| Over the last5to10years evidence has been accumulating in different countries thatthe incidence of hepatocellular carcinoma (HCC) is rising. HCC is the third cause ofcancer related death, next to lung cancer and stomach cancer. It is estimated that700,000-1,000,000cases are diagnosed worldwide annually, of which China contributesmore than half. The five-year survival rate of HCC patients is very low, with a poorprognosis. Although in recent years surgeons have developed selection criteria,hepatectomy, and liver transplantation techniques, tumor recurrence rate exceeds70%at5years after operation due to intrahepatic and extrahepatic recurrence. Therefore,identification of easily measurable biomarker that has strong correlations with clinicaloutcomes, especially for early stage HCCs, has important significance.On the other hand, we detected the proteins interacting withaspartyl-(asparaginyl)-b-hydroxylase (ASPH) by mass spectrometry analysis andco-immunoprecipitation in our privious study. We have confirmed that ASPH plays acritical role in HCC invasion and is associated with worse surgical outcome in previousstudies. The interaction between ASPH and ADP-ribosylation factor4was confirmed andfurther research indicated that ARF4expression level was associated with better surgicaloutcome. ADP-ribosylation factor4(ARF4) is a member of the ARFs family, which aresmall20kDa guanine nucleotide binding proteins and originally identified as a protein thatstimulates ADP-ribosyltransferase activity of cholera toxin in vitro. ARFs are involved inseveral cellular processes, including vesicular trafficking, cytokinesis, cell adhesion, andtumor cell invasion, and are known activators of phospholipase D. In recent years, severalgroups have examined the role of ARF4in human cancer cells; the exact cellular functionsof ARF4and the regulatory mechanism of ARF4expression, however, remain unclear. Inthis study, we investigated the effect of ARF4in HCC, and found that there was a stronginverse correlation between ARF4expression level and tumor growth, as well as HCCpatients prognosis. These findings will help us to further study the hepatocarcinogenesisand provide potential therapeutic targets. This study proceeded from the following three aspects:1)A total of247adult patientswith HCC undergoing hepatectomy were enrolled and tissue microarrays were used todetect the ARF4expression level; then retrospective analysis was done to investigate therelationship between ARF4expression and patient survival. Moreover, patients werestratified according to Barcelona Clinic Liver Cancer stage classification, especially forBCLC0/A patients. Cancer-specific survival outcomes were expressed by applying theKaplan–Meier method for all patients. The log–rank test was used to compare theprognostic significance of individual variables on survival. Cox proportional hazardsmodel was used in a multivariate analysis to identify the independent predictors of survival.2) Establishing stable ARF4-overexpressing or low expression HCC cell lines，includingHepG2-GFP, HepG2-ARF4, HepG2-siARF4-779, Huh7-GFP, Huh7-ARF4, andHuh7-siARF4-779. The effect of ARF4was tested on cell growth, clone formation,transwell chambers and cell cycle progression in vitro to detect growth and invasion ability.Moreover, different cell lines were planted in Nude/SCID mouse to observe theirtumorigenic ability.3) To investigate the role of protein related to cell cycle andinteractions between ARF4and interactive proteins. Cell immunofluorescence was chosento investigate their effect on cell proliferation.Part1Expression and clinical significance of ARF4in patients with HCC.The purpose of this part was to investigate the relationship between ARF4expression,tumor recurrence, and patient survival, especially for early HCC. A total of247adultpatients with HCC undergoing hepatectomy were enrolled and tissue microarrays wereused to detect the ARF4expression level; then retrospective analysis was done toinvestigate the relationship between ARF4expression and patient survival. Moreover,patients were stratified according to BCLC stage classification, especially for BCLC0/Apatients. Univariate analysis and multivariate analysis were done to determine whetherARF4was an independent prognostic factor influencing patient survival.The results of tissue microarrays, real time PCR and Western blotting showedincreased ARF4expression in some HCC samples. We further examined the relationshipbetween ARF4expression levels in tumor tissues and the clinico-pathologicalcharacteristics of all patients. Correlation regression analysis indicated that overexpressionof ARF4was significantly correlated with liver cirhosis, tumor number, portal vein tumorthrombus and BCLC staging system. High expression of ARF4was associated with