| Malignant tumor is a serious threat to human health, among them, digestive system tumors represented by esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer and others are the greatest threat to patients. It is of great significance to further understand the mechanism of tumor development and progression in order to find new therapeutic targets. Immunotherapy for cancer is a new direction of tumor therapy following surgery, radiotherapy and chemotherapy. B7-H3(CD276), an immunoglobulin, is a new member of the B7 co-stimulatory molecule family. At present, it is found that B7-H3 is highly expressed in many kinds of tumor tissues, and is closely related to the progression of tumor, and is a potential therapeutic target for tumor.The research group has always been dedicated to the study of the expression of B7 co-stimulatory molecules in tumor. Our earlier stage study found that the co-stimulatory molecule B7-H3 was highly expressed in human esophageal cancer tissues, and its expression was negatively correlated with the number of infiltrated CD8+T lymphocytes. In vitro, target interference to esophageal cancer cell B7-H3 expression can promote the transformation of phorbol ester(PMA) induced THP-1 cells to M2 type macrophages. These findings suggest that B7-H3 may be involved in the progression of esophageal cancer. In this study, we first examined the expression of B7-H3 in human esophageal carcinoma cell strains KYSE-170, Eca-109, TE-13, TE-1, Eca-9706 and human normal esophageal epithelial cells(HEEC), found that the expression of B7-H3 in esophageal carcinoma cell line was significantly increased. Then we studied the effect of knockdown B7-H3 on the biological characteristics of esophageal cancer cells through gene transfection. Finally, we screened B7-H3 expression related genes through the Agilent expression profile gene chip and to explore the possible mechanism of action of B7-H3 involved in the progression of esophageal cancer. Part I Effect of co-stimulatory molecule B7-H3 on the biological characteristic of esophageal cancer cellsObjective: Study on the effect of knockdown B7-H3 expression on the biological characteristics of esophageal cancer cells.Methods:1 Application of real-time fluorescence quantitative PCR(qRT-PCR) and Western blot for the detection of B7-H3 mRNA and protein expression in human esophageal carcinoma cell strains KYSE-170, Eca-109, TE-13, TE-1, Eca-9706 and human normal esophageal epithelial cells(HEEC).2 Application transfected method of HuSH 29 mer shRNA to established B7-H3 stable low expression esophageal carcinoma Eca-109 cell line, and detected transfection efficiency and silencing efficiency by real time PCR, Western blot, fluorescence microscope observation and flow cytometry(FCM).3 MTS assay and clone formation assay was used to detect the effect of down-regulation of B7-H3 expression on the influence of proliferation of esophageal cancer Eca-109 cells in vitro.4 Scratch test and transwell chamber invasion assay was used to detect the effect of down-regulation of B7-H3 expression on the influence of migration and invasion of esophageal cancer Eca-109 cells.5 Flow cytometry was used to detect the effect of down-regulation of B7-H3 expression on the influence of cell apoptosis and cell cycle of esophageal cancer Eca-109 cells.6 Angiogenesis assay was used to detect the effect of down-regulation of B7-H3 expression on the influence of vascular endothelial cells angiogenesis ability esophageal cancer Eca-109 cells.7 Subcutaneous xenotransplanted tumor model in nude mice was used to detect the effect of down-regulation of B7-H3 expression on the influence of proliferation of esophageal cancer Eca-109 cells in vivo.Result:1 Real time fluorescence quantitative PCR results showed that B7-H3 mRNA exists at different levels of expression in esophageal carcinoma cell line KYSE-170(1.000±0.000), Eca-109(3.210±0.609), TE-13(1.919±0.067), TE-1(1.399±0.136), Eca-9706(1.447±0.319), and expression levels were higher than human normal esophageal epithelial cells(0.151±0.057), the difference was statistically significant(P<0.05). The results of Western Blot are consistent with the results of Real Time PCR. Because the expression of B7-H3 in esophageal cancer cell line Eca-109 is the highest, so we choose Eca-109 to carry on the following experiment.2 Successfully established B7-H3 silencing plasmid p-GFP-V-RS shRNA and control ineffective plasmid p-GFP-V-RS TR30007(TR37) stable transfection esophageal cancer Eca-109 cells, and named as Eca-109 shB7-H3 and TR37 Eca-109. Fluorescence microscopy and flow cytometry analysis showed that the transfection rate was more than 90%. Real-time PCR results showed that compared with Eca-109 cells(1.000±0.000) and Eca-109 TR37 cells(1.156±0.198), Eca-109 shB7-H3 cells(0.197±0.042) B7-H3 mRNA expression was decreased significantly and silencing efficiency is up to 80%, the difference is statistically significant(P<0.05), and