The Molecular Mechanism Of FBXO22 Regulates Renal Cell Carcinoma Cell Metastasis

Objective To study the effect of FBX022 gene on the migration,invasion,proliferation and angiogenesis ability of renal cancer cells,and to explore the related molecular mechanism of its role.Methods The expression of FBX022 in 277 renal cell carcinoma tissues and 35 normal kidney tissues was detected by immunohistochemical SP method.Renal cancer 786-0 and ACHN cells were transfected with negative control siRNA-Control and FBX022 small interfering siRNA-FBX022,respectively.The effect of siRNA-FBX022 on the expression of FBX022 protein in two renal cancer cells was detected by Western Blot.The FBX022 gene was silenced by sh-FBX022 lentivirus to construct sh-FBX022 stably transfected cell lines of 786-0 and ACHN kidney cancer cells,and sh-Control was used to construct stable cells in control group;CCK8 cell proliferation experiment observed silence The effect of FBX022 gene on the proliferation of two renal cancer cells;Transwell migration and invasion assay to detect the migration and invasion of 786-0 and ACHN kidney cancer cells after silencing FBX022 gene;The effect of silencing FBX022 gene on the secretion of MMP-9 by 786-O and ACHN cells was studied by gelatin zymography.Silencing FBXO22 gene was used to detect N-cadherin,MMP-9 and TIMP-1 and VEGF protein expression in 786-O and ACHN cells by Western Blot assay;then using HUVEC cells for CCK8 cell proliferation assay and angiogenesis experiment,observe the effect of silencing FBX022 gene on angiogenesis of renal cancer cells,and detect VEGF in transfected two renal cancer cells by ELISA Secretion changes.In vivo experiment:FBX022 gene was silenced by sh-FBX022 lentivirus to construct 786-0 sh-FBXO22 stably transfected cell line,and sh-Control was used to construct a stable cell line of control group,and the nude mice were injected into the tail vein to construct metastatic nude mice model.Six weeks later,the bioluminescence of lung metastasis in nude mice was detected by small animal living imaging system.The role of FBXO22 in tumor metastasis was studied,and the survival of nude mice was observed.Results 1.The high expression level of FBX022 was observed in 157 cases(56.7%)of RCC tissues and 30 cases(85.7%)of normal kidney tissues;the high expression rate of FBX022 in the tumor diameter>7cm group was significantly lower than that in the ?7cm group,and the difference was statistical Significance(P=0.047);the high expression rate of FBXO22 in the distant metastasis group was significantly lower than that in the distant metastasis group(P=0.020);the low expression of FBXO22 and poor 5-year overall survival(P=0.001)and 5 years Disease-free survival(P=0.003)was associated;2.Western Blot experiments showed that FBXO22 was silenced compared with siRNA-Control group.The expression of FBXO22 was decreased in 786-0 and ACHN kidney cancer cells.3.The proliferation of CCK-8 cells showed that silencing FBXO22 gene had no effect on the proliferation of 786-O and ACHN renal cell carcinoma cells.There was no statistical significance(P>0.05).4.Cell migration and invasion experiments showed that silencing FBX022 gene promoted cell migration and invasion in 786-O and ACHN cells compared with the corresponding control group,the difference was statistically significant(P<0.001);5.Gelatin zymography results showed that compared with the control group,the enzyme activity of MMP-9 secreted by 786-0 and ACHN cells in siRNA-FBX022 group was increased,and the difference was statistically significant(P<0.05).Western Blot showed that the protein expression levels of N-cadherin,MMP-9 and VEGF in 786-0 and ACHN cells of siRNA-FBXO22 group were significantly higher than those of siRNA-Ctrl group,while the expression of TIMP-1 was significantly decreased.The difference between the two groups was statistically significant(P<0.01).7.The proliferation of HUVEC and its angiogenesis experiments showed that compared with the siRNA-Ctrl group,the siRNA-FBX022 group 786-0 and ACHN two kinds of renal cancer cell supernatants.The proliferation and angiogenesis ability of HUVEC increased significantly and the difference between the two groups was statistically significant(P<0.01).8.ELISA experiments showed that the level of VEGF secreted by 786-0 and ACHN cells was significantly increased after silencing FBX022 gene,and the difference was statistically significant(P<0.01).In vivo experiments:Bioluminescence imaging showed that nude mice carrying sh-FBX022 were more likely to metastasize than mice carrying sh-Control cells.Survival curves showed that the survival time of sh-FBX022 nude mice was significantly shorter than that of sh-Control group,and the difference was statistically significant(P<0.01).Conclusion 1.The expression of FBX022 gene is down-regulated in renal cell carcinoma.2.The expression of N-cadherin was up-regulated after silencing FBX022 gene in vitro,influencing the progression of EMT in renal cell carcinoma,and promoting the expression and secretion of VEGF,which ultimately promotes angiogenesis in renal cancer cells.3.In vivo experiments,silencing FBX022 promotes lung metastasis of renal cell carcinoma.Therefore,the FBX022 gene may be a new molecular marker for judging renal cancer metastasis and will play an important role in future research and treatment.