| Objective: 1.To study the expression of FGF2 in malignant choroidal melanoma.2.It has been proved that the stimulation of human invasive choroidal melanoma(MUM2B)cell line by FGF2 makes tumor cells larger and more likely to migrate.This process is achieved through calcium storage channel(SOCE)regulation of calcium release and activation of calcineurin(Orai1)and stromal cell reaction molecule 1(STIM1)expression.3.Verify that calcium channel blocker inositol triphosphate receptor antagonist(2-APB)reverses the changes of tumor cells induced by FGF2.Methods: 1.To collect the general information of patients with malignant choroidal melanoma and the clinical data of tumor tissue(carcinoma in situ and metastasis to various organs and lymph nodes).HE staining and immunohistochemistry were used to detect the expression of fibroblast growth factor 2(FGF2)in tumor,and the relationship between the level of fibroblast growth factor 2 and clinical stage was analyzed.Meanwhile,the expression of Orai1 and STIM1 protein in the samples were compared.2.MUM2 B cells were divided into three groups: tumor cell group(control group),tumor cell + FGF2 group(FGF2 group),tumor cell + FGF2 +2-APB group(FGF2/2-APB group).Fluo3-AM was used as calcium indicator to stain the intracellular calcium ions,and then the content of calcium ions in the three groups was detected by flow cytometry.3.Western-Blot method was used to detect the expression of Orial and STIM1 proteins in control group,FGF2 group and FGF2/2-APB group.The relative concentration of reaction band was analyzed by Odyssey far-infrared detector,and the immunoreaction band was monitored by Ecl protein imprinting substrate image.4.The wound healing test,chemotaxis test in vitro,cell adhesion test and cytoskeleton staining test were used to observe and evaluate the migration and adhesion of the cells in the control group,the FGF2 group and the FGF2/2-APB group.5.Nude mice were randomly divided into three groups: control group,fibroblast growth factor 2 group and fibroblast growth factor 2/2-APB group.Each group had 5 mice.All the three groups were subcutaneously injected with 3 *106 MUM2 B cells.The fibroblast growth factor 2 group was injected with fibroblast growth factor 2,and the fibroblast growth factor 2/2-APB group was injected with fibroblast growth factor 2 and2-APB.The metastasis and FGF2 expression of tumor tissues were observed by HE staining and immunohistochemistry.6.Statistical analysis: SPSS21.0 statistical analysis software was used to analyze the data.Use Graphpad to make statistical chart.Normality test was carried out on the measured data,and the measured data conforming to the normal distribution were described by mean standard deviation.The mean values of two samples were compared by independent sample t test,and the mean values of multiple samples were compared by one-way ANOVA.The relationship between FGF2 expression and clinicopathological parameters was evaluated by Fisher experiment.All statistical analyses were bilateral test,and P<0.05 was statistically significant.Results: 1.HE staining and immunohistochemical staining showed that18(56.3%)of the 32 UM specimens were positive for FGF2.The metastasis rate of the patients with positive expression of FGF2(10/12,83.3%)was higher than that of the patients with negative expression of FGF2(2/14,14.3%).The expression of FGF2 was closely related to pathological classification and tumor metastasis.The expression of FGF2 was positive in epithelioid(16/20,80%)and negative in spindle cell(2/12,16.7%).The expression of FGF2 was higher in lung metastases(12/20,60%)than in primary tumors(8/20,40%)(P < 0.05).Orai1 and STIM1 protein were detected in 32 human UM samples.The expression of Orai1(13/17,76.5%)and STIM1(11/13,84.6%)were higher in the positive expression of FGF2(P <0.05).Orai1 expression(4/17,23.5%)and STIM1expression(2/13,15.4%)were weak in samples with negative expression of FGF2.2.Fluo-3AM flow cytometry showed that the average fluorescence density of calcium ions added with FGF2 in MUM2 B cells was significantly higher than that in blank control group and FGF2/2-APB cells(P < 0.05).3.Western-Blot experiments show that FGF2 has a positive correlation between Orai1 and STIM1 protein expression of two.Compared with the blank control group,the expression of Orai1 and STIM1 protein increased significantly in group FGF2(P<0.05).The expression of Orai 1 and STIM 1 protein in FGF2/2-APB group was significantly lower than that in FGF2 group(P < 0.05).4.Compared with the blank control group,there were significant differences in the distance of movement and the speed of wound repair between the two groups(P < 0.05).In the cell adhesion test,FGF2 caused the decrease of adhesion between MUM2 B cells.In the chemotaxis test in vitro,FGF2 caused the decrease of adhesion between MUM2 B cells.The cell chemotaxis ability was strengthened.The addition of 2-APB weakened the horizontal and vertical migration ability of MM2 B cells stimulated by FGF2,decreased the adhesion ability of MM2 B cells stimulated by FGF2,and decreased the chemotactic ability of cells.In cytoskeleton staining experiment,the stimulation of fibroblast growth factor 2(FGF2)induced cell polarization and cytoskeleton aggregation,resulting in the pseudopodal process of MUM2 B cells.2-APB prevented the aggregation of MUM2 B cytoskeleton.Compared with the control group,the motor ability of MUM2 B cells stimulated by FGF2 was significantly lower in the group of FGF2/2-APB.5.In the nude mice model of subcutaneous xenotransplantation of MUM2 B cells,the tumor volume growth curve showed that the tumor mass of the stimulation group with FGF2 was higher than that of the control group and the treatment group with FGF2/2-APB.HE and immunohistochemical staining showed that one liver and lung metastasis,one lung metastasis and two neovascularization invasion were found in the fibroblast growth factor 2 group,but no metastasis site was found in the fibroblast growth factor 2/2-APB group.Moreover,the expression of Orai1 and STIM1 increased significantly in group FGF2.In the blank control group and FGF2/2-APB group,the tumor tissues were not expressed weakly(P<0.05).Conclusion: 1.There is a high expression of FGF2 in tumor tissues of MU patients.The expression of FGF2 is closely related to pathological classification and metastasis.2.FGF2 promotes tumor cell growth and migration by controlling the expression of Orai1 and STIM1 mediated by SOCE.3.Calcium channel blocker 2-APB prevented the growth and migration of tumor stimulated by FGF2,and decreased the expression of Orai1 and STIM1. |
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