Vascular Endothelial Growth Factor-C Gene Promoted Human Gastric Cancer Lymphangiogenesis And The Identification Of MiRNAs Associated With It

Background and objective:Gastric carcinoma is one of the most common malignant tumors of the serious threat to human health. In recent decades, although the incidence of gastric carcinoma has declined, but in most parts of the world, especially in eastern Asia, absolute numbers of new cases every year has been on the increase. At the present, the mortality of gastric carcinoma all over the world accounted for 10% have been in the second place of malignant tumors after lung cancer. The lymph node metastasis is a major way of early gastric carcinoma patients with distant metastasis, also is the main reason of death in patients. However, because of the absent of specific markers for lymphatic endothelial cells, the study was relatively blind for a long time, the mystery of lymphatic metastasis mechanism was a bottleneck to overcome gastirc carcinoma, therefore, lymphangiogenesis has become one of the hot research field for tumor metastasis currently. The increased expression of VEGF-C in gastric carcinoma has been reported, which suggested that VEGF-C may have an effect on lymph node metastasis in gastric cancer. Although there are numerous studies on VEGF-C gene, but only with the increased secretion of VEGF-C and its direct effect is not sufficient to explain how VEGF-C promote lymphangiogenesis in lymph node metastasis of gastric carcinoma, which prompted us to look for its regulatory mechanisms. In this study, by constructing the overexpression of VEGF-C gene human gastric cancer cell, the effects of VEGF-C gene in human gastric carcinoma was observed, with the help of lymphatic endothelial cells induced by the human gastric cancer cells and untreated lymphatic endothelial cells as the research object, the regulatory mechanism of gastric cancer lymphatic generated was explored; The miRNA expression profile of induced lymphatic endothelial cells and untreated lymphatic endothelial cells of was compared by application of miRNA chips, so as to look for human miRNAs associated with gastric cancer lymphangiogenesis, in order to further clarify regulatory mechanism of human gastric cancer lymphangiogenesis.Methods:The differences of VEGF-C mRNA and protein expression levels among BGC-823、NCI-N87、SGC-7901、MKN-45、MKN-28 and GES-1 gastric cancer cell lines were tested by the qRT-PCR method and Western blot method. By constructing overexpression of growth factor-C gene gastric cancer cells, the human lymphatic endothelial cells tube formation was assayed. The miRNA expression profile of induced lymphatic endothelial cells and untreated lymphatic endothelial cells of was compared by application of miRNA chips, preliminary candidate miRNA-related gastric lymphangiogenesis were validated in gastric cancer tissues by using qRT-PCR.Results:1. It was different of the VEGF-C gene mRNA and protein expression detected by qRT-PCR and Western Blot in all of the six groups cell lines, the minimum expression of VEGF-C was found in MK.N-45 cell line, and the maximum expression in SGC-7901 cell line;2. The over expression of VEGF-C gene lentivirus was constructed and infected human gastric cancer cell MKN-45 successfully:the efficiency up to 90% observed by a fluorescence microscope and confirmed at the gene and protein levels;3. The results of endothelial tube formation experiment showed:There were respectively statistics significance about the tubular number and the length of tube in the experimental group, control group and blank group;4. The results of miRNA chips essay showed:threre were 89 differentially expressed miRNA by different induced ways, tumor-induced lymphatic endothelial cells had relatively 47 miRNA upregulated, and 42 downregulated miRNA for untreatd lymphatic endothelial cells;5. Gastric cancer tissue with lymphatic metastasis and without lymphatic metastasis by qRT-PCR showed:miR-648, miR-5002-3p, miR-4754, miR-4760-5p, miR-4491, miR-4252, miR-5007-3p and miR-647 expression was up regulated, miR-3178, miR-593-5p, miR-4485, miR-135a-3p, miR-17, miR-1469 and miR-124-5p expression was down regulated, results were consistent with the miRNA chips.Conclusions:1. The gastric carcinoma cell line of VEGF-C overexpression was successfully established;2. The over expression of VEGF-C gene in gastric cancer cells has ability to promote lymphangiogenesis;3. Screening and partial identification of lymphatic metastasis of miRNAs related with lymphangiogenesis in gastric cancer.