Preliminary Screening Of The Receptor Information Of Vascular Endothelial-cell-specific Binding Peptide GEBP11 In Gastric Cancer

【Background】Tumor angiogenesis is related closely to growth, invasion, metastatic and prognosis of tumor. The researches of tumor angiogenesis provide new strategy and perspective for the diagnosis and treatment of tumor. However, the status of diagnosis and treatment of tumor by angiogenesis is not satisfactory due to the deficiency of target molecules. Researches indicated that the genetic component of vascular endothelial cells changed in the tumor microenvironment and the endothelial cells expressed heterogeneity molecules, which would probably provide target molecules for diagnosis and therapeutics of tumor. The heterogeneity of tumor vascular depends on that of tumor and decided by the type and microenvironment of tumor. The co-culture of gastric cancer cells and vascular endothelial cells in vitro could simulate tumor microenvironment and induce the expression of the heterogeneity molecules. In previous studies, we obtained a peptide GEBP11 specifically binding to the co-cultured human umbilical vein endothelial cells (Co-HUVECs) through establishing an in vitro co-culture model of gastric cancer cell line SGC7901 and HUVECs and screening in phage display peptides library. Initial studies suggested that GEBP11 peptide has the ability in targeting to gastric cancer vascular and the biological activity in inhibiting angiogenesis. It is important to screen the receptor of GEBP11 for the mechanistic study of the targeting and angiogenesis inhibiting activities of GEBP11. Therefore, the aim of this study is screening the information about the receptor of GEBP11 and setting a foundation for obtaining the receptor.【Objectives】1. To obtain the localizing information of the receptor of GEBP11 in gastric cancer cell lines, normal tissues and multiple organ cancers; 2. To detect the changed genes of Co-HUVECs and pathways influenced after treated by GEBP11, which could provide information for screening the receptor; 3. To screen the receptor of GEBP11 and settle a foundation for elucidating the mechanism and function of GEBP11.【Methods】1. Immunohistochemistry and immunocytochemistry were performed to detect the expression of the receptor of GEBP11 in normal tissues, multiple organ cancers and gastric cancer cell lines.2. The result of gene array was verified by RT-PCR and Western blot; bioinformatics was used to analyze the different genes and pathways in Co-HUVECs after interfered by GEBP11.3. The receptor of GEBP11 was screened by immunoprecipitation and mass spectroscopy.【Results】1. The expression of the receptor of GEBP111) The expresstion of the receptor of GEBP11 in cell lines. The result of immunocytochemistry revealed that GEBP11 was stained on the cytoplasm of Co-HUVECs and MKN45, but not MKN28, SGC7901, AGS, GES.2) The expresstion of the receptor of GEBP11 in multiple organ cancers.The result of immunohistochemistry showed that GEBP11 was stained on the vascular of gastric cancer, pancreatic cancer and hysterocarcinoma, but not other cancers including cancer of lung, bladder, suprarenal epithelioma, colon, esophagus, rectum, liver.3) The expresstion of the receptor of GEBP11 in normal tissues.The result of immunohistochemistry showed that GEBP11 was not stained on the vascular of normal tissues including esophagus, liver, pancreas, hypophysis, thyroid, parathyroid gland, cardiac muscle, lung, kidney, thymus, marrow, spleen, mammary gland, ovary, tonsil, testis, sialaden, stomach, small intestine, colon.2. The changed genes of Co-HUVECs and pathways after treat by GEBP11The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.3. The screening of the receptor of GEBP11We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Compared with the result of gene microarray, we found that the expression of candidant protein ZCCHC11 was upregulated about 3 folds after treated by GEBP11.【Conclusions】1. The receptor of GEBP11 expressed in some poorly differentiated gastric cancer cell lines and vascular of some other cancers, but not that of normal tissues.2. The results of RT-PCR and Western blot were consistent with gene microarray. The screening of differential genes and analysis of GoTerm and pathway provided information for the receptor of GEBP11.3. We obtained 12 candidant proteins by using the methods of immunoprecipitation and mass spectrography. Candiant protein ZCCHC11 was upregulated about 3folds after treated by GEBP11.