The Significance Of SET Domain Gene Family In Renal Cancer And The Mechanism Of SETD8 Promoting CcRCC Progression

1 IntroductionRenal cell carcinoma(RCC)is the most common renal solid malignant tumor in the urinary system.It accounts for 2.2%of the world’s cancer and the rate of its incidence is on the rise.RCC has many histological subtypes and each subtype has unique molecular characteristics.The most common one is clear cell renal cell carcinoma(ccRCC).Researchers discovered that epigenetic changes often occur in ccRCC.The regulatory information imposed by this process is called "epigenetics",which plays an important role in the initiation and progression of ccRCC.One of them is covalent modification of histones.The histone methylation is mainly regulated by histone methyltransferases.The human genome can encode more than 50 kinds of lysine methyltransferases.Among them,genes that can encode SET domain containing proteins and have the identical nomenclature belong to the same gene family,mainly including SETD1A,SETD1B,SETD2,SETD3,SETD4,SETD5,SETD6,SETD7,SETD8,SETDB1 and SETDB2.At present,a large number of studies have confirmed that they play a vital role in the tumorigenesis and progression of various tumors.However,there are few studies relating to their expression level and clinical value in RCC,and their values in RCC has not been fully revealed.Abnormal accumulation of lipids,often forming perirenal adipose layer,is one of the typical characteristics of ccRCC.The accelerating growth of ccRCC leads to the enhancement of cell metabolic capacity and the corresponding increase in various metabolic needs.Abnormally accumulated lipid droplets have become indispensable materials for them to deal with the harsh condition.Nowadays,the specific molecular mechanism of lipid metabolism in ccRCC is ambiguous.Therefore,we will explore the clinical significance of SETDs gene family in RCC and reveal the deep mechanism of abnormal expression of lipid metabolism in ccRCC and the crucial role of SETD8 in this process.2 The clinical significance of SETDs gene family in RCCMETHODS:GEPIA2 database was used,in this study,to analyze the protein structures and mRNA expressions of SETDs gene family members,and UALCAN database was employed for further verification;The human protein atlas database(HPA)was adopted to assess the expressions of proteins encoded by SETDs family genes in RCC.In addition,cBio cancer genomics portal database is also used to visualize the geneic alterations of all genes in clinical samples,explore the frequency of genetic alterations,and comprehensively analyze the genomic changes and specific biological characteristics in a single sample,such as gene mutation,homozygous deletion,gene amplification,increase or decrease of mRNA,etc.What’s more,Kaplan Meier plotter survival analysis,OncoLnc website and UALCAN database were utilized to evaluate the prognostic value of SETDs family genes for the overall survival of RCC patients.RESULTS:The SET domain which is the core structure exists in all protein structures of SETDs gene family members.Compared with renal normal tissues,the mRNA expression level of SETD1A,SETD7,SETD8 or SETDB1 in RCC significantly increased,whlie that of SETD2,SETD3 or SETDB2 in tumor tissues decreased.For SETD4 and SETD5,there was no alteration between cancerous and normal tissues.All SETDs family genes have a variety of mutation types in renal tumors,mainly including missense mutation,amplification,deep deletion,increased mRNA or protein expression level,decreased mRNA expression level and multiple mutation fusion.Their mutation frequency is 48%.In other words,one or more mutations exist in at least one gene of this family for almost half of RCC patients.The high expression of SETD8 was positively correlated with the short overall survival,indicating patients would have a poor prognosis;patients with higher expression of SETD3 have longer overall survival.Other family genes have no prognostic value on the overall survival of patients.In conclusion,the results suggest that SETD3 and SETD8 may play an important role in the progression of RCC,and provide new strategies for the treatment of renal tumors,indicating they have potential clinical value.3 The expression of SETD8 and its clinical significance in ccRCCMETHODS:In this study,the expression of SETD8 in ccRCC and its correlation with tumor stage and grade were analyzed by using UALCAN online database.IHC and Western blot were employed to further clarify the expression level of SETD8 in ccRCC;The correlation between the expression level of SETD8 and the overall survival of patients was evaluated by the Kaplan Meier plotter survival analysis and the survival data of TMA patients.CCK-8,Transwell,WB,qPCR and colony formation assays were performed to detect