PHF8 Promotes Clear Cell Renal Cell Carcinoma Progression By Upregulating GLUL In Lipid Deposition

Background:Clear cell renal cell carcinoma is(CCRCC)the most common subtype of renal cancer with its cytoplasm filled with lipids and glycogen.The characteristics of renal clear cell carcinoma include gene mutations in the hypoxia signaling,metabolic disorders,enhanced angiogenesis,intratumoral heterogeneity,and interference with the tumor microenvironment.Metabolic reprogramming plays an important role in the development of tumors.Renal clear cell carcinoma is a metabolic disease,usually accompanied by reprogramming of glucose metabolism,fatty acid metabolism,amino acid metabolism such as tryptophan,arginine and glutamine and the tricarboxylic acid cycle.This study aims to explore the role of PHF8 in the lipid deposition and further clarify the mechanism of metabolic reprogramming in the development of renal clear cell carcinoma.Thus,provide evidences for prognosis prediction and targeted therapy of CCRCC.Material and methods:1.Public clinical databases: TCGA database;CPTAC database2.Cell lines: human renal tubular epithelial cell HK-2,human renal cell carcinoma cell line 786-O,ACHN,RCC4.3.Organoid: established from fresh renal clear cell carcinoma tissue specimens and treated with MSO.4.Animal models: 4 weeks old nude mice were used for subcutaneous xenograft formation and tail vein injection migration model experiment,respectively.5.Clinical specimens: A.Collect the cases and paraffin specimens diagnosed as renal clear cell carcinoma in our hospital,and construct tissue microarray for immunohistochemical staining;B.Collect the fresh tissues of CCRCC diagnosed in our hospital.They were used to evaluate the expression level of PHF8 or for establishing organoid.6.Experimental methods: This study used bioinformatics analysis,Western-blot,PCR,immunohistochemistry,cell transfection,RNA sequencing,oil red O staining,lipidomics,metabolite determination and other experiments.Results:1.The expression of PHF8 in renal cancer cells is higher than that of normal renal tubular epithelium.In clinical specimens,the expression of PHF8 protein in CCRCC is higher than that in adjacent normal tissues.The results of immunohistochemical staining analysis suggest that the expression level of PHF8 is related to pathological grade and clinical stage.The higher the expression level of PHF8,the worse the patient’s prognosis.2.Knocking down or knocking out PHF8 can inhibit the proliferation,migration and invasion of renal cancer cells in vitro and in vivo.Knocking out PHF8,26% of the differential expression genes(DEGs)were related to metabolism.In the results of pathway analysis,the alanine-aspartate-glutamate metabolic pathway was most affected.In this pathway,the GLUL gene was regulated by PHF8.The lipidomics results revealed that 75 of the 255down-regulated lipids belonged to triglycerides in the knockout group.3.Glutamine synthetase is highly expressed in CCRCC,and its expression level is related to the prognosis.Glutamine synthetase inhibitors and everolimus have a synergistic inhibitory effect on tumors.4.The levels of CPT1 A and CPT2 in CCRCC can be used as prognostic markers and potential therapeutic targets.The combination of CPT2 expression level and TNM staging can significantly improve the accuracy of the prognosis of renal clear cell carcinoma.5.The accumulation of the succinate has multiple epigenetic effects.Succinate dehydrogenase-deficient renal cell carcinoma is a good model for studying the aberrant metabolism and epidemics of renal cell carcinoma.Conclusion:PHF8 promotes renal clear cell carcinoma lipid deposition by up-regulating glutamine synthetase.Inhibiting the expression of PHF8 or its downstream GLUL can inhibit the growth of renal cancer.PHF8 and GLUL as markers or synergistic treatment targets are of great significance for the treatment of renal clear cell carcinoma.