The Potential Mechanism Of Centrosome Redundancy In HBV Positive HCC Tissues And The Level Of Serum Cripto-1 In HCC Patients

Hepatocellular carcinoma(HCC)is a major health problem worldwide,and its global incidence is increasing because of the dissemination of hepatitis B or C virus infection.Most cases of HCC derived from HBV or HCV infection caused chronic hepatitis and cirrhosis and non-viral factors including alcoholic and nonalcoholic caused fatty liver disease.Some cases of HCC were caused by aflatoxin exposure and genetic factors.Cirrhosis patients got high risk of hepatocarcinogenesis.HCC could be cured at early stage,but the treatment of HCC patients at late stage is very difficult and prognosis is poor.There were no specific symptoms at the early stage of HCC and it brought difficulty in early diagnosis.So exploration of HCC biomarker for early detection is of great importance.The first part: Centrosome is the major microtubule organizing center in cell.The defects in the regulation of centrosome duplication might lead to tumorigenesis by abnormal cell division and resulting chromosome missegregation.Therefore,the accuracy of maintaining centrosome number is very important for cell fate.Clarify the regulatory mechanisms of centrosome is crucial for understanding the causal relationship between overduplication of centrosome and tumorigenesis.And it might lead to the exploration of more effective anti-tumor treatment.The abnormal increased centrosome number known as centrosome amplification is the major form of centrosome abnormity,existing in a variety of human tumors.Autophagy is an evolutionarily conserved process that cytoplasm and damaged organelles are degraded by hydrolytic enzyme in lysosomes for energy recycling.It provides a survival pathway in response to nutrient deficiency and other stimulation.In recent years,there are lots of progressions in the study of the relationship between autophagy an tumor and autophagy defects are associated with tumorigenesis.Studies about centrosome in HCC are rare.In this study,we explored the condition of centrosome and autophagy in HCC tissues by immunofluorescence and immunohistochemistry.There were 21 cases of normal liver tissues and 77 cases of HCC tissues including 63 cases were HBV positive and 14 cases were HBV negative.And we explored the underlying relationship between the abnormal centrosome and autophagy by the use of HCC cell lines.Results are as follows in the first part:The number of centrosome in HCC tissues was significantly higher than it in normal liver tissues,while the expression of autophagy defection marker p62 in HCC tissues was higher than normal liver tissue,and the number of centrosome was positively correlated with p62 expression in HBV positive HCC tissues.However,the number of centrosome didn't have significant correlation with tumor histological grade and TNM stage in HCC tissues.We observed the condition of centrosome in HepRG,HepG2 and HepG2.2.15 cells.We found that abnormal centrosomes mainly shown as number abnormity were existed in HepG2 and HepG2.2.15 cells and the centrosome in HepRG cell was normal.LC3 was transfected into HepG2 and HepG2.2.15 cells and p62 expression was shown by immunofluorescence staining.The LC3 and p62 fluorescence intensity were significantly higher in HepG2.2.15 cells containing HBV genome,suggesting the initiation of autophagy is high but defective,so the process of autophagy couldn't complete.We also found that abnormal centrosome and overexpression of p62 could be observed at the same time in HBV or HBx transfected HepG2 cells and the extra centrosome colocalized with p62.This indicating that extra centrosome couldn't degrade at least partly because of HBV or HBx induced autophagy defection.HepG2 cells treated with starvation to induce autophagy initiation showed that the centrosome number was decreased compared with normal HepG2 cells.The reason might be the extra centrosomes degraded by autophagy to cell survival.In conclusion,these results suggest that increased number of centrosome in liver tissues could be severed as a biomarker for HCC.HBV could induced initiation of autophagy but failed to complete in HCC cells,the abnormal centrosomes couldn't be removed by autophagy,this might be the potential mechanism about the presence of abnormal centrosomes in HBV related HCC.The second part:Cripto-1 is cell membrane-anchored protein that has been shown to be important in embryonic development and also has been shown to play an important role in the formation and progression of various human tumors.It also could stimulate cell proliferation,epithelial to mesenchymal transition,migration and tumor angiogenesis.Some studies have demonstrated that cell fate regulation in the embryonic development share the same signaling pathways with cell transformation during oncogenesis,indicating that losing control of the activated embryonic signaling pathways might lead to cell transformation and tumor progression in adult tissues.Cripto-1 has its activity as a membrane-associated co-receptor and its soluble forms have been reported to be active in some different in vitro and in vivo assays.Cripto-1 has been detected to be overexpression in various cancer tissues than normal tissues.In recent research,Cripto-1 was also found to be elevated in the plasma or serum level of some human cancer.However,the serum level of Cripto-1 in patients with liver disease especially in HCC was unknown.In our study,serum CR-1 level was Sandwich-type ELISA detected in 330 patients with liver diseases including HCC,cirrhosis and chronic hepatitis and 50 volunteers without HBV or HCV infection as control.Results are as follows in the second part:The serum CR-1 level was significantly higher in HCC patients than volunteer controls and it was also significantly higher in HBV related HCC than HCV related HCC.In addition,serum CR-1 level was correlated with serum alfa-feta-protein(AFP)in HBV related HCC patients.The serum CR-1 was also higher in cirrhosis and chronic hepatitis than volunteer controls.The serum CR-1 in HBV related cirrhosis was higher than chronic hepatitis B,but there was no significant difference between HCV related cirrhosis and chronic hepatitis C.In conclusion,serum CR-1 was higher in HCC patients but its diagnostic sensibility in HBV related HCC was low.So it might only serve as a complementary biomarker to clinical diagnosis of HBV related HCC.The high level of serum CR-1 in HBV related liver disease might be partly attributed to HBV infection.