The Function And Mechanism Of USP39 In Regulating VEGF-A Variable Splicing In Promoting Malignant Proliferation And Angiogenesis Of Renal Cell Carcinoma

Background and ObjectivesRenal cell carcinoma(RCC)is the most common tumor of the urinary system following prostate cancer.In recent years,with the development of imaging technologies such as ultrasound and tomography,more and more early renal cancer patients have been found and received timely surgery.But still one third of patients have had distant metastasis when first diagnosed,losing the chance of radical surgery.Advanced renal cancer has poor sensitivity to radiotherapy,chemotherapy,interferon and other treatments,the prognosis of patients have not been improved significantly.The advent of tyrosine kinase inhibitors targeted drugs has brought hope to the treatment of patients with advanced renal cancer,significantly improving the patient's survival rate,prolonging the survival time.Therefore most patients will develop to targeted drug resistance after about 1 year of medication,and patients with drug resistance have a rapid disease progression and poor prognosis,so exploring the molecular mechanism of RCC,looking for new targets for the treatment in order to improve the survival prognosis of patients have become urgent problems for clinicians in urology.USP39,also known as ubiquitin specific polypeptide enzyme 39,is similar to deubiquitination enzyme in structure,but does not have the activity of deubiquitination enzyme.Instead,it is an essential molecule in the assembly process of spliceosome microribonucleoprotein(sn RNP)during pre-m RNA maturation,which is involved in the regulation of m RNA maturation of multiple functional genes.In osteosarcoma and melanoma cells,knockdown of USP39 can inhibit the mitosis and growth of tumor cells.In prostate cancer,USP39 can promote the malignant proliferation of tumor cells,suggesting that USP39 is an important proto-oncogene.We used lentiviral vectors to overexpress USP39 in human-derived 293 T cells and performed co-immunoprecipitation combined with mass spectrometry analysis to screen USP39 interacting proteins serine threonine splicing factor 1(SRSF1)and serine protein kinase 1(SRPK1).The literatures report that SPRK1 directly acts on SRSF1 to promotes vascular endothelial growth factor-A(VEGF-A)splicing to form VEGF-A165 and VEGF-A165 b splice isomers,the former promotes angiogenesis and the latter inhibits angiogenesis which plays an important role in the occurrence and development of tumors.However,the mechanism of its interaction and function in renal cell carcinoma is still unclear,we aim to explore the function and mechanism of USP39 in regulating VEGF-A variable splicing in promoting malignant proliferation and angiogenesis of renal cell carcinoma.Methods1.The expression level of USP39 in renal cell carcinoma and its relationship with survival and prognosis were detected in the gene database.The ONCOMINE gene database was used to analyze the expression level of USP39 in renal cell carcinoma and normal renal tissue,and to analyze the correlation between its expression level and the clinical characteristics of renal cell carcinoma.2.Stable knocking down cells construction and detection the proliferation ability and cell cycle in renal cell carcinoma cells.786-o and ACHN cells were infected with lentivirus containing sh USP39 and sh CON.The infection efficiency was verified by the proportion of green fluorescence and the silence efficiency was confirmed by Western Blot and Real-time PCR.After knocking down USP39,MTT experiment and cell cycle detection were carried out to detect the proliferation ability and clone formation assay was carried out to detect the clone situation of renal carcinoma cells.3.Detection of angiogenesis ability of HUVEC with stable USP39 knockdown.On the basis of stable knockdown of USP39,the vascularization of HUVEC cells was detected by tubule formation experiment.4.Exploring the molecular mechanism of USP39 interacting with SRSF1 and SRPK1.Using the lentiviral vector to overexpress USP39 in human 293 T tool cells and performing co-immunoprecipitation combined with mass spectrometry analysis,it was confirmed that USP39 interacts with SRSF1 and SRPK1,and the USP39 short-cut mutant was co-transfected with SRSF1 and SRPK1 plasmids to verify the interaction sites of USP39 with SRSF1 and SRPK1.5.Exploring the effects of USP39 knockdown and overexpression on the expression of VEGF-A variable spliceosome.After knockdown and overexpression of USP39 in 786-O cell line,Western-Blot was carried out to detect the expression of VEGF-A165 b.Results 1.Analysis of m RNA expression levels in renal cancer and normal kidney tissues and their correlation with clinical characteristics using ONCOMINE databaseThrough meta-analysis of ONCOMINE database,the expression level of USP39 m RNA in CCRCC is obviously higher than normal kidney tissue,and the expression level is negatively correlated with the survival rate of renal cancer patients,which is an independent risk factor for patient survival.2.USP39 knockdown significantly inhibits the bioprocess of proliferation and colony formation of RCC cells and the cell cycle stagnated in the S phase.After stable knockdown of USP39,the growth curve of renal cacinoma cells was significantly inhibited compared with the control group,and the number of cell clones after knockdown was significantly decreased compared with the control group(P < 0.001)and the cell cycle stagnated in the S phase.3.Knockdown and overexpression of USP39 can correspondingly inhibit or promote tubule formation in vascular endothelial cells.Tubulogenesis experiments were carried out after knocking down and overexpressing USP39 in HUVEC cells,The number of tubules and branches formed by HUVEC cells increased significantly after overexpressing USP39,while the number of tubules and branches decreased significantly after USP39 knocking down.4.The USP39(101-565)fragment plays a biological role by directly binding to SRSF1 and SRPK1.The direct interaction between USP39 and SRSF1/SRPK1 was detected by coimmunoprecipitation combined with mass spectrometry,and the co-transfection of USP39 with SRSF1 and SRPK1 plasmids by variant shortened USP39 indicated that USP39 plays a biological role by binding to SRSF1 and SRPK1 with fragment(101-565).5.Knockdown and overexpression of USP39 can respectively up-regulate and down-regulate VEGF-A165 b expression.Proteins were collected after USP39 was knocked down and over-expressed in 786-O cell line.The expression of VEGF-A165 b was detected by Western Blot.It was shown that knockdown of USP39 up-regulated the expression of VEGF-A165 b,and over-expression of USP39 down-regulated the expression of VEGF-A165 b.Conclusion 1.USP39 is significantly up regulated in RCC tissues,and the expression level is negatively correlated with survival and prognosis of renal carcinoma patients.2.USP39 knockdown significantly inhibits RCC malignant biological processes including proliferation and angiogenesis.3.USP39 inhibits VEGF-A splicing to VEGF-A165 b variable spliceosome by directly binding to SRSF1 and SRPK1 to promote tumor malignant proliferation.4.As an important tumor-promoting factor,USP39 plays an important role in the occurrence and development of renal cell carcinoma and malignant biological processes.