistically. Results Of the 73 ESCC patients studied, 39 cases (53.4%) were strongly positive for VEGF-C. VEGF-C expression correlated with histological grade (P=0.005), depth of tumor invasion (pT) (P=0.021), lymphnode metastasis (pN) (P=0.002) and lymphatic invasion (P=0.008). In univariate analysis by log-rank test, histological grade, pN, stage, lymphatic invasion and VEGF-C were significant prognostic factors (P=0.047, 0.007, 0.018, 0.002 and 0.003, respectively). In multivariate analysis, high VEGF-C expression (P=0.0451) maintained its independent prognostic influence on overall survival, as well as pN status (P=0.0029). Conclusions Expression of VEGF-C is related to histological grade, pT, pN and lymphatic invasion, and is a prognostic indicator for ESCC.Part three: Construction of vascular endothelial growth factor-C overexpression and silencing vectors and their interference effectsObjective: Vascular endothelial growth factor-C overexpression and silencing vectors were constructed to provide bases for research on influence of VEGF-C expression on esophageal cancer proliferation. Methods: For VEGF-C overexpression vector, a fragment containing human VEGF-C coding region was amplified by PCR, and then inserted into the pEGFP-N1 vector. For VEGF-C silencing vectors, pGPU6/GFP/Neo vectors containing short hairpin interfering RNA (shRNA) against VEGF-C mRNA were constructed. The human ESCC cell line TE-1 transfected with VEGF-C overexpression and silencing vectors was maintained to generate stable VEGF-C-overexpressing or silencing clones, and VEGF-C mRNA expression was analysed by real time PCR, while protein levels were determined by immunostaining and enzyme-linked immunosorbent assay. Results: The overexpression vector pEGFP-VEGF-C and two silencing vectors (shRNA-1 and shRNA-2) were constructed. VEGF-C transcription, translation and secretion were significantly increased in the VEGF-C-overexpressing TE-1 cells compared with cells transfected with shRNA negative control. Cells transfected both of the shRNA vectors showed reduced VEGF-C transcription, translation and secretion levels. By contrast, shRNA-2 has more potently suppressed VEGF-C level than shRNA-1. Conclusion: The overexpression vector of VEGF-C increased the transcription, translation and secretion of VEGF-C, which were decreased by the silencing vector.Part four: In vitro effects of vascular endothelial growth factor-C on esophageal cancer proliferation in stably transfected TE-1 cell lines and in vivo effects on esophageal tumor growth in nude miceObjective: To analysis in vitro effects of vascular endothelial growth factor-C on esophageal cancer proliferation using TE-1 cell lines stably transfected with VEGF-C overexpression or shRNA vectors. And to analysis in vitro effects of vascular endothelial growth factor-C on esophageal cancer tumor growth using nude mice bearing TE-1 cells xenografts as a model. Methods: In vitro growth was measured using CCK-8 kit. To evaluate the effect of VEGF-C on chemosensitivity, the proliferation index was calculated after cisplatin treatment. Migration assay and focus formation assay were conducted under a microscope. Quantitative chromatin immunoprecipitation was carried out to determine the downstream effector of VEGF-C/VEGFR-3 signaling. Male mice at 4 weeks of age were injeted with tumor cells transfected with siRNA-NC, shRNA-2 or pEGFP-VEGF-C vector, respectively. Tumor diameters were measured at regular intervals with digital calipers, the tumor volume was calculated and tumor growth curve was then constructed. Results: pEGFP-VEGF-C transfected TE-1 cells exhibited a 1-fold significant increase in cell proliferation rate, 70 percents increased migrated cells in supernatant, unchanged resistance to cisplatin, 1.1-fold increase colonies numbers and promoted CNTN-1 transcription. In contrast, shRNA-2 transfection inhibited TE-1 cell growth to 72 % of the control, decreased migrated cells in supernatant by 50 percents, did not change resistance to cisplatin, while reduced colonies numbers and CNTN-1 transcripts. A significant increased or decreased tumor size was observed in nude mice injected with pEGFP-VEGF-C-transfected or VEGF-C shRNA-transfected cells, respectively. Conclusion: In vitro, VEGF-C overexpression vector increased cell proliferation, migration and focus formation, which were decreased by VEGF-C silencing vectors. And in vivo, gene silencing could also decreased ploliferation of tumor in nude mice. Besides, CNTN-1 may be the potential downstream effector of VEGF-C.
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