BAG2 Inhibits The Migration And Invasion Of Renal Clear Cancer Cells By Promoting YBX3 Ubiquitination

Objective: Renal clear cell cancer is one of the common urinary system tumors,the incidence of male is higher than that of female,some patients have lost the opportunity of surgery at the time of diagnosis,advanced renal cancer patients have fewer treatment options,high mortality and poor prognosis.For advanced renal clear cell cancer,targeted drugs mainly focus on anti-VEGF and m TOR inhibitors,which are not highly effective.It is very important to study the molecular mechanism of renal clear cell cancer in the treatment of renal cancer.BAG2 is a member of the Bcl-2 associated athanogene(BAG)protein family.Members of the BAG family all contain at least one BAG domain,which acts through its conserved BAG domain binding to the ATPase domain of the Hsp70/Hsc70 heat shock protein and is therefore also known as cochaperone proteins.BAG familiy is a multifunctional protein that can be involved in regulating various physiological processes,including apoptosis,neuronal differentiation,tumorigenesis,stress response and cell cycle.BAG2 is highly expressed in a variety of different tumors.Many studies have shown that BAG2 affects the proliferation,migration and invasion of tumor cells.Multiple databases have also shown that high expression of BAG2 in multiple tumors is associated with poor prognosis.Although these results suggest that BAG2 has a potential function in the tumor progression,its role in renal cancer has not been studied.In our previous study,we found that BAG2 expression was lower in renal clear cell cancer tissues than its paired paratumoral tissues,which was inconsistent with its role in other tumors,and the mechanism remains to be clarified.YBX3 is a member of the Y-box protein family.In recent years,there has been increasing evidence that YBX3 plays a very important role in the progression of various diseases,such as cancer,kidney disease and gastrointestinal diseases.It plays a role in tumor or other diseases related to cell cycle regulatory factors,such as cyclin D1,PCNA,etc.It is also reported that YBX3 can directly regulate SLC7A5 to regulate the occurrence and development of tumors.YBX3 is considered as a marker of poor prognosis in many tumors.YBX3 is an independent prognostic factor for renal cancer.With the increase of YBX3 expression,patients have later tumor stage and worse prognosis.In the preliminary study of this experiment,proteins interacting with BAG2 were screened by mass spectrometry,and it was found that YBX3 may be one of the potential proteins interacting with BAG2.Thereby,BAG2 may play a role in renal clear cell cancer by regulating YBX3,and its regulatory mechanism needs to be further analyzed.Methods:1.Lentivirus transfection was used to construct Caki1 and Caki2 cell lines with stable knockoff or overexpression of BAG2,and Western blot was used to detect the efficiency.2.The changes of cell proliferation in Caki1 and Caki2 cell lines were detected by direct cell count assay and Edu cell proliferation assay,and the effects of BAG2 on the migration and invasion ability of Caki1 and Caki2 cells were detected by Transwell transfer chamber assay.3.IP combined with mass spectrometry was used to screen the interacting proteins with BAG2,and the role of the interacting proteins with BAG2 was analyzed by bioinformatics database.4.Interaction between BAG2 and YBX3 was verified by co-IP and PLA.5.Western blot and q RT-PCR were used to detect the effects of BAG2 knockoff or overexpression on YBX3 protein and m RNA expression levels,respectively.6.Caki1 and Caki2 cells were transfected with Flag-BAG2 and Myc-YBX3 recombinant plasmids,and the transfection efficiency was detected by Western blot.The effects of BAG2 and YBX3 on the migration and invasion of renal clear cell carcinoma cells were detected by Transwell migration chamber.7.Lysosome inhibitor E64D+pepstatin or proteasome inhibitor MG132 was used to detect the degradation pathway of YBX3 protein.8.The effect of BAG2 overexpression on YBX3 ubiquitination level was detected by ubiquitination experiment.9.Through database(PLMD,http://plmd.biocuckoo.org/)YBX3 amino end to find possible ubiquitin modifications of sites,ubiquitin experimental verification.10.The possible E3 ubiquitin ligase with the highest score was found through the software prediction,and the corresponding si RNA was transfected.The transfection efficiency was