PSMB4 Promotes The Proliferation Of Clear Cell Renal Cell Carcinoma Via NF-?B Signaling Pathway

Introduction: Renal cell carcinoma(RCC)is the second most common urological cancer,and accounts for nearly 3% of malignant neoplasms in the world.More than 90% of kidney tumors are renal cell carcinomas,arising from the epithelial lining of the proximal convoluted tube.Among RCC subtypes,clear cell renal cell carcinoma(ccRCC)is the most common one(70-80%),and histologically is the most aggressive one.Proteasome function alterations are connected with tumor cell progression.The proteasome consists of ATP-dependent proteases,with caspase-,trypsin-and chymotrypsin-like activities on ubiquitinated proteins,and is crucial to the degradation of proteins in cells by the ubiquitin-proteasome pathway.The proteasome is important to the regulation of cellular processes such as proliferation,apoptosis,migration,gene transcription,and immune responses.The most common proteasome form is the 26 S,which contains nearly 60 subunits and comprises approximately 2% of cell protein.It is crucial to many vital biological functions.The 26 S proteasome consists of a 20 S core particle and is bound to one or two 19 S regulatory particles.Proteasome subunit beta type-4(PSMB4),the b7 subunit of the 20 S core complex,regulates the assembly of the proteasome,which is the rate-limiting procedure for 20 S proteasome assembly.PSMB4 are highly expressed in breast,lung,ovarian,skin,prostate tumor,hepatocellular carcinoma,myeloid leukemia,and gliomas.PSMB4 is identified as the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo.Evidence shows that the proteasome is vital for several proliferative and anti-apoptosis signaling pathways,indicating the importance of the proteasome in the progression of tumors.Up to now,there is no aricle to illustrate the relationship between ccRCC and PSMB4.The study of PSMB4 may have implications for the treatment of renal cancer.Proteasome inhibitors are therapeutic candidates in tumor treatment.Methods: We collected the expression levels of PSMB4 as well as the clinical information of patients from TGCA database.With bioinformatic analysis,we detected the expression difference of PSMB4 in ccRCC tissues,and in their adjacent normal tissues.We also found the correlation between PSMB4 and several clinical characteristics.With GSEA analysis,we showed the relationship between PSMB4 and several signaling pathways downsteam,including NF-?B axis.Q-rt PCR and Western Blotting were conducted to measure the expression level of PSMB4 in ccRCC tissues and cell lines,as well as in cancner tissues and normal tissues,including the adjacnet normal tissues.Becides,si-RNA was used to downregulate the expression of PSMB4 in ccRCC cells.The animal model was aslo estabished.The affection of PSMB4 on the proliferation of ccRCC was uncovered in vitro and in vivo by a variety of proliferation assays,including CCK-8 assay,colony formation assay,EdU assay and RTCA assay.Besides,PCNA,which was related to the proliferation of cells,was aslo measured with Western Blot.Long-term si-RNA was applied to knock down PSMB4 persistantly in ACHN cells.The cells and NCs were injected into balb/c nude mice.After 6 weeks' observation,the sizes and weights of tumors were recorded.The expression level of PSMB4 in tumors were measured as well.Finallly,using TNF-? as a signaling pathway activator,combined with the quantification of P50 and P65,we proved PSMB4 affected the proliferation of ccRCC via NF-?B signaling pathway.The percedure was controlled by miR-524-5p.Results: Bioinformatic analysis showed that high level of PSMB4 expression correlated with TNM stages,grades of ccRCC.High PSMB4 expression levels correlated with low overall survival rate of ccRCC patients.GSEA analysis showed that several signaling pathways,including NF-?B signaling axis were enriched in PSMB4 high expression phenotypes.Detected by PRC and Western Blotting,the expression level of PSMB4 was up-regulated in ccRCC tissues and cell lines.PSMB4 knock-down by si-RNA suppressed the proliferation of ccRCC cells both in vitro and in vivo.The down-regulation of PSMB4 supressed the expression of P50 and P65 in cell nucleus of ccRCC cell lines,which were the core molecules of NF-?B pathway.TNF-?,which was a NF-?B signaling pathway activator,was used to conform that the PSMB4 affected the cell proliferation via NF-k B axis.The percedure was controlled by miR-524-5p.Conclusion: High level of PSMB4 expression correlated with the progression of ccRCC and poor overall survival rate of ccRCC patients.PSMB4 was upregulated in RCC tissues and cell lines.PSMB4,controlled by miR-524-5p,promoted ccRCC cell proliferation by activating the NF-?B axis.