The Role Of G3BP1 On Renal Cell Carcinoma Proliferation And Metastasis

Objectives and Contents:Renal carcinoma also called renal adenocarcinoma or renal cell carcinoma(Renal cell carcinoma,RCC),originated in the renal parenchyma urinary tubular epithelium system,is the most common urological cancer.Many cases of RCC are symptomless until the condition is malignant,and about one-third of RCC patients develop local invasive or metastatic diseases when they are initially diagnosed.Though surgical intervention followed by immunotherapy is emerging as a therapeutic option for RCC with metastasis.However,it is still failed to demonstrate a clear benefit of this therapeutic strategy for the overall survival of RCC patients.Thus,it is urgent to understand the molecular mechanisms of RCC progression and metastasis,which is critical for development of novel targeting therapies for advanced RCC.G3BP1(Ras-GTPase-activating protein SH3 domain binding protein,G3BP1)is overexpression in human cancers,including breast,gastric,colon and liver carcinomas,suggesting the oncogenic and functional role of G3BP1 in tumorigenesis.However,it remains unknown whether and how G3BP1 is participated in RCC progression and metastasis.In this report,we explored the expression of G3BP1 in primary RCC and its association with clinicopathological parameters.Functionally,we investigated the effects of G3BP1 on RCC cell proliferation,migration and invasion in vitro and in vivo.We further defined the molecular mechanism of G3BP1 in RCC.Methods: 1.Western blot and Immunohistochemistry staining assay were applied to detect the expression of G3BP1 in RCC tissues and their corresponding para-tumor tissues;statistically analyzed the correlation between the expression of G3BP1 and clinicopathological features.2.G3BP1 knockdown stable renal carcinoma cells ACHN and A498 were constructed by lentivirus mediated RNAi technology,Western blot and Real time PCR were applied to detect its protein and mRNA expression.3.CCK8 assay was used to examine the effect of G3BP1 knockdown on proliferation ability of renal carcinoma cells ACHN and A498.4.Western blot was used to detect the effect of G3BP1 knockdown on the proteins associated with cell proliferation.5.Migration assay,Matrigel invasion assay and EGF induced chemotaxis assay were respectively used to detect the effects of G3BP1 knockdown on migration ability,Matrigel invasion ability and EGF induced chemotaxis ability of renal carcinoma cells ACHN and A498.6.Western blot was used to detect the effect of G3BP1 knockdown on the proteins associated with EMT.7.Luciferase assay was used to screen the change of related signaling pathways on G3BP1 knockdown.8.Western blot was used to detect the molecular mechanism on the progression and metastasis of renal cell carcinoma.9.Western blot was used to detect the expressions of G3BP1,IL-6 and p-STAT3 in renal cell carcinoma,statistically analyzed the correlation between the expression of G3BP1 and IL-6 or p-STAT3.10.In vivo imaging assay was ussed to investigate the effect of G3BP1 knockdown on the form of orthotopic tumor and metastasis of liver and lung in mice.Results: 1.Compared with their corresponding para-tumor tissues,the protein expression of G3BP1 was higher in renal cell carcinoma tissues,and the expression level of G3BP1 in RCC patients was associated with higher levels of TNM stages and Fuhrman grade(*P<0.05).Meanwhile,no statistically significant correlations were identified between the expression of G3BP1 and any other clinicopathological characteristics,including patient's age,gender,tumor size,tumor side and necrosis.2.The lentivirus-mediated RNAi technology had been successfully used to establish G3BP1 knockdown stable cell lines ACHN and A498,the protein and mRNA expression of G3BP1 was significantly decreased(**P<0.01),which was detected by Western blot and Real time PCR.3.After G3BP1 knockdown,the proliferation ability of renal carcinoma cells ACHN and A498 was significantly decreased than control(**P<0.01).4.After G3BP1 knockdown,compared to control group,the protein expression of c-myc and cyclin D1 was significantly decreased in renal carcinoma cells ACHN and A498.5.After G3BP1 knockdown,the migration ability,Matrigel invasion ability and EGF induced chemotaxis ability of renal carcinoma cells ACHN and A498 were significantly decreased(**P<0.01).6.After G3BP1 knockdown,the protein expression of epithelial cell marker E-cadherin was significantly increased,while the protein expression of mesenchymal cell markers N-cadherin,Vimentin,Snail and Snail was significantly decreased than control in renal carcinoma cells ACHN and A498.7.After G3BP1 knockdown,the signaling pathway such as p-STAT3 was inhibited in renal carcinoma cells ACHN and A498(**P<0.01).8.After G3BP1 knockdown,the expression of p-STAT3 was significantly decreased than control in renal carcinoma cells ACHN and A498.Moreover,knockdown of G3BP1 dramatically impaired the signaling connection of IL-6 stimulation and downstream STAT3 activation,eventually inhibited migration and Matrigel invasion ability of renal carcinoma cell(**P<0.01).Stattic which is the inhibitor of STAT3 did not affect the expression of G3BP1.9.Compared with their corresponding para-tumor tissues,the protein expression of G3BP1 was higher in renal cell caicinoma tissues(*P<0.05),and the expression of G3BP1 was significantly associated with the expression of p-STAT3 and IL-6(**P<0.01).10.After G3BP1 knockdown,the fluorescence intensity of tumor sites on the kidney,liver and lung metastasis were reduced,and the kidney tumor volume was smaller,in addition,the formation of liver and lung metastasis numbers were also significantly decreased than control(**P<0.01).Conclusions: 1.Compared with their corresponding para-tumor tissues,the expression of G3BP1 was higher in renal carcinoma tissues,and G3BP1 promoted the proliferation and metastasis of renal cell carcinoma.2.G3BP1 knockdown inhibited cell proliferation by inhibiting the expression of c-myc and cyclin D1.3.G3BP1 knockdown inhibited cell metastasis by inhibiting EMT.4.After G3BP1 knockdown,the expression of p-STAT3 was significantly decreased.Moreover,knockdown of G3BP1 dramatically impaired the signaling connection of IL-6 stimulation and downstream STAT3 activation,eventually inhibits migration and Matrigel invasion ability of renal carcinoma cell.5.Compared with their corresponding para-tumor tissues,the G3BP1 expression was higher in renal cell carcinoma tissues.Meanwhile,the expression of G3BP1 was significantly associated with the expression of p-STAT3 and IL-6 expression in renal cell carcinoma tissues.