detected by Western blot,and the E3 ubiquitin ligase was searched in the ubiquitin experiment.11.Wild type and H260 Q mutant STUB1 plasmid were constructed,and ubiquitination experiments were conducted to find the functional domain of STUB1.12.co-IP experiment verified the interaction between BAG2,YBX3 and STUB1.13.Lentivirus transfection was used to construct Caki1 and Caki2 cell lines with stable knockoff or stable overexpression of BAG2,and Western blot was used to detect the stability efficiency and the effect on the expression of YBX3 and STUB1.Results:1.BAG2 knockoff promotes the migration and invasion of renal clear cancer cell lines.Lentivirus infection was used to construct Caki1 and Caki2 cells with BAG2 stable knockoff.Direct cell counting and Edu cell proliferation experiments were conducted to observe that BAG2 knockoff had no effect on the survival and proliferation of renal clear cancer cells.Transwell migration and invasion experiment observed that BAG2 knockoff promoted the migration and invasion of renal clear cancer cells.2.Overexpression of BAG2 reduces the migratory and invasive abilities of renal clear cancer cells.Lentivirus infection was used to construct Caki1 and Caki2 cells with stable BAG2 overexpression.Direct cell counting and Edu cell proliferation experiments were conducted to observe that BAG2 overexpression had no effect on the survival and proliferation of renal clear cancer cells.Transwell migration and invasion assay observed that BAG2 overexpression inhibited the migration and invasion of renal clear cancer cells.3.BAG2 interacts with YBX3.It was found that YBX3 may be the interacting protein of BAG2 by using IP combined mass spectrometry,and the interaction between BAG2 and YBX3 was verified by experiments of co-IP and PLA.4.Overexpression of BAG2 reduces the migration and invasion ability of renal clear cancer cells,which is related to the downregulation of YBX3.Transwell migration and invasion of Caki and Caki2 cells showed that: When BAG2 was overexpressed,cell migration ability was weakened,and when YBX3 was overexpressed alone or simultaneously with BAG2,migration and invasion ability were enhanced,suggesting that YBX3 was the downstream target molecule of BAG2,and BAG2 might regulate the migration and invasion ability of renal clear cell carcinoma through YBX3.Overexpression of YBX3 can reverse the inhibition of BAG2 overexpression on migration and invasion ability of renal clear cell carcinoma.5.BAG2 knockoff promotes migration and invasion of renal clear cancer cell lines,which is related to YBX3 upregulation.Transwell migration and invasion experiments of Caki and Caki2 cells showed that when BAG2 was knocked down,cell migration and invasion were enhanced;when YBX3 was knocked down,cell migration and invasion were weakened;when BAG2 and YBX3 were knocked down simultaneously,cell migration and invasion were weakened.It was further confirmed that BAG2 regulates the migration and invasion ability of renal clear cell carcinoma through YBX3,and knocking down YBX3 can reverse the promotion of BAG2 knocking down on the migration and invasion ability of renal clear cell carcinoma.6.BAG2 regulates the stability of YBX3 through the ubiquitin-proteasome system.Caki1 and Caki2 cells stably overexpressed with BAG2 were treated with E64D+pepstatin and MG132 to block the lysosome degradation pathway and proteasome degradation pathway,respectively.The results suggested that BAG2 regulated the expression level of YBX3 through the proteasome pathway.The possible ubiquitization sites of YBX3 were searched in the database,and the ubiquitization experiments were conducted,suggesting that BAG2 affects the ubiquitization modification of YBX3 protein at K90 site.7.STUB1 is the E3 ubiquitin ligase for YBX3 ubiquitination.The website was used to predict the potential E3 ubiquitin ligase by mediating YBX3 ubiquitination.The ubiquitin experiment confirmed that STUB1 was the E3 ubiquitin ligase of YBX3 ubiquitination,and the interaction between the three was further verified.co-IP experiment confirmed that BAG2 enhanced the interaction between YBX3 and STUB1.Conclusion:1.BAG2 expression is lower in renal clear cell carcinoma,and is negatively correlated with YBX3 expression.Low BAG2 expression promotes the invasion and metastasis of renal clear cell carcinoma.2.BAG2 promotes ubiquitination degradation of YBX3 to regulate the stability of YBX3.STUB1 is the E3 ubiquitin ligase of YBX3.