| Objective: Renal cell carcinoma is not only the most common malignant tumor in the urinary system,but also one of the most common malignant tumors among all solid tumors.The incidence rate of renal cell carcinoma is increasing during these decades in China and even around the world,and the new estimated death cases are also gradually increasing.Renal clear cell carcinoma(cc RCC)is the most common pathological type of renal cell carcinoma,which is prone to demonstrate invasion and distant metastasis in advanced stages.Patients in the early stage of renal cell carcinoma often demonstrates no obvious specific clinical symptoms,and many patients who once diagnosed as renal cell carcinoma are confirmed to be in the advanced cancer stages.At present,laparoscopic surgery is the main method of clinical treatment for renal cell carcinoma,but at least 30%of renal cell carcinoma patients have recurrence or continued metastasis even after surgery.Metastatic renal cell carcinoma is not sensitive to radiotherapy,chemotherapy,and immunotherapy.Although great progress has made in the clinical targeted therapy nowadays,the overall prognosis of renal cell carcinoma patients is still not ideal.Therefore,the study of the pathogenesis mechanism and molecular targets of renal clear cell carcinoma is of great significance for the early detection of renal cell carcinoma and the improvement of the prognosis of patients with advanced metastasis.FBP protein is involved in the assembly of SKP1-CULLIN1-F-box E3 ubiquitin ligase,and several members of the family have been reported to participate in the regulation of intracellular and extracellular protein ubiquitination.FBXO30 is involved in the regulation of intracellular ubiquitination of various substrates during mitosis and muscle growth.Among tumors,FBXO30 has significant correlation with prostate cancer and nasopharyngeal carcinoma.However,the expression of FBXO30 in renal cell carcinoma and its specific mechanism are still unclear.As a member of the hZIP family,the hZIP1 protein is encoded by the SLC39A1 gene and is involved in maintaining the homeostasis of zinc ions in and out of cells.Among tumors,hZIP1 showed significantly low expression in prostate cancer,ovarian cancer,colon cancer,gastric cancer,and lung mucinous adenocarcinoma.Therefore,the expression of hZIP1 in tumor cells has significant specificity,but its specific molecular biological mechanism is still unclear.Hypoxia is one of the typical characteristics of solid tumors,and renal cell carcinoma is no exception.In normal oxygen state,HIF-1α is rapidly degraded by ubiquitin proteasome pathway after binding with p VHL E3 ubiquitin ligase,and p VHL inactivation under hypoxia leads to HIF-1α Enrichment.However,a variety of novel E3 ubiquitin ligases have been reported to participate in HIF-1α ubiquitination degradation in normoxic tumors,such as Parkin,TNF receptor-related factor 6 and FBXW7.Our previous studies showed that the expression of hZIP1 in renal clear cell carcinoma was significantly lower than that in normal tissues adjacent to the cancer,and was negatively correlated with the malignancy of renal clear cell carcinoma.After silencing the expression of hZIP1 in renal clear cell carcinoma cells,the proliferation and invasion ability of the cells was significantly improved.HIF-1 α The protein expression in renal clear cell carcinoma was higher than that in normal tissues adjacent to the cancer.After silencing the expression of hZIP1 in renal clear cell carcinoma cells,the proliferation,migration and invasion of the cells were significantly reduced.Overexpression of hZIP1 in renal clear cell carcinoma cells significantly inhibited HIF-1 α And its specific mechanism remains to be explored.The purpose of this study is to explore the difference of expression of FBXO30 in renal clear cell carcinoma and its role in tumor progression,and to explore the role of hZIP1/FBXO30/HIF-1α axis in the genesis and development of renal clear cell carcinoma,to identify a new E3 ubiquitin ligase targeted HIF-1α,and to provide new molecular biological basis and evidence for the clinical treatment and diagnosis of renal clear cell carcinoma.Methods: 1.Based on the transcriptome data of kidney renal cell carcinoma(KIRC)in the TCGA database,predict the differential expression pattern of FBXO30 in renal clear cell carcinoma and normal renal tissue through bioinformatics analysis,and draw the survival curve of FBXO30 expression and the prognosis of renal clear cell carcinoma patients The expression level of FBXO30 in renal clear cell carcinoma tissues from clinical patients and corresponding normal tissues adjacent to cancer was detected by real-time fluorescence quantitative PCR and immunohistochemical staining;2.The expression of FBXO30 in renal clear cell carcinoma and adjacent normal tissues was detected by Western blotting assay,real-time quantitative PCR assay and immunohistochemistry staining assay,and then the correlation between the expression of FBXO30 and the prognosis of renal clear cell carcinoma patients was detected by bioinformatics analysis based on transcriptome data in TCGA database;2.The renal clear cell carcinoma cell line with overexpressing FBXO30 was constructed.The proliferation,migration and invasion of the cells were detected by Ed U assay,wound-healing assay,and Transwell assay,and the effect of FBXO30 on tumor formation in vivo was detected by subcutaneous tumorigenesis in nude mice;3.Western blotting assay,real-time quantitative PCR,zinc supplementation assay were carried out to explore the regulatory relationship among hZIP1,FBXO30 and HIF-1α;4.Protein half-life assay,MG132 inhibition assay and co-immunoprecipitation assay to verify that FBXO30 mediates HIF-1α Ubiquitination degradation.Results: The expression of FBXO30 at mRNA and protein levels in renal clear cell carcinoma was significantly lower than those in adjacent normal tissues;FBXO30 can directly inhibit the proliferation,migration and invasion of renal clear cell carcinoma cells,and suppresses the formation of subcutaneous tumors in nude mice;In mechanism,hZIP1 promotes the protein level of FBXO30 and promotes the direct combination between FBXO30 and HIF-1α,and then fostering the degradation of HIF-1α via ubiquitin-proteasome pathway,thus finally demonstrating tumor inhibition effect.Conclusion: FBXO30 plays a role as a tumor suppressor gene in renal clear cell carcinoma;hZIP1 up-regulates the expression of FBXO30 and inhibits HIF-1αexpression by recruiting zinc ions;FBXO30 directly bounds to HIF-1α and mediates its degradation through ubiquitin-proteasome pathway;hZIP1/zinc ion/FBXO30/HIF-1α axis may be the key regulatory mechanism to inhibit the malignant progression of renal clear cell carcinoma. |
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