| 1. The prognosis of recurrent HCCObjective: To clarify the prognosis of liver transplantation(LT) for recurrent HCC through a graft survival analysis.Methods: A retrospective analysis of the clinical and graft survival data of patients who underwent LT between Jan 1st 2000 and Dec 31 st 2011 was conducted. Finally 1554 patients were studied, including 1392 primary HCC and 162 recurrent HCC.Results: In the analysis of all the included patients, there was a significant difference in survival between the primary HCC and the recurrent HCC groups(P=0.030). The result of Cox regression analysis showed that primary/recurrent HCC(P=0.049), MELD score(P<0.001), Milan criteria(P=0.022) and UCSF criteria(P<0.001) were the independent prognostic factors for graft survival. In the subgroup analysis, Cox regression analysis found that Milan-UCSF criteria grading was the only independent prognostic factor for graft survival in the recurrent HCC group(P=0.023). When the survival analysis was performed among the patients with HCC under Milan criteria, the difference in survival was not significant between primary and recurrent HCC groups(P=0.292). In contrast, the difference in survival was still significant between primary and recurrent HCC groups, when the analysis was preformed among patients with HCC between Milan and UCSF criteria(P=0.046).Conclusion: The LT for recurrent HCC had an independent impact on the graft survival. In the LT for recurrent HCC, the stage of intrahepatic recurrent lesion before LT was associated with the graft survival.2. The correlation between HCC recurrence after LT and tumor growthObjective: To explore the relationship between tumor growth and tumor recurrence after LT through a correlation analysis between time to recurrence(TTR) and tumor growth of pulmonary metastasis.Methods: In Tianjin first central hospital medical record system, case selection was conducted among patients who underwent LT between Jan 1st 2010 and Sep 31st 2013 and suffered from pulmonary metastasis after LT. Finally, 32 cases of pulmonary metastasis were included and 26 paraffin HCC samples from excised liver were used for immunohistochemical staining of Ki-67.Results: There was a significant correlation between TTR and the diameter increasing of target lesion(n=32, P=0.021). And the correlation between TTR and Ki-67 expression also reached statistical significant(n=26, P=0.045).Conclusion: The HCC recurrence after LT was correlated with HCC growth.3. Ki-67 and IRF-1 predict the prognosis of liver transplantation for HCCObjective: To assess the predictive value of biomarkers for HCC recurrence after LT.Methods: The patients who underwent LT in Tianjin first central hospital between Jan 1st 2012 and Dec 31 st 2014 and matched inclusion criteria were involved in this study. Several biomarkers were leveled by the results of immunohistochemical staining. Patients were grouped by the levels of each biomarker and the comparisons of recurrence free survival(RFS) were conducted between groups. The prognostic value of each biomarker was determined in this way.Results: 127 cases of HCC were included. After comparisons between biomarker levels(after Benjamini-Hochberg correction), the significant differences in RFS were found in Milan-UCSF criteria grades(q<0.001), with/without tumor microemboli groups(q<0.001), Ki-67 negative/positive groups(q<0.001) and Ki-67 low/high groups(q<0.001). The results of Cox regression analysis showed that Milan-UCSF criteria(P<0.001), tumor microemboli(P<0.001), Ki-67(P<0.001) were independent prognostic factors for RFS. In the subgroup analysis, a significant difference in RFS was also found between IRF-1 negative and positive groups(q<0.001). Among patients with HCC under UCSF criteria, there was a significant difference in Ki-67 positive expression between primary and recurrent HCCs(P=0.022).Conclusion: Ki-67 can effectively predict the recurrence of HCC after liver transplantation, and can be used in candidate selection before LT. Among patients with HCC under UCSF criteria, there was a difference in Ki-67 expression between the primary and the recurrent HCCs, which may be one explanation for the discrepancy in graft survivals of them. IRF-1 was not a good marker for HCC recurrence after LT, but IRF-1 expression was associated with lower HCC recurrence after LT, in patients with HCC beyond Milan criteria.4. Role of IRF-1 on apoptosis and autophagy in HCC cellObjective: To explore the roles of IRF-1 on apoptosis and autophagy in HCC cells, through in vitro experiments on human HCC cell line SK-Hep1.Methods: In experiment 1, the validities of cells were tested after IFN-γ stimulation by MTT method. In experiment 2 and 3, SK-Hep1 cells were grouped into control group and IFN-γ group. Proteins related to autophagy and apoptosis were tested by Western Blot analysis. Apoptosis after IFN-γ stimulation was confirmed by PI staining and flow cytometry. Double labeled LC3 transfected SK-Hep1 cells were stimulated with IFN-γ and observed under fluorescence microscope. Ultrastructure of SK-