The Role Of NDRG1 Gene In Rats Model Of Hepatocellular Carcinoma By Targeting Wnt/?-catenin Pathway

Objective: Liver cancer is one of the most common malignant tumor of digestive tract,the incidence of which has remained stubbornly high.There is no obvious clinical manifestation of early liver cancer patients.Most patients are in the advanced stage once founded,who has lost the best treatment time.Although there are lots of the etiology of liver cancer,the exact pathogenesis is still not clear.In recent years,the study of the molecular mechanisms of liver cancer gradually deepened.NDRG1,differentiation related factor,is a newly discovered signaling pathway regulatory molecule in recent years.NDRG1 gene is a member of NDRG(N-myc downstream-regulated gene)family.The family is a newly discovered gene family which is closely related to tumorgenesis and development in recent years.NDRG1 gene is involved in the occurrence and development of many tumors.It plays a different role in different tumors.Some researches show that the role of NDRG1 in the occurrence and development of HCC is still unclear.Wnt/?-Catenin pathway is the classical Wnt signaling pathway.More and more studies have shown that the abnormal activation of Wnt/?-Catenin pathway plays an important role in the occurrence and development of hepatocellular carcinoma.?-Catenin is the core factor of Wnt signaling pathways,the increased expression of which is closely related to the abnormal activation of Wnt signaling pathways.As the downstream target of Wnt signaling pathways,the expression of Cyclin D1 in malignant tumor tissues is significantly higher than that in normal tissues.Now,there is a great controversy about the correlation between the expression of NDRG1 and Wnt/?-Catenin pathway in the occurrence and development of hepatocellular carcinoma.Studies have shown that human NDRG1 gene and mouse NDRG1 genehas 86% homology in the coding region.In this study,we established hepatoma model of SD rats.We studied the correlation between NDRG1 gene expression and Wnt/?-Catenin pathway in animal level.To clarify the occurrence and development of liver cancer,and to provide a theoretical basis for the diagnosis and treatment of liver cancer.Methods: 1.50 male SD rats were randomly divided into control group(20)and model group(30).Rats in the model group were treated with DEN(Diethylnitrosamine)to establish hepatoma model of SD rats.The rats were killed every 4 weeks and sacrificed at the end of 16 weeks.Pathological slice technique was used to evaluate the success of model making.2.q RT-PCR echnology was used to detect the expression of NDRG1,?-Catenin and Cyclin D1 at the m RNA level of each group.3.Western Blot was used to detect the expression level of NDRG1 and ?-Catenin gene in protein.4.Data analysis: SPSS19.0 statistical software was used for data analysis.The experimental results were compared using one-way analysis of variance(ANOVA).P<0.05 was considered statistically significant.Results: 1.The general situation of liver tissue and the rate of cancer formation in rats: With the prolongation of DEN intake in rats,the surface of liver tissue gradually became rough,and color gradually changed from light to dark red.The texture of the tissue hardened,and the edge blunted.At the 12 th week,different size of gray white nodular lesions began to appear on the liver surface.When it came to the end of the 16 th week,the liver surface was covered with hard cancerous nodules.The rate of cancer formation in model group was 100%.There were 6 rats died in the model group at 14 weeks.The main causes of death were ascites,lung infection and multiple metastases.The mortality of rats in model group was 20%.2.Counted the result according to the rat culture cycle(the 4th,8th,12 th,16th week).q RT-PCR showed that the expression levels of NDRG1gene m RNA in the control group and the model group were respectively: 1.01�0.08,1.07�0.10,1.01�0.10,1.00�0.09;1.03�0.03,1.03�0.11,8.34�0.27,13.53�0.22.There was no significant difference between the model group and the control group in both the 4th and 8th week(P>0.05).The rest of the model group were significantly higher than those of the control group.The difference was statistically significant(P<0.01).The highest expression level of NDRG1 gene was the group of the 16 th week.The expression levels of ?-Catenin m RNA in each group were 1.04�0.10,1.03�0.08,1.02�0.09,1.04�0.11;1.00�0.04,1.07�0.13,5.92�0.47,9.90�0.45.Compared with the control group,there was no significant difference in both the 4th and 8th week in the model group(P>0.05).The others of the model group showed a significant upward trend(P<0.01).And the expression levels of Cyclin D1 m RNA in each group were as follows: 1.03�0.09,1.05�0.12,1.00�0.06,1.03�0.09;0.99�0.06,1.06�0.13,5.99�0.39,9.97�0.56.The results showed that there was no significant difference between the model group and the control group in both the 4th and 8th week(P>0.05).The rest of the model group were significantly higher than those of the control group(P<0.01).The highest expression level of Cyclin D1 gene was in the 16 th week.3.The expression levels of NDRG1 and ?-Catenin protein in rat liver tissue of all groups were detected by Western Blot: Counted the result according to the rat culture cycle(the 4th,8th,12 th,16th week).The expression levels of NDRG1 protein in the control group and the model group were respectively: 0.31�0.05,0.31�0.02,0.32�0.03,0.34�0.03;0.32�0.05,0.35�0.06,0.71�0.06,0.95�0.07.The expression levels of ?-Catenin protein were as follows: 0.39�0.04,0.44�0.05,0.42�0.05,0.41�0.02;0.43�0.04,0.45�0.06,0.76�0.05,1.06�0.11.The expression levels of NDRG1 and ?-Catenin protein were consistent with the results of PCR.There was no significant difference between the model group and the control group in both the 4th and 8th week(P>0.05).Both of them showed an upward trend in the 12 th week and the 16 th week,which were higher thanthose in the control group.The difference was statistically significant(P<0.01).Conclusion: 1.Liver cancer model was successfully established by Small dose intermittent oral administration of DEN.2.NDRG1 gene was highly expressed in liver cirrhosis and liver cancer tissues.Its expression was significantly higher in HCC tissues than that in normal liver tissues and liver benign lesions.3.NDRG1 gene which is high expression can promote the occurrence and development of primary liver cancer.Its mechanism may be associated with up regulation of ?-Catenin and abnormal activation of Wnt/?-Catenin signaling pathway.