| BackgroundIκBαserves as a central anchoring molecule in the sequestration of NF-κB transcription factor in the cytoplasm. Upon the signal of upstream, ubiquitination-mediated IκBαdegradation immediatedly precedes and is required for NF-κB nuclear translocation and activiation. However, the precise mechanism for the deubuiquitination of IκBαis still not fully understood.Metastatic clear cell renal cell carcinoma (ccRCC) has a poor prognosis and unpredictable course, and resistant to radiotherapy and chemotherapy. A full understanding of the molecular genetics and signaling pathways involved in the metastatic process of ccRCC is important for early detection of ccRCC, prediction of metastasis and the development of innovative treatment options.MethodsUbquitin-specific proteases (USPs) expressional library was established. Using proteomic approach and reporter gene assay, we screened this library and got the potential candidate, which can inhibit NF-κB activation and bind with IκBα. Either for the endogenous or overexpression of USP, the physical binding with and functional deubiquitination of IκBαwere detected in vivo and in vitro. In stable knockdown of USP 11 cells, the expression and modification of the protein related to TNFa induced IKBa ubiquitination and NF-κB activation were detected. Moreover, IL6, the NF-κB target gene, were detected at the transcriptional and expressional level.Metastatic (RCC05TXJ) or non-metastatic (RCC05ZYJ) ccRCC cell lines were established by our lab. Differentially expressed genes and significant signal pathway, which was related to metastatic process and differentially expressed genes were involved in, were analyzed by cDNA microarray and bioinformatics approach. The expression of the molecule of our interest, which were related to the key signal pathway relative to metastatic process, were detected at different time point by real time quantitative PCR. After knocking down USP11 expression in RCC05ZYJ cells, the expression of molecule of interest were detected. ResultsUSP11 is associated with IκBαin vivo and in vitro. USP11 is an IκBαassociated deubiquitinase. Overexpression of USP11 strongly inhibits IκBαubiquitination and NF-κB activation. Recombinant of USP11 catalyzes deubiquitination of IκBαin vitro. Further, suppression of USP11 expression of enhances TNFa induced IκBαubiquitination and NF-κB activation. Suppression of USP11 enhances TNFa induced NF-κB-dependent IL6 gene expression.In the differentially expressed gene profile, up-regulation of CA9,VEGF, MMP2, LAMA4,TNF reflected the characteristics of ccRCC and tumor invasion and metastatics. Up-regulation of cytochrome P450 family members indicated the association of metabolism of environmental agents and ccRCC progression. VEGF, Hypoxia and angiogenesis signal pathway enriched the differentially expressed genes (p<0.05, Benjamini<1). RhoC was up-regulated at 48h after passage in RCC05TXJ cells compared to RCC05ZYJ cells (p<0.05), which indicated the genetic background of metastatic ccRCC. The expression of HIF1αwas up-regulated in RCC05TXJ and RCC05ZYJ cells, but no significant difference was detected in these two cell lines. The expression of TNFa,IL6,VEGF, MMP2 at 72h after passage was up-regulated in RCC05TXJ cells compared to RCC05ZYJ cells (p<0.05), and the up-regulated expression of TNFa,IL6,VEGF was also detected (p＜0.05) in RCC05ZYJ cells of which USP11 expression was knocked down to-50%. It strongly indicates the NF-κB signaling pathway and VEGF pathway is related to the progression of ccRCC invasion and metastatics. Further study will be focused on the interaction of these two pathways.ConclusionUSP11 negatively regulates TNFa induced NF-κB activation by deubiquitination of IκBα; NF-κB signal pathway and VEGF, Hypoxia signal pathway play an important role in the process of ccRCC invasion and metastatics.
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