Role And Regulation Of OPN In Gastric Cancer

Osteopontin (OPN) was firstly described by Senger in 1979 as a phosphoprotein secreted by transformed malignant epithelial cells. OPN is a secreted glycoprotein that is rich in aspartate and sialic acid residues and contains several functional domains. OPN functions by mediating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. Recently, substantial data have linked OPN with the regulation of metastatic spread, angiogenesis and prevention of apoptosis by tumor cells. However, the role and regulation of OPN in gastric cancer are incompletely understood. Our study casts some light on the role and regulation of OPN in gastric cancer. It enriches our knowledge of gastric carcinogenesis.[Objectives] (1) To examine the expression of OPN in gastric cancer tissues and cell lines and its correlation with the characteristics of gastric cancers; (2) To investigate the regulation and possible mechanisms of OPN in gastric cancer cells; (3) To explore the possible roles of OPN on the malignant phenotye and angiogenesis of gastric cancer cells.[ Methods ](1) Expression of OPN in gastric cancer tissues was examined by immunohistochemistry and its correlations with the characteristics of gastric cancers, COX-2, VEGF and MVD were analysed by statistics; (2) Expression of OPN mRNA and protein in gastric cancer cell lines were examined by semi-quantitatively RT-PCR and Western blotting; (3) siRNA expression vectors of OPN were constructed. Stable transfectants were obtained by G418selection. mRNA and its corresponding protein in the transfected cells were examined; (4)RT-PCR and Western blotting analysis were performed to examine the expression change of OPN after gastric cancer cell lines were administrated with PI3K inhibitor LY294002, selective COX-2 inhibitor celecoxib and PGE2. (5) MTT assay was used to draw the growth curves of the transfected cells and control cells. (6) Flow cytometry (FCM) was used to examine the cell cycle distribution of the transfected cells and their control counterparts; (7) The ultrastructure of the transfected cells and control cells was observed using electromicroscope; (8)Wound-healing assay was used to examine the migration ability of the transfected cells and control cells; (9) Colony formation assay was carried out to evaluate the proliferation of transfectants; Anchorage-independent growth as a parameter of in vitro tumorigenicity was assessed by soft agar clonogenic assay; (10)Xenograft experiment was used for analysis of the tumorigenicity of transfected cells in vivo; (11)The expression of endothelium-specific angiogenic factors in gastric cancer cell lines was measured by semi-quantitative RT-PCR; (12)Conditioned culture media from cells SGC7901, SGC7901/mU6pro and SGC7901/OPN-siRNA (OPN-siRNA transfected cells) were used to stimulate EC, and the proliferation, apoptosis, migration and tube-formation ability of EC were assessed upon stimulation of the conditioned medium; (13)The micreovessel density (MVD) of the tumor grafts from the transfected cells and control cells was assayed by VIII factor positive blood vessels counting. [Results] (1) Compared with normal mucosa, OPN, COX-2 and VEGF were over-expressed in cancerous tissues. Positive expression of OPN, COX-2 and VEGF in cancerous tissues was 75.5% (40/53), 83.0% (44/53) and 84.9% (45/53). Moreover, the results indicate co-expression of OPN, COX-2, and VEGF in gastric cancer. Levels of OPN, COX-2, and VEGF were all significantly correlated with MVD, TNM stage, lymph node metastasis and distant metastasis; (2) OPN was over-expressed in gastric cancer cell lines GC9811, SGC7901, MKN45 at the levels of protein and mRNA while it was expressed at very low level in AGS cell line and it was not detected inMGC803 cell line; (3) The eukaryotic expression vector of mU6pro/OPN-siRNA, which down-regulated mRNA and protein of OPN in the transfected gastric cancer cells, was successfully constructed; (4) The level of COX-2 had not been changed when SGC7901 cells were transfected with OPN siRNA while the expression of OPN was down-regulated when SGC7901 cells were transfected with anti-sense COX-2 cDNA. The expression of OPN was down-regulated by Celecoxib and LY294002 and it was up-regulated by PGE2. LY294002 could inhibit the effect of PGE2 on OPN; (5) MTT assay revealed that proliferation of transfected cells wasn't altered significantly; (6) No significant changes of cell cycle were found in all transfectants; (7) The ultrastructures of the transfected cells and control cells had not differences; (8) Wound-healing assay showed the migration ability of the transfected cells and control cells was similar; (9) Colony formation assay and soft agar clonogenic assay indicated that the proliferation and Anchorage-independent growth of transfectants were not altered significantly; (10) Tumorigenesis assay showed that SGC7901/OPN-siRNA group grew slower than other groups and the mean tumor graft weight (mg) 30 days afterimplantation in nude mice were (;r±SD): SGC7901 group(638.12±45.76);SGC7901/mU6pro group(652.67±58.13); SGC7901/OPN-siRNA group (232.11±22.52). Statistics results showed a significant growth suppression of OPN siRNA vector transfected cells (P<0.01); (11) Semi-quantitative RT-PCR showed the expression of endothelium specific angiogenic factors in gastric cancer cell lines had not been changed; (12) When incubated with the conditioned medium from SGC7901/OPN-siRNA, proliferation, migration and tube-formation ability of EC were inhibited (P<0.05, P<0.01,P<0.01) and apoptosis increased, compared with those from parental and empty-vector transfected cells; (13) The tumor graft of SGC7901/OPN-siRNA cell in nudemice had less vessel formation with MVD (,x±SD) 4.21±1.07, comparedwith SGC7901 group(8.28±1.56) and SGC7901/mU6pro group(8.57±1.62) (P<0.01).