Using Quantitative Proteomics To Predict New Potential Targets For Kidney Cancer Drugs

Objective:The incidence and mortality of kidney cancer increase year by year,and the recurrence rate after traditional surgery is also very high.Therefore,the purpose of this experiment is to predict new targets of anticancer drugs for kidney cancer and to find effective anti-renal cancer drugs.Methods:Five pairs of renal cancer tissues and paraneoplastic kidney tissues were obtained from surgical resection and analyzed using the QExactive Plus and nanoLC systems with unlabeled quantitative proteomics to screen for differentially expressed proteins.Differentially expressed proteins,such as PYGL,ANXA4,PSMB8,and ATP5B,were verified on a small sample level by qRT-PCR,Western blotting,and IHC.Further testing at large sample levels included the absolute quantification of ANXA4,PYGL,PSMB8,and ATP5B in kidney cancer tissue and its adjacent paraneoplastic kidney tissues from fifty kidney cancer patients using the new technique,high-throughput immunoblot(QDB).In vivo validation,siPYGL was transfected with renal cell carcinoma cells(786-0),and transfected cells were tested for cell proliferation and migration using the xCELLigence Real-Time Cell Analyzer(RTCA)-DP system.Results:A total of 2091 proteins were detected by mass spectrometry,of which 532 proteins were significantly different in renal cell carcinoma and paraneoplastic kidney tissues.In 50 pairs of tissues,the median absolute quantificationn of PYGL was significantly higher in RCC tissues than in adjacent paracancerous tissues(1.21 nmol/g vs.0.16 nmol/g,P<0.0001,paired t-test).ROC analysis of PYGL showed AUC 0.93(P<0.001;95%CI 0.87-0.99).When the PYGL cut-off value was 0.35 nmol/g,the sensitivity of PYGL was 92%,the specificity was 92%,and the overall accuracy was 96%.The overall accuracy of ANXA4,PSMB8 and ATP5B was 92%,82%,and 70%,respectively.Cell experiments showed that after PYGL knockdown,cell proliferation and migration were reduced.Conclusion:We detected renal cancer tissue by quantitative proteomics methods.The differentially expressed proteins in renal cancer tissues and paraneoplastic kidney tissues were tested in molecular biology experiments and validated at the population level,and proteins with distinct and stable expression were screened out.It has been analyzed that PYGL may become a new target for anticancer drugs for kidney cancer.