there is no statistical significance differences of B7-H3 mRNA expression between Eca-109 cells and Eca-109 TR37 cells(P>0.05). The results of Western blot are consistent with the results of real time PCR. Therefore, we chose Eca-109 shB7-H3 cells and Eca-109 TR37 cells for follow experiments.3 The results of MTS assay and clone formation experiment showed that there is no significant difference of proliferation ability of Eca-109 shB7-H3 cells and Eca-109 TR37 cells in vitro(P>0.05). Flow cytometry showed that there is no significant difference of cell cycles between two kind of cells(P>0.05), suggesting that knockdown B7-H3 expression had no effect on the proliferation ability of esophageal cancer cells in vitro.4 The scratch test results showed that compared with control group of Eca-109 TR37 cells, the plane migration ability Eca-109 shB7-H3 cells decreased significantly and wound healing of scratches at 12 h and 24 h was significantly slower, the difference is statistical significance(P<0.05), indicating knockdown B7-H3 expression can reduce the planar motion ability of esophageal carcinoma Eca-109 cells.5 Transwell chamber invasion assay results showed that compared with control group of Eca-109 TR37 cells, the number of through artificial basal membrane of Eca-109 shB7-H3 cells was decreased significantly, the difference is statistically significant(P<0.05), suggesting that knockdown B7-H3 expression can reduce the invasion ability of esophageal carcinoma Eca-109 cells.6 Flow cytometry analysis results show that compared with control group of Eca-109 TR37 cells, the apoptosis rate of the and control group of Eca-109 shB7- H3 cells was significantly increased, the difference was statistically significant(P<0.05), suggesting that knockdown B7-H3 expression can promote apoptosis of esophageal carcinoma Eca-109 cells.7 Angiogenesis experiment results show that using cultured Eca-109 shB7-H3 cells 48 h supernatant culture human vascular endothelial cells after 8 h, human vascular endothelial cells formed reticular tubular length was significantly lower than that of the control group, the difference is statistically significant(P<0.05), indicating knockdown B7-H3 expression can reduce proangiogenic ability of esophageal carcinoma Eca-109 cells.8 The results of tumor bearing experiment in nude mice show that 28 days after tumor bearing, there is no significant difference of average tumor growth size(P>0.05) between experimental group(injection of Eca-109 shB7-H3 cells) and control group(injection of Eca-109 TR37 cells), indicating knockdown B7-H3 expression has no effect on esophageal carcinoma Eca-109 cells proliferation ability in vivo.Conclusion:1 B7-H3 expression was significantly up-regulated in esophageal carcinoma cells, suggesting that B7-H3 is involved in the tumor progression of esophageal cancer.2 No significant effect of knockdown B7-H3 expression on proliferation ability of esophageal carcinoma cells in vitro and in vivo, but could inhibit esophageal carcinoma cells migration, invasion and proangiogenic ability in vitro, promote apoptosis of esophageal carcinoma cells. Part ? Study on the mechanism of silencing B7-H3 gene expression inhibits invasion of esophageal carcinoma cellsObjective: To explore the mechanism of knockdown B7-H3 inhibited the invasion of esophageal carcinoma cellsMethods:1 The application of Agilent Sure Print G3 Human Gene Expression chip(8×60K Design ID:039494) for analysis of differentially expressed genes on pre-established Eca-109 shB7-H3 and Eca-109 TR37 cells, screened a variety of genes associated with B7-H3 expression.2 Application of Real time PCR, ELISA and Western Blot to verify the screened invasion related genes combined with the experimental results.Result:1 126 differentially expressed genes were screened between the experimental group Eca-109 shB7-H3 cells and the control group of Eca-109 TR37 cells, including 49 up-regulated genes and 77 genes down-regulated genes, fold difference >=2.0, P<0.05.2 ELISA results showed that compared with the control group of Eca-109 TR37 cells, the secretion level of IL-6, VEGF of supernatant of Eca-109 shB7-H3 cells was decreased significantly, the difference was statistically significant(P<0.05).3 Real time PCR and Western Blot results showed compared with the control group of Eca-109 TR37 cells, the expression level of phosphorylated Jak-2, phosphorylated STAT-3, MMP-9 and MMP-7 of experimental group Eca-109 shB7-H3 cells was significantly decreased and the expression level of TIMP-1 and EFEMP-1 was significantly increased, the difference was statistically significant(P<0.05).Conclusion:Knockdown B7-H3 protein expression can inhibit the secretion of IL-6 thereby reduced the activation of Jak2/STAT3 signaling pathway, decrease the expression of MMP-7, MMP-9 and VEGF thus inhibit the invasion and metastasis of esophageal carcinoma cells. |
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