| Objective: Primary hepatocellular carcinoma(PHC)is one of the most common malignant tumor in the world,the main risk factors include hepatitis B virus and / or hepatitis C virus chronic infection,alcohol abuse,aflatoxins,toxic chemicals and nonalcoholic fatty liver disease(NAFLD).In recent years,the incidence of NAFLD increases rapidly and is closely related to PHC.The pathogenesis of this disease remains to be elucidated.In this paper,through the establishment of fatty liver and liver cancer dynamic models,in the condition of accumulation of liver fat accumulation of fatty liver,2-fluorenyl acetamide(2-FAA)induced liver cell malignant transformation.In this process,screening differentially expressed genes in hepatocellular carcinoma by gene expression microarray and making further bioinformatic analysis,focused on molecular markers of hepatocellular carcinogenesis and mitochondrial enzymes,a potential target for immune and the relationship between abnormal malignant transformation of hepatocytes and lipid metabolism.Methods: Sprague-Dawley(SD)male rats divided into normal control group,fatty liver group and cancer induction group were fed with normal,high fat(HF),and HF containing 2-fluorenylacetamide(2-FAA,0.05%)diet for 14 weeks,respectively.1 rat from the model group,1group from the fatty liver groups and 1group from the cancer induction groups were sacrificed every 2weeks and preserve liver tissue and serum.Rats according to the Hematoxylin and Eosin staining of livers were divided into control,fatty liver,degeneration,precancerous,and cancerous groups.Liver lipids were dyed with the Oil Red O.Levels of total cholesterol(Tch),triglyceride(TG),and amino-transferase(ALT,AST)were determined by the routine methods.RNA of each group was extracted and then c RNA was synthesized.The c RNA was labeled,hybridized and scan.Differentially expressed genes for pairwise comparison of control,fatty liver,degeneration,precancerous,and cancerous groups were screened,and analyzed further by GO term analysis,KEGG Pathway analysis and protein interaction analysis.Finding the markers of hepatocyte malignant transformation and detect the changes of mitochondrial genes such as carnitine palmitoyl transferase-II(CPT-II)by immunohistochemistry and quantitative analysis.Results:The fatty liver and hepatocyte malignant transformation rat models were successfully prepared.After the rats intaked with HF and/or HF+2-FAA diets,the rat livers appeared mass lipid accumulation.The lipid levels in the control group was significantly lower than in fatty liver group(t=-11.556,P<0.001),degeneration group(t=-4.847,P=0.04),precancerous group(t=-13.652,P=0.005)and cancerous group(t=-10.896,P=0.008)。 The changes of serum TG and Tch levels were abnormally increasing 2 ~ 3 times more than those in the controls(P<0.05).During the rat liver morphological changes from normal to cancer development process with hepatocyte injury,serum AST and ALT levels were significantly higher(4~8 times,P<0.05)than those in the control group.The change of gene expression profiling chip.10 groups differentially expressed genes were found by Pairwise comparison through the gene expression profiling chip,among which fatty liver group vs normal group there were 163 differentially expressed genes,liver injury in vs normal group there were 934 differentially expressed genes;precancerous of vs normal group there were 1452 differentially expressed genes;hepato-cellular carcinoma vs normal group there were 1738 differentially expressed genes.The changes of related signaling pathways.Steroid biosynthesis pathway,p53 signaling pathway,Cell cycle pathway were the main signaling pathways found that related to the rat hepatocyte malignant transformation under lipidaccumulation.Besides,the Cyp51 and Tm7sf2 in the sterol synthesis pathway were significantly down regulated in the whole process of malignant transformation of hepatocyte.Ccnb1 and CDK1 are not only in the P53 pathway but also in the cell cycle.At the same time,all of these are in the key position in the protein interaction network.The change of CPT-II levels.The hepatic CPT-II levels(CPT-II,ng/mg,liver protein,ng/mg,P)was significantly higher in the control group than those in the fatty liver(t=2.641,P=0.035),denaturation(t=7.559,P<0.001),precancerous(t=5.504,P<0.001),and cancerous group(t=6.825,P<0.001),respectively.Besides,the CPT-II levels in the fatty liver group was significantly higher than those denaturation(t=10.210,P<0.001),precancerous(t=4.721,P=0.001),and cancerous group(t=7.100,P<0.001),respectively.It can be seen that the specific concentration of liver CPT-II expression was progressively decreasing during hepatocyte malignant transformation.The immunohistochemistry of CPT-II find that average optical density of hepatic CPT-II expression in the control group was significantly higher than those in the fatty liver(t=2.648,P=0.025),denaturation(t=9.071,P<0.001),precancerous(t=8.397,P<0.001),and cancerous group(t=8.836,P<0.001),respectively.Besides,the average optical density in the fatty liver group was significantly higher than those denaturation(t=6.976,P<0.001),precancerous(t=6.166,P<0.001),and cancerous group(t=6.698,P<0.001),respectively.From these data,the decreasing specific concentration of liver CPT-II expression was discovered during rat hepatocyte malignant transformation.The changes of genes related to hepatocyte malignant transformation.At the transcriptional level,GPC3 and CD44 m RNA were significantly lower than those in denaturation,precancerous and cancerous groups.AFP m RNA was significantly lower than in precancerous(t=-4.051,P=0.003)and cancerous groups(t=-2.836,P=0.021).At the protein level,AFP and GPC3 were significantly lower than those in denaturation,precancerous and cancerous groups in serum concentration(ng/m L)and liver specific concentration(ng/per mgwet tissues).The serum CD44 was significantly lower than those in denaturation(t=-3.562,P=0.015),precancerous(t=-9.347,P<0.001)and cancerous group(t=-7.131,P=0.001).The liver specific concentration of CD44 was significantly lower than in precancerous(t=-6.82,P<0.001)and cancerous group(t=-6.836,P<0.001).The results showed that the content of AFP,CD44 and GPC3 in liver tissue and serum increased gradually,which were consistent with the gene level,during the process of hepatocyte malignant transformation.Conclusions: Through the hepatic fat accumulation and hepatocarcinoma dynamic double models,observe the hepatocyte malignant transformation under the condition of fat accumulation involving multiple genes.many genes enriched in cholesterol synthesis,P53,cell cycle related pathways.Abnormal expression of cell markers in hepatocyte malignant transformation,suggesting that the molecular mechanism of hepatocyte malignant transformation induced by lipid accumulation was an extremely complex process,which is valuable to study more. |
PDF Full Text Request