The Mechanism Of Interaction Between YBX1 And G3BP1 Promotes Renal Cell Carcinoma Cell Metastasis Via SPP1

Objectives:Renal cell carcinoma(RCC)is one of the most common urological tumors.RCC is insidious,with no typical clinical manifestations and diverse manifestations in the early stage,resulting in a low diagnostic rate.RCC is resistant to traditional radiotherapy and chemotherapy.Although recent targeted therapies have improved the overall survival of RCC patients,the prognosis of RCC remains poor.Therefore,it is necessary to elucidate the molecular mechanisms of RCC progression to discover new prognostic markers and develop new targeted therapeutics.Y-box binding protein1(YBX1)is a transcription factors which can specifically bind to the Y-box element(CTGATTGGCCAA)of target gene promoter and enhancer to regulate their transcription and translation.YBX1 is abnormally expressed in various tumors including breast cancer,prostate cancer,nasopharyngeal carcinoma and lung adenocarcinoma,and is associated with tumor prognosis.The STRING database was used to predict the interaction of YBX1 with Ras-GTPase activating protein SH3 domain binding protein 1(G3BP1).G3BP1 is a 68 k Da cytoplasmic protein.It was identified through its ability to immunoprecipitate with the SH3 domain of Ras-GAP and involved in downstream Ras signaling pathways.The present study investigate the mechanism of YBX1 interaction with G3BP1 upregulated their downstream target secreted phosphoprotein 1(SPP1),which led to an activated NF-?B signaling pathway,regulated RCC progression,and provided experimental basis for exploring the drug resistance,new prognostic markers and anti tumor drug targets.Methods:1.YBX1 stable knockdown RCC cell lines ACHN and A498 were established via lentiviral-mediated RNA interference technology,the efficiency of YBX1 knockdown was determined by real-time PCR and western blot.The effects of YBX1 in RCC adhesion ability,migration ability and invasion ability were detected by adhesion assay,migration assay and Matrigel invasion assay,respectively.2.The effects of YBX1 knockdown on G3BP1 m RNA and protein levels and the effects of G3BP1 knockdown on YBX1 m RNA and protein levels were detected by real-time PCR and western blot.The interaction between YBX1 and G3BP1 was predicted by the STRING database.Co-immunoprecipitation assay was used to detect the interaction between YBX1 and G3BP1,and the interaction between different domains of YBX1 and G3BP1.Immunofluorescence was performed to detect the localization of YBX1 and G3BP1 in RCC cells.3.The downstream genes regulated by YBX1 were identified using a microarray.Real-time PCR and western blot were used to verify the genes and proteins expression after YBX1 or G3BP1 knockdown.The effects of YBX1 or G3BP1 on NF-?B transcriptional activity were determined by dual-luciferase reporter gene assay.The effects of YBX1 or G3BP1 knockdown on the NF-?B protein expression and phosphorylation were examined by western blot.Western blot was used to detect the effect of SPP1 knockdown on the activation of NF-?B signaling pathway induced by the upregulation of YBX1 or G3BP1.4.The effects of SPP1 down-regulation on the RCC migration and invasion regulated by YBX1 or G3BP1 were examined by migration assay and Matrigel invasion assay.5.The expressions of YBX1,G3BP1 and SPP1 in RCC and adjacent tissues was detected by western blot and immunohistochemistry.The correlation of YBX1 expression with G3BP1 and SPP1 expression was analyzed statistically.6.A mouse sub-renal capsule xenograft model was constructed,and the bioluminescence intensity of the primary tumor as well as liver and lung metastasis were observed by live imaging system.Liver and lung metastasis were detected by HE staining.Immunohistochemistry was used to detect the effect of G3BP1 knockdown on the expression levels of YBX1 and SPP1.Immunofluorescence was used to detect the localization of YBX1 and G3BP1 in the tissue section cells of the mouse xenograft models.Results:1.YBX1 stable knockdown RCC cell lines ACHN-sh YBX1/Scr and A498-sh YBX1/Scr were successfully constructed by lentiviral-mediated RNA interference technology.The m RNA(***P<0.001)and protein levels of YBX1 were significantly decreased,which were determined by real-time PCR and western blot.Compared with the control group,YBX1 knockdown significantly inhibited the adhesion ability,migration ability and invasion ability in both ACHN and A498 cells(***P<0.001).2.YBX1 knockdown did not affect the m RNA and protein levels of G3BP1.Similarly,there were no significant effects on YBX1 m RNA and protein levels following G3BP1 knockdown.YBX1 was predicted to interact directly with G3BP1 by the STRING database.Co-immunoprecipitation further confirmed the interaction between YBX1 and G3BP1.Meanwhile,YBX1C-terminal domains(aa130-205)were involved in the complex formation between YBX1 and G3BP1.Immunofluorescence revealed that the colocalization of YBX1 and G3BP1 in RCC cell cytoplasm.3.We screened differentially expressed genes in the microarray data and further confirmed that the m RNA(***P<0.001)and protein levels of SPP1 were significantly decreased after YBX1 knockdown.In addition,the m RNA(***P<0.001)and protein levels of SPP1 also decreased following G3BP1 knockdown.NF-?B transcriptional activity was decreased after YBX1(***P<0.001)or G3BP1(*P<0.05)knockdown.Compared with the control group,the phosphorylation of p65 together with the total amount of p65 protein levels were decreased after YBX1 or G3BP1 knockdown.SPP1 knockdown abolished YBX1/G3BP1-induced activation of NF-?B signaling.4.SPP1 knockdown attenuated the cell migration and invasion induced by the upregulation of YBX1 or G3BP1(*P<0.05).5.Compared with the corresponding adjacent normal tissues,the expressions of YBX1,G3BP1 and SPP1 elevated in RCC tissue(*P<0.05),and YBX1 expression was positively correlated with G3BP1 and SPP1 expression(**P<0.01).6.After G3BP1 knockdown,fluorescence intensity of primary tumors as well as liver and lung metastasis were decreased.HE staining revealed that the metastatic nodules of liver and lung were reduction(**P<0.01).Immunohistochemistry confirmed that the expression of YBX1 remained unchanged,while the expression SPP1 was significantly decreased after G3BP1 knockdown.Immunofluorescence confirmed that YBX1 and G3BP1 were co-localization in mouse xenograft models.Conclusions:1.YBX1 knockdown significantly inhibited RCC cell adhesion ability,migration ability and invasion ability.2.YBX1 interaction with G3BP1 upregulated their downstream target SPP1,which led to an activation of NF-?B signaling pathway.3.Compared with the corresponding adjacent normal tissues,the expressions of YBX1,G3BP1 and SPP1 elevated in RCC tissue,and YBX1 expression was positively correlated with G3BP1 and SPP1 expression levels in clinical RCC samples.4.YBX1 and G3BP1 co-localization in the cytoplasm in xenograft mouse models and G3BP1 knockdown regulated RCC cell metastasis through YBX1/G3BP1–SPP1 signaling pathway.