Clinical Significance Of VPS33B And Its Mechanism In Renal Cell Carcinoma

BackgroundRenal cell carcinoma(RCC)is one of the most common types of renal cancer in clinical practice,and its incidence is second only to bladder cancer in the urogenital malignancies.With the deepening of molecular biology research,molecular targeting drugs targeting VEGF,VEGFR and mTOR have significantly improved the therapeutic effect of patients with renal cell carcinoma,but they are easy to produce drug resistance.Therefore,the search for new therapeutic targets is urgent.Vacuolar protein sorting protein 33B(VPS33B)is a β-subunit binding protein widely expressed in human tissues.Studies have shown that VPS33B plays a crucial role in establishing the polar structure and function of liver cells,and can regulate the foreign autosecretion signal to mediate hematopoietic,thus delaying the development of leukemia,and may become a potential target for gene replacement therapy.But its regulatory role in renal cell carcinoma remains unclear.Therefore,we mainly studied the expression level of VPS33B in renal cancer tissues and analyzed its clinical significance,as well as its influence on the proliferation,migration and invasion ability of renal cancer cells and related molecular mechanisms.MethodsPart Ⅰ:In order to clarify the expression level and clinical significance of VPS33B in renal cell carcinoma,we first used UALCAN online bioinformatics prediction website to mine TCGA public database,and predicted and analyzed the expression of VPS33B in renal cell carcinoma tissues and normal tissues.The relationship between the expression level and T stage,clinical stage and prognosis of patients was analyzed.Next,immunohistochemical analysis(IHC)was used to detect the expression status of VPS33B protein in renal cell carcinoma tissues and paracancer tissue chips.The relationship between VPSS33B protein expression level and the clinicopathologic features of patients with renal cell carcinoma,such as age,sex,T stage,clinical stage,renal venous invasion,relapse-free survival rate and overall survival(OS),was analyzed statistically.Finally,Cox regression analysis was used to investigate the prognostic factors of patients with renal cell carcinoma.Part Two:In order to clarify the regulatory role of VPS33B in the malignant biological behavior of renal cancer cells,we first constructed 786-O and A498 cell lines with stable overexpression of VPS33B by lentivirus transfection.To verify the effect of VPS33B overexpression on proliferation,invasion and migration of renal carcinoma cells.Meanwhile,Western blot was also used to detect the expression of VPS33B overexpression on EMT-related molecules,so as to preliminarily explore the relationship between VPS33B and EMT.In addition,in order to further confirm the regulatory role of VPS33B in vivo,we used VPS33B overexpression and renal carcinoma cells of control group to construct renal cell carcinoma model in nude mice,and observed the tumorformation of mice in each group.Part Three:In order to further explore the molecular mechanism of VPS33B regulating the function of renal cell carcinoma and considering the important role of PI3K/AKT signaling pathway in a variety of tumors,we discussed the relationship between VPS33B and PI3K/AKT pathway,and to verify this mechanism,We overexpressed VPS33B and treated it with PI3K pathway agonist 740Y-P.MTT assay,EDU assay,Transwell assay and Western blot assay confirmed that VPS33B affected the progression of renal cell carcinoma by regulating PI3K/AKT signaling pathway.ResultsPart Ⅰ:Analysis results of UALCAN database based on statistical analysis of TCGA data showed that VPS33B was abnormally low expression in primary renal cell carcinoma,and low expression of VPS33B was closely related to patients’ later clinical stage and shorter OS.Immunohistochemical results showed that VPS33B was mainly expressed in cytoplasm.Compared with paracancer tissues,VPS33B expression level in renal cell carcinoma tissues was significantly decreased,and the later the clinical stage,the lower the expression level of VPS33B.Further analysis showed that low expression of VPS33B was negatively correlated with T stage,clinical stage,and renal vein invasion,but not with patient age,sex,and tumor size.Kapaln-Meier analysis indicated that patients with low VPS33B expression had significantly lower OS than those with high VPS33B expression.Cox regression analysis showed that low expression of VPS33B was an independent factor for poor prognosis in patients with renal cell carcinoma.These results suggest that VPS33B may play an important role in the occurrence and development of renal cell carcinoma.Part Ⅱ:The results of MTT and EDU experiments showed that the overexpression of VPS33B could inhibit the proliferation of renal cell carcinoma.The results of Transwell experiment showed that overexpression of VPS33B could significantly inhibit the migration and invasion of renal cell carcinoma.In vivo tumor carrying experiments of nude mice,it was found that overexpression of VPS33B could inhibit the tumorigenesis of renal cell cancer cells in vivo,and inhibit the expression levels of proliferation-related molecules Ki67 and PCNA in vivo.The results of Western blot indicated that overexpression of VPS33B could inhibit the expression of EMT-promoting proteins Vimentin and N-cadherin.These results suggest that overexpression of VPS33B can inhibit the proliferation,migration,invasion and other malignant biological behaviors of renal cell carcinoma,and may play a regulatory role in the process of EMT.Part Ⅲ:Western blot results showed that VPS33B could inhibit the expression level of p-PI3K and p-AKT.The results of MTT,EDU and Transwell experiments showed that PI3K/AKT pathway agonist 740Y-P could antagonize the inhibition of VPS33B overexpression on the proliferation,invasion and migration of renal cancer cells,and 740Y-P could antagonize the inhibition of VPS33B overexpression on EMT-related proteins.ConclusionsVPS33B is abnormally low expressed in renal cell carcinoma,and its low expression is closely related to poor prognosis of patients with renal cell carcinoma.VPS33B may inhibit the proliferation,invasion and migration of renal carcinoma cells by inducing the inactivation of PI3K/AKT signaling pathway.