| Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in China.According to the latest data released by the National Cancer Center: the number of new cases of HCC in China has reached 370,000,and the death toll has reached 326,000.It is high mortality that make HCC as the second lethal factor among all malignant in China.Currently,the overall prognosis of Hepatocellular carcinoma patients gradually improved due to the appearance of the multi-disciplinary team(MDT).However the primary method in MDT of HCC is surgical treatment until now.The high recurrence rate of HCC is the main factor affecting the quality of life of the patients after hepatic resection and aggravating the disease burden.Therefore,it is necessary to screen molecular biomarkers associated with recurrence and metastasis of HCC in order to generate the adjustment of clinical treatment strategies of the patients,which can effectively improve the prognosis of patients and reduce the disease burden of patients.NEDD8 is a small molecule with 81 amino acids and highly homologous to ubiquitin.It is a post-translational modified protein that completes the neddylation process in cells.Studies have shown that it is highly expressed in colon cancer and kidney cancer.Although it has been shown that it is associated with recurrence and metastasis of HCC,the exact molecular mechanism about recurrence and metastasis is not clear at present.Objective:1.To inspect the expression of NEDD8 in the tissues of HCC patients after liver resection.To estimate the value of NEDD8 as the molecular markers in predicting recurrence and metastasis of HCC,which was analyzed through the clinical and pathological data;2.To investigate the effect of NEDD8 related neddylation on the migration,invasion and stemness of HCC cells,and further investigate the molecular mechanism of NEDD8 regulating the recurrence and metastasis of HCC.Methods:1.46 patients’ HCC tissues and paired adjacent tissues were constructed into tissue microarrays which were used to detect the expression of NEDD8 protein by immunohistochemistry.Kaplan-Meier analysis was used to evaluate the value of NEDD8 in predicting postoperative recurrence and metastasis of HCC.Pearson chisquare test was used to analyze the correlation between the expression of NEDD8 and clinicopathological factors.2.After treated with NEDD8 inhibitor MLN4924,the expression of NEDD8 and related neddylation were detected by Western blot.Transwell chamber assay was used to measure the migration and invasion ability.And the migration ability was further verified by scratch test.The tumor sphere formation assay and western blot were performed to evaluate the stemness of HCC cells.Result:1.The immunohistochemical results showed that NEDD8 was higher expression in HCC tissues compared with their adjacent tissues(p <0.0001).Using Kaplan-Meier analysis,it concluded that HCC patients with obviously high expression of NEDD8 had a lower recurrence-free survival(p =0.023).In HCC after hepatic resection,it is easy to find that NEDD8 expression was an independent risk factor for recurrence free survival through COX multivariate risk analysis(p=0.022).Further analyzing NEDD8 expression and clinical proportionality in HCC tissues,the results showed that the expression of NEDD8 was related with AFP(p =0.006)and tumor differentiation level(p <0.001);2.Whole HCC cell neddylation was suppressed after treated with MLN4924,an inhibitor of NEDD8,and the ability of migration and invasion of HCC cells were significantly inhibited.In addition,the data of tumor sphere formation assay and western blot indicated that MLN4924 inhibited the stemness of HCC cells which decrease expression of OCT4,Nanog,SOX2.Besides,MLN4924 can inhibited the occurrence of EMT.Conclusion:1.NEDD8 was highly expressed in HCC and associated with poor prognosis of HCC.It is also an independent risk factor for postoperative recurrence of HCC,which could be used as a potential biomarker for risk assessment of recurrence and metastasis of HCC;2.Neddylation mediated by NEDD8 can affect the migration and invasion ability of HCC cells,which also inhibit the stemness of HCC cells and promote the occurrence of EMT in tumor cells. |
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