| Objective:Notch signaling pathway is an evolutionarily highly conserved mechanism for cell-cell communication, which keeps cell proliferation, differentiation and apoptosis, and plays an important role in embryogenesis and many types of cell differentiation fate determination. There are reports showing that Notch signaling is also involved in the development of some cancers. The deregulation of Notch receptors and ligands and the aberrant activation of Notch signaling are found in a series of malignant tumors. Until now, the role of Notch signaling in HCC and its underlying mechanism are still unknown. In this study, the Real time-PCR was used to investigate the expressional levels of genes of Notch receptors and ligands in human primary hepatocellular carcinoma (HCC), adjacent non-cancerous cirrhotic hepatic tissue, hepatic adenoma tissues and normal hepatic tissue, and to understand the role of Notch signaling in the development and progression of hepatocellular carcinoma, and to analyze the influence of the survival of postoperative HCC patients with the expressional levels of genes of Notch receptors and ligands.Methods :1 Samples collection: 40 specimens of HCC tissue, 40 specimens of adjacent non-cancerous cirrhosis hepatic tissue, 10 specimens of hepatic adenoma tissues and 10 specimens of normal hepatic tissue were obtained from the fourth hospital of Hebei Medical University without clinical treatment before operation.2 The expressional levels of Notch1 mRNA, Notch2 mRNA, Notch3 mRNA, Notch 4 mRNA, Jagged 1 mRNA, RBP-J mRNA in HCC tissue, adjacent non-cancerous cirrhosis hepatic tissue, hepatic adenoma tissue and normal hepatic tissue were measured by Real time-PCR.3 Following up the survival information of postoperative HCC patients, the clinical and pathological features (sex, age, tumor diameter, serum AFP, HBsAg, HBeAg, clinical staging, pathological claasification, tumor thrombosis or metastasis) of postoperative HCC patients were collected with all the survival information. The relationship between the expressional levels of genes of Notch receptors and ligands and the survival information of postoperative HCC patients was analyzed.Results :1 The expressional levels of Notch1 mRNA (P=0.001), Jagged1 mRNA (P=0.003), RBP-J mRNA (P=0.005) in HCC tissues were significantly higher than those in normal liver tissue; but the expressional levels of Notch2 mRNA (P=0.069), Notch3 mRNA (P=0.535), Notch4 mRNA (P=0.491) in HCC tissue were no statistical significance compared with those in normal liver tissue.The expressional levels of Notch1 mRNA(P=0.000), Notch2 mRNA(P=0.005)in HCC tissues were significantly higher than those in adjacent non-cancerous cirrhosis hepatic tissue; but the expressional levels of Notch3 mRNA (P=0.209), Notch4 mRNA (P=0.068), Jagged1 mRNA (P=0.141), RBPJ mRNA (P=0.388) in HCC tissue were no statistical significance compared with those in adjacent non-cancerous cirrhosis hepatic tissue.The expressional level of Notch1 mRNA (P=0.036)in HCC tissue was significantly higher than that in hepatic adenoma tissues; but the expressional levels of Notch2 mRNA (P=0.611), Notch3 mRNA (P=0.696), Notch4 mRNA (P=0.343), Jagged1 mRNA (P=0.335), RBPJ mRNA (P=0.727) in HCC tissue were no statistical significance compared with those in hepatic adenoma tissues.The expressional level of Jagged1 mRNA (P=0.036) in adjacent non-cancerous cirrhosis hepatic tissue was significantly higher than that in normal liver tissue; but the expressional levels of Notch1 mRNA (P=0.532), Notch2 mRNA (P=0.275), Notch3 mRNA (P=0.925), Notch4 mRNA (P=0.123), RBPJ mRNA (P=0.152) in adjacent non-cancerous cirrhosis hepatic tissue were no statistical significance compared with those in normal liver tissues.The expressional levels of Notch1 mRNA (P=0.882), Notch2 mRNA (P=0.150), Notch3 mRNA (P=0.490), Notch4 mRNA (P=0.176), Jagged1 mRNA (P=0.782), RBPJ mRNA (P=0.917) in adjacent non-cancerous cirrhosis hepatic tissue were no statistical significance compared with those in hepatic adenoma tissues.The expressional levels of Notch1 mRNA (P=0.648), Notch2 mRNA (P=0.427), Notch3 mRNA (P=0.685), Notch4 mRNA (P=0.097), Jagged1 mRNA (P=0.446), RBPJ mRNA (P=0.195) in hepatic adenoma tissues were no statistical significance compared with those in normal liver tissues.2 The relationships between the expression of Notch1 mRNA, Notch2 mRNA, Notch3 mRNA, Notch4 mRNA, Jagged1 mRNA and RBP-J mRNA in HCC tissue and the clinical and pathological features in postoperative HCC: the expression of Notch1 mRNA, Notch3 mRNA, Notch4 mRNA, Jagged1 mRNA and RBP-J mRNA in HCC was independent on sex, age, tumor diameter, serum AFP, HBsAg, HBeAg, the tumor thrombosis or metastasis, clinical staging and pathological claasification. The expression of Notch2 mRNA in HCC was independent on sex, age, tumor diameter, serum AFP, HBsAg, HBeAg, the tumor thrombosis or metastasis, clinical staging and pathological claasification, but the expression of Notch2 mRNA in HCC with the tumor diameter larger than 5cm or equal to 5cm was stronger than that less than 5cm, and was positively correlated with tumor diameter (χ2=5.000, P=0.025; r=0.354, P =0.025).3 Survival analysis of the expressional levels of genes of Notch receptors and ligands in