The Effect Of FBXO22 Gene On The Migration, Invasion And Angiogenesis Of Melanoma Cells And The Study Of HIF-1?/VEGF Signaling Pathway

Objective To investigate the effect of FBXO22 gene on melanoma cell metastasis and angiogenesis and thier molecular biological mechanism.Methods The expression of FBXO22 in human melanoma was observed by immunohistochemistry with tissue microarray.Human melanoma cells MV3 and A375 were transfected with siRNA-Control,siRNA-FBXO22 and Vector,FBXO22-OE plasmid,and melanoma cells MV3 and A375 were transfected with Control-shRNA,FBXO22-shRNA,respectively.The effects of siRNA-FBXO22 and FBXO22-OE plasmid on the expression of FBXO22 and HIF-1?,VEGF in the two types of melanoma were detected by Western Blot.FBXO22 gene was interfered by lentivirus FBXO22 shRNA to construct MV3 stable cell lines,while Control-shRNA was used to to constructed stable cell line that was set as control group.CCK8 assay was conducted to observe the effect of FBXO22 gene interference on the proliferation of MV3 and A375 melanoma cells.Transwell cell migration assay was conducted to investigate the effect of overexpression and interference of FBXO22 gene on the migration ability of MV3 and A375 melanoma cells.The effect of overexpression of FBXO22 gene and interference on the invasion ability of MV3 and A375 in melanoma cells was studied by cell invasion assay.Steady cell FBXO22 was injected into the underarm fat pad of nude mice.After 1 week,the dead mice were stripped of subcutaneous tumor tissue and their angiogenesis was observed.The tumor tissue were made into wax blocks and identified by HE staining and immunohistochemical staining.Then the effect of FBXO22 on melanoma angiogenesis in vitro was observed.Finally,Western Blot,cell migration and invasion experiments were used to investigate the mechanism by which FBXO22 affects the angiogenesis and cell metastasis of melanoma through regulating HIF-1?.Results hrough the comprehensive analysis of immunohistochemistry statistics and clinical data of melanoma microarray,it was found that the expression of FBXO22 in melanoma tissues was significantly higher than that in adjacent and normal tissues,and FBXO22 expressed in metastatic melanoma was significantly higher than primary melanoma(P<0.001).In vitro experiments,Western Blot showedthat compared with the Vector group,the expression level of FBXO22 protein in MV3 and A375 cells was significantly increased after OE-FBXO22 was stable,and HIF-1? and VEGF proteins were also increased,with statistically significant differences(P<0.001).Compared with Control-siRNA group,both siRNA-FBXO22#1and siRNA-FBXO22#2 transfected cells reduced the expression of FBXO22 protein in MV3 and A375 cells,as well as the expression of HIF-1? and VEGF protein,with statistically significant differences(P ?0.001).Compared with the Control-shRNA group,FBXO22-shRNA transfection significantly reduced the expression of FBXO22 protein in MV3 and A375,as well as the HIF-1?(P ?0.001).The results of CCK8 showed that compared with the Control group,the proliferation capacity of MV3 and A375 cells in the transient transfection FBXO22-siRNA group showed no significant change,and the difference had no statistic significance(P>0.05).The migration and invasion results showed that the migration and invasion ability of MV3 and A375 cells transfected with siRNA-FBXO22#1 and siRNA-FBXO22#2 were significantly reduced compared with the Control group.Compared with the Control group,the migration and invasion abilities of MV3 and A375 cells in the OE-FBXO22 group were significantly enhanced.The angiogenesis experiment results showed that compared with the siRNA-Control group,the angiogenesis abilities of MV3 and A375 cells transfected with siRNA-FBXO22#1 and siRNA-FBXO22#2 were significantly reduced.Results of subcutaneous angiogenesis experiment showed that after removing subcutaneous tumor from the nude mouse model for 1 week,the angiogenesis ability of melanoma cells was significantly reduced after the interference of FBXO22 was observed under the microscope.The immunohistochemical staining results of the removed tumor showed that the expressions of FBXO22,HIF-1? and CD31 in the subcutaneous tumors were significantly decreased after the interference of FBXO22.Conclushon 1.FBXO22 was highly expressed in melanoma tissues,and its expression in metastatic melanoma was higher than that in non-metastatic melanoma.2.In vitro experiments showed that interfering with FBXO22 reduced the angiogenesis,migration and invasion ability of melanoma cells,and overexpressionof FBXO22 increased the migration and invasion ability of melanoma cells.3.In vivo studies have shown that interfering with FBXO22 can reduce HIF-1 expression and thereby decrease the angiogenesis and metastasis of melanoma cells.