Overactivated Neddylation Pathway And Its Specific Substrates In Hepatocellular Carcinoma

Objective: The aim of this research is to prove the important role of the neddylation pathway in the tumorgenesis and development of human hepatocellular carcinoma. In addition,we want to reveal its value to estimate the survival of the HCC patients. Moreover, we aim to discover the specific substrates of the pathway in HCC.Content: Due to the basic biological experiments and the statistic methods, we proved the upregulation trend of the whole neddylation pathway, which was related to the survival of the HCC patients. What’s more, we also found the CES1 and ENO1 as the specific substrates of the neddylation pathway. With the use of the inhibitor of neddylation pathway, the expression of these two substrates was altered.Methods: First, the expression of NEDD8 and the survival situations were analyzed by the student’s t test, Wilcoxon signed-rank test, χ2 test and Kaplan-Meier analysis with the HCC tissue microarray. The univariate analysis and multivariate analysis were made to analyze the tissue microarray. Second, the quantitative real-time PCR was used to confirm the m RNA expression of the key enzymes in the HCC tumor tissues and the corresponding distant normal tissues include NAE1, UBC12 and UCHL1. The Western Blot was used to detect the expression of the enzymes and then the results were analyzed by the statistic methods mentioned above. And the last, to discover the specific substrates of the pathway, we performed the Co-IP experiment and Western Blot. Moreover, to test the expression of CES1 and ENO1 in the cell lines treated with the neddylationpathway inhibitor, Western Blot was performed.Results: Firstly, we defined that the expression of NEDD8 was hopefully upregulated in the HCC tumor tissues compared to the corresponding distant normal tissues(P<0.001,N=278)which was related to the HCC’s multifocal growth(P=0.006,N=278), pathology grade(P=0.014,N=278), vascular invasion(P<0.001, N=278) and HBe Ag(P<0.012, N=278). And with the Kaplan-Meier analysis and univariate analysis and multivariate analysis, we confirmed that the expression of NEDD8 could affect the survival of HCC patients and also could be an independent risk of the survival for the HCC patients(HR/95%CI: 1.717(1.214-2.428)P=0.02). Secondly, the three key enzymes named NAE1, UBC12, and UCHL1 were upregulated in both m RNA(PNAE1<0.001,PUBC12<0.001,PUCHL1<0.001,N=126) and protein(PNAE1=0.037,PUBC12=0.017,PUCHL1=0.013,N=10)level. And the higher m RNA expression of the three enzymes was related the poorer overall survival of HCC patients(PNAE1=0.015,PUBC12=0.040,PUCHL1=0.040,N=126). Finally, we figured out the two specific substrates of the pathway that were upregulated in the HCC tumor tissues compared to the corresponding distant normal tissues that turned to be much more increased in the cell lines when treated with the neddylation pathway inhibitor with the Co-IP experiment, mass spectrum and Western Blot technique.Conclusion : Our research had proved the upregulation of the entire neddylation pathway including the enzymes in every stage which were related to the overall survival to the HCC patients. And we also confirmed the NEDD8 expression as an independent risk factor of the survival to HCC patients. Which illuminate the value of neddylation pathway system to predict and evaluate the survival of HCC patients. And at the same time, we defined the specific substrates of neddylation pathway in HCC which were upregulated while treated with inhibitor of neddylation pathway which indicated that the neddylation pathway may affect the tumorgenesis and development of HCC due to its modification of the two new defined substrates CES1 and ENO1.