postoperative HCC patients: the result of univariate survival analysis was showed that the patients in whom Notch1 mRNA was positively expressed had 21.10 months' mean survival time, and the 6 months' cumulative survival rate was 75%, the 12 months' cumulative survival rate was 65%, the 18 months' cumulative survival rate was 55%. The patients in whom Notch1 mRNA was negatively expressed had 20.55 months' mean survival time, and the 6 months' cumulative survival rate was 85%, the 12 months' cumulative survival rate was 80%, the 18 months' cumulative survival rate was 50%. the 24 months' cumulative survival rate was 43.7%, the expressional levels of Notch1 mRNA were irrespective of life span in postoperative HCC patients(P=0.670).The patients in whom Notch2 mRNA was positively expressed had 19.33 months' mean survival time, and the 6 months' cumulative survival rate was 75%, the 12 months' cumulative survival rate was 62.5%, the 18 months' cumulative survival rate was 45.8%. The patients in whom Notch2 mRNA was negatively expressed had 23.06 months' mean survival time, and the 6 months' cumulative survival rate was 87.5%, the 12 months' cumulative survival rate was 75%, the 18 months' cumulative survival rate was 62.5%. the 24 months' cumulative survival rate was 54.7%, the expressional levels of Notch2 mRNA were irrespective of life span in postoperative HCC patients(P=0.387).The patients in whom Notch3 mRNA was positively expressed had 20.47 months' mean survival time, and the 6 months' cumulative survival rate was 78.9%, the 12 months' cumulative survival rate was 68.4%, the 18 months' cumulative survival rate was 47.4%. The patients in whom Notch3 mRNA was negatively expressed had 21.11 months' mean survival time, and the 6 months' cumulative survival rate was 81%, the 12 months' cumulative survival rate was71.4%, the 18 months' cumulative survival rate was 57.1%. the 24 months' cumulative survival rate was 50.8%, the expressional levels of Notch3 mRNA were irrespective of life span in postoperative HCC patients(P=0.869).The patients in whom Notch4 mRNA was positively expressed had 18.94 months' mean survival time, and the 6 months' cumulative survival rate was 76.5%, the 12 months' cumulative survival rate was 64.7%, the 18 months' cumulative survival rate was41.2%. The patients in whom Notch4 mRNA was negatively expressed had 18.93 months' mean survival time, and the 6 months' cumulative survival rate was 80%, the 12 months' cumulative survival rate was 66.7%, the 18 months' cumulative survival rate was 53.3%. the 24 months' cumulative survival rate was 42.7%, the expressional levels of Notch4 mRNA were irrespective of life span in postoperative HCC patients(P=0.784).The patients in whom Jagged1 mRNA was positively expressed had 18.40 months' mean survival time, and the 6 months' cumulative survival rate was 80%, the 12 months' cumulative survival rate was 60%, the 18 months' cumulative survival rate was 40%. The patients in whom Jagged1 mRNA was negatively expressed had 21.19 months' mean survival time, and the 6 months' cumulative survival rate was 80%, the 12 months' cumulative survival rate was 74.3%, the 18 months' cumulative survival rate was 54.3%. the 24 months' cumulative survival rate was 50.9%, the expressional levels of Jagged1 mRNA were irrespective of life span in postoperative HCC patients(P=0.598).The patients in whom RBPJ mRNA was positively expressed had 17.83months' mean survival time, and the 6 months' cumulative survival rate was 75%, the 12 months' cumulative survival rate was 58.3%, the 18 months' cumulative survival rate was 50%. The patients in whom RBPJ mRNA was negatively expressed had 21.30 months' mean survival time, and the 6 months' cumulative survival rate was 82.1%, the 12 months' cumulative survival rate was 71.4%, the 18 months' cumulative survival rate was 53.6%. the 24 months' cumulative survival rate was 49.7%, the expressional levels of RBPJ mRNA were irrespective of life span in postoperative HCC patients(P=0.805).The result of the multifactor Cox proportional hazard model analysis was showed that the risk factor of influencing survival rate of HCC was the clinical staging (P=0.008), RR was 0.240, (95%CI 0.084-0.685). we did not find that sex, age, tumor diameter, serum AFP, HBsAg, HBeAg, the tumor thrombosis or metastasis, pathological claasification and the expressional levels of Notch1 mRNA, Notch2 mRNA, Notch3 mRNA, Notch4 mRNA, Jagged1 mRNA and RBPJ mRNA were apparent correlative with the survival rate of postoperative HCC patients.Conclusions :1 The expression of Notch1 mRNA in HCC tissue was significantly higher than that in normal hepatic tissue, adjacent non-cancerous cirrhosis hepatic tissue and hepatic adenoma tissues. It prompted that Notch1 could promote the development and progression of hepatocellular carcinoma and certified further that the activation of Notch signaling pathway was found to be closely related with the development and progression of hepatocellular carcinoma.2 The expressional levels of genes of Notch receptors and ligands were irrespective of the influence of life span in postoperative HCC patients, but could be highly significant of early discovery of HCC.
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