better 5-year survival than low expression, while recurrence rate was opposite, especially forstage0/A patients. There was no statistically significant association of ARF4expressionlevel with prognosis in stage B/C patients. Univariate analysis of18survival-related andrecurrence-related clinico-pathological variables revealed that serum AFP level, albumin,tumor diameter, tumor number, portal vein tumor thrombus, cell differentiation grade,BCLC stage, TNM stage, ARF4expression level were statistically correlated with bothrecurrence and survival. Multivariate Cox proportional hazards model indicated that ARF4expression level was also an independent and significant factor, and it could affect therecurrence and survival of HCC patients. Finding1: ARF4expression level was associatedwith prognosis and was a protective factor for HCC patients.Part2Effect of ARF4on cell proliferation and invasion in HCC cell lineThe purpose of this part was to investigate the relationship between ARF4expressionlevel and malignant degree, to explore the Effect of ARF4on cell proliferation andinvasion in HCC cell line. Firstly, we established stable ARF4-overexpressing or lowexpression HCC cell lines，including HepG2-GFP, HepG2-ARF4, HepG2-siARF4-779,Huh7-GFP, Huh7-ARF4, and Huh7-siARF4-779. Secondly, the effect of ARF4was testedon cell growth, clone formation, transwell chambers and cell cycle progression in vitro todetect growth and invasion ability. Lastly, different cell lines were planted in Nude/SCIDmouse to observe their tumorigenic ability.The results of real time PCR and Western blotting showed increased ARF4expressionlevel in both HepG2-ARF4and Huh7-ARF4cell lines, and decreased ARF4expressionlevel in both HepG2-siARF4-779and Huh7-siARF4-779, compared with HepG2-GFP orHuh7-GFP, suggesting that cell line had been successfully established. Cell grow analysisshowed that the number of ARF4-overexpressing cell lines（HepG2-ARF4、Huh7-ARF4）was markedly reduced compared with that of the cells infected with GFP, while the numberof ARF4-lowexpressing cell lines（HepG2-siARF4-779, Huh7-siARF4-779）were in theopposite way. In colony formation assays, infection with ARF4markedly inhibited theclonogenic ability of both HepG2and Huh7cells, as reflected by the size and number ofcolonies. Flow cytometry was then used to determine the cell cycle profiles. The resultssuggested that overexpression of ARF4arrested HCC cells at the G2/M phase of the cellcycle. Transwell experiments confirmed that there was no obvious change on cellmigration in different cell lines. The similar results were achieved in Nude/SCID mice, which showed decreased tumorigenic ability in ARF4-overexpressing cell lines andincreased tumorigenic ability in ARF4-lowexpressing cell lines. Finding2: ARF4expression level was negatively associated with cell proliferation, colony formation andtumorigenic ability, but not with cell migration.Part3The mechanism for the effect of ARF4on cell proliferationIn this part we discussed the reason why overexpression of ARF4arrested HCC cellsat the G2/M phase of the cell cycle by detecting proteins related to ARF4expression. Wealso explored the relationship between protein kinaseC and ARF4overexpression. Firstly,microarray expression of225HCC patients was analyzed by statistical method to explorethe possible regulatory molecules for ARF4. Co-immunoprecipitation andimmunohistochemical staining were used for further validation. Secondly, stress fiberexpression and multinucleate cells were observed by cell immunofluorescence to explainthe mechanism why overexpression of ARF4arrested HCC cells at the G2/M phase.Thirdly, PMA, PDBu, Go6976and Go6983were used to explore the reason why ARF4over expression could be observed in some hepatocellular carcinoma.The statistical analysis results of microarray expression showed a close connectionbetween Rho A and ARF4expression (pearson coeff=0.64)；The stress fiber and Rho Aplayed crucial roles in the process of cell proliferation, which should be the researchfocus in future. There was decreased stress fiber expression level in ARF4overexpressioncell lines, but increased expression level in ARF4knockdown cells as well as an increasedmultinucleate cell. Another important finding of this study was that PKC signal pathwaystimulated by PMA or PDBu resulted in an increased expression level of ARF4whileGo6976or Go6983lead to the opposite result.To sum up, we conclude that ARF4may play an important protective role in cellproliferation and tumorigenicity via regulation of G2/M phase transition, and mayinfluence the prognosis of HCC patients. This molecule is likely to provide a more preciseprognostic predictor in early stage HCCs, and may be a potential therapeutic target to treathepatocellular carcinoma. |
PDF Full Text Request