the effects of the loss of SETD8 expression on the proliferation,migration and invasion of ccRCC cell lines.In addition,we also verified the effect of SETD8 on the growth of ccRCC cells in vivo by constructing a stable cell line with shSETD8 expression and simulating tumorigenesis in vivo via subcutaneous implantation.RESULTS:The mRNA expression of SETD8 in ccRCC was greatly higher than that in adjacent normal tissues,and the high mRNA expression of SETD8 in ccRCC was positively correlated with the high tumor grade and stage.The higher the expression of SETD8 in renal cancer tissues,the worse the outcome of patients in the future,which indicated SETD8 might be an independent predictor for the prognosis of ccRCC patients.In addition,the reduction of the SETD8 mRNA expression by gene silencing or molecular inhibitors could effectively impede the proliferation and colony formating ability of ccRCC cells,as well as the migration and invasion ability of tumor cells.At last,the result of tumor xenotransplantation model(CDX model)showed that the gene silencing of SETD8 could significantly slow down the tumorigenesis and progression of ccRCC cells in vivo.4 SETD8 promotes the progression and metastasis of ccRCC via directly modulating SREBP1 mediated lipogenesisMETHODS:In this part,we analyzed the RNA-seq data by GSEA analysis,and identified the functional pathways regulated by SETD8 in ccRCC cells.Through the correlation analysis of genes,qPCR and Western blot assays,we explored the relationship between SETD8 and three key enzymes in lipid metabolism pathway(ACACA,FASN and SCD1)and the relationship between SETD8 and transcription factor SREBF1,respectively.The specific molecular mechanism of SETD8 modulating SREBF1 was confirmed by ChIP-qPCR assay.We also verified the effect of SETD8 on the phenotypic function of ccRCC cells in proliferation,colony formation,migration and invasion via regulating the expression of SREBP1 by CCK-8,colony formation,Transwell,WB,ORO and rescue experiments.By using the GEPIA2 database and IHC assay,we further clarified the correlation between SETD8 and three key enzymes,and the correlation between SREBF 1 and three key enzymes,respectively.Furthermore,qPCR,WB and IP experiments were conducted to verify the mechanism in which USP17 regulated SETD8 expression.RESULTS:GSEA analysis showed that SETD8 was involved in the regulation of lipid metabolsim pathway,and SETD8 silencing could reduce the expression of three key enzymes(ACACA,FASN and SCD1)at the same time.Spearman correlation analysis displayed that there was a positive correlation between the mRNA abundance of SETD8 and SREBF 1.The deletion or overexpression of SETD8 could lead to the decrease or increase of SREBP1 expression.As was expected,the result of ChIP-qPCR assay demonstrated that SETD8 could change the chromatin pattern of SREBF1 promoter region by mediating H4K20me1 signal,so as to directly activate the gene transcription of SREBF1.The results of rescue experiments revealed that SETD8 promoted the lipogenesis of ccRCC and accelerated the growth and metastasis of cells by activating the expression of SREBP1.GEPIA2 analysis disclosed that SETD8 and SREBF1 were both positively correlated with three key enzymes,respectively.The result of IHC assay verified the effect of SETD8 on the expression of these key enzymes and reduction of the KI67 expression in sh-SETD8 tumor tissues,indicating that SETD8 impaired the proliferation of ccRCC cells in vivo.Besides,IP experiments confirmed that USP17 stabilized SETD8 proteins through deubiquitination and maintained its high expression in ccRCC.Moreover,USP17 regulated the lipid metabolism pathway by stabilizing the protein expression of SETD8,and then promoted the progression and metastasis of ccRCC.CONCLUSIONS1.SETD3 and SETD8 may play a major role in the progression of RCC and indicate the prognostic outcome of RCC patients,which can provide new strategies for the treatment of renal tumors and have the potential clinical value.2.SETD8 is highly expressed in ccRCC and a predictor of poor prognosis for ccRCC patients.3.SETD8 can modulate the lipid metabolism through directly regulating SREBF1 with mediating H4K20me1 signal,so as to promote the progression and metastasis of ccRCC.SIGNIFICANCESWe unraveled the expression and genetic mutations of SETDs family genes in RCC through bioinformatics analysis and analyzed the clinical value on patient prognosis for each gene,which indicated that SETD3 and SETD8 might be potential molecular markers for RCC.By carrying out a series of cell functional experiments in vitro and tumor exnograft models in vivo,we clarified the clinical significance of SETD8 in ccRCC and the crucial role of USP17/SETD8/SREBP1 pathway in the lipid metabolism,progression and metastasis of ccRCC.