Abnormality Of Signal Transduction In Hepatocellular Carcinoma And Effects Of HCV Core Protein On Biological Characteristics And Signal Transduction Pathways Of Human Hepatocytes

Hepatocellular carcinoma is one of common malignant tumors, which severelyharm human health. The epidemiologic study has revealed that the crucial etiologicfactors are infection of hepatitis viruses, intake of aflatoxins and insobriety. Thedevelopment of molecular biological techniques at full speed and the deeply researchon signal transduction pathways have given an important impulse for elucidation ofhepatocellular carcinogenesis. It was indicated that neoplastic genesis is adevelopment process of multiple factors and multiple stages, which involves in cellcycle, cell proliferation, apoptosis and signal transduction etc. Especially, the relationof signal transduction to diseases has become hot point and front edge in the field oflife science research. In this study, the phosphorylation of p44/42^MAPK and stat3, andthe expression of c–fos and c-jun proteins were detected by immunohistochemicaltechnique in HCCs, their surrounding liver tissues and normal liver tissues. Theircorrelation and clinical significance were investigated at first. It was indicated that the negative expression of phosphorylated p44/42^MAPK, c-fosand c-jun protein was in all nine cases of normal tissue, and p-stat3 positive signalwas only in one case. In HCCs, the positive expression rate of p-p44/42^MAPK, p-stat3,c-fos and c-jun proteins were 87.3%(48/55), 74.5%(41/55), 76.4%(42/55)and 87.3%(48/55)respectively. Their positive signals were mainly located in the nucleus. Thedistributive patterns of positive cells were clustered and /or diffused. Inpericarcinomatous liver tissues (PCLT), the positive expression rate of p-p44/42^MAPK,p-stat3, c-fos and c-jun proteins were 32.7%(18/55), 23.6%(13/55), 34.5%(19/55)and 29.1%(16/55) respectively. Their positive signals were nuclear/cytoplasm type,and the distributive patterns of positive cells were scattered. The positive rates andexpression levels of p-p44/42^MAPK, p-stat3, c-fos and c-jun protein in HCCs weresignificantly higher than those in PCLTs. The expression intensity of c-fos and c-junproteins were positive correlation to those of p-p44/42^MAPK and p-stat3 respectively,but there was no significant correlation between p-p44/4^MAPK and p-stat3 in bothHCCs and PCLTs. The results suggest that abnormalities of Ras/Raf/MAPK andJAKs/Stat3 cascade reactions may contribute to malignant transformation ofhepatocytes. Hepatocytes with p-p44/42MAPKand stat3 expressions may be apre-cancerous cell with potential malignant. Activation of MAPK and stat3 proteinsmay be an early event in hepatocellular carcinogenesis.Hepatitis virus C (HCV) is one of important causative agents of HCC, and itscarcinogenesis mechanism is not well understood until now. HCV, as a positive strandRNA virus, does not have reverse transcriptase activity, and fails to integrate with thehost genome to activate oncogene, because of replicating only in cytoplasm of hostcells. Most researches have indicated that the malignant transformation of host cellsmay occur through the expression products of HCV gene, in which HCV NS3, NS5and core proteins may play an essential role in hepatocellular carcinogenesis.HCV core protein consists of 191aa(1-191aa), It can be localizated in cytoplasmand/or nucleus, and suppress immune response of host. Especially, HCV core proteinpromotes primary human hepatocytes to an immortalized phenotype by introductionof the core genomic region into primary human hepatocytes. In transgenic mice, theexpression HCV core protein could result in hepatic steatosis and HCC. The ratembryonic fibroblasts co-transfected with HCV core genome and H-ras oncogenedisplayed tumorigenic phenotype. As a hepacivirus, HCV natural host is thehepatocyte of human or chimpanzee. But the cell lines and transgenic mice used bymost researches were not based on HCV natural infection process, so the resultsacquired may be hardly convinced. In order to simulate the HCV infection modelmore clearly, non-tumorigenic human liver cell line QSG7701 was chosen andtransfected with eukaryon expression plasmid of HCV core protein--pcDNA3.1-core.The biological characteristics of transfected QSG7701 cells were observed.This study indicated that the cell line transfected with plasmid of expressing HCVcore protein (pcDNA3.1-core/QSG7701) displayed growth inhibiting. The cloningratio of cells transfected with pcDNA3.1-core was significantly lower than those ofcells transfected with pcDNA3.1 and nontransfected(32.25%, 47.5% and 42.5%respectively, χ2=20.025,p<0.01). Flow cytometry analysis indicated that thepcDNA3.1-core /QSG7701 cells were arrested in G1/G0 phase, and S phase cellsdecreased;Its apoptosis ratio (1.04%) was lower than those of the other two( 1.68%and 3.7%, respectively). Immunohistochemical labeling confirmed that the expressionlevel of activated caspase-3, an important executing molecular for apoptosis, wasdecreased in pcDNA3.1-core/QSG7701 cells. Cell proliferation assay found that thepopulation doubling time in pcDNA3.1-core /QSG7701 cells was much longer thanthose in cells transfected with pcDNA3.1 and non-transfected (36h, 27h, 28hrespectively). MTT detection showed that the survival rate ofpcDNA3.1-core/QSG7701 cells was lower than those of other two groups. The resultssuggest that HCV core protein may inhibit cell proliferation and apoptosis.Cell biological characteristics are precisely regulated by signal transductionpathways, in which MAPK cascade is essential for regulation of cell proliferation andapoptosis. The effects of HCV core protein on MAPK cascade inpcDNA3.1-core/QSG7701 cells were detected by western blotting. The resultsshowed that there was no differences of the expression levels of P44/42MAPK, p38 andJNK in pcDNA3.1-core/QSG7701, pcDNA3.1/QSG7701 and QSG7701 cells, but theexpression levels of phosphorylated-P44/42MAPK, -p38 and -JNK inpcDNA3.1-core/QSG7701 cells were much lower than those in the other two groups,suggesting that HCV core protein may suppress hepatocyte proliferation by inhibitingphosphorylation of p44/42MAPK and JNK, and suppress hepatocyte apoptosis throughinhibiting phosphorylation of p38MAPK. Anti-proliferation and anti-apoptosis functionsof HCV core protein may benefit the survival of hepatocytes infected with HCV andcontribute to the selective advantage for HCV replication, which may result in thepersistence of HCV infection and development of chronic hepatitis C, liver cirrhosisand HCC.Biological behavior of cells is regulated by signal transduction through nucleartranscript factors to affect the genome of cell. For this reason, the effects of HCV coreprotein on the expression and activity of NF-κB, AP-1 and stat3 were researched bywestern blotting, luciferase assays, immunocytochemistry and EMSA. In comparisonof pcDNA3.1-core/QSG7701 cells with pcDNA3.1 /QSG7701 and QSG7701 cells,there was on difference for total proteins of p50, p65, c-fos, c-jun and p-stat3, but thenuclear proteins extracted from pcDNA3.1-core/QSG7701 cells were significantlydecreased. The positive signals of p50, p65, c-fos, c-jun and p-stat3 inpcDNA3.1-core/QSG7701 cells were weaker and located in cytoplasm and nucleus,but they were strong and mainly located in nucleus of pcDNA3.1/QSG7701 andnontransfected cells. Luciferase assay indicated that the activities of NF-κB andAP-1 in pcDNA3.1-core/QSG7701 cells were lower than those in other two groups.EMSA showed that DNA binding activities of NF-κ B, AP-1 and stat3 inpcDNA3.1-core/QSG7701 cells were weaker than those in pcDNA3.1/QSG7701 andnontransfected cell. These results suggest that HCV core protein may suppress cellproliferation through inhibiting the activation, nuclear translocation and DNA bindingactivity of NF-κB, AP-1 and stat3, and especially lead to resistant to interferon byinhibiting JAK/stat3 signal pathway, and persistence of HCV infection andoccurrences of liver cirrhosis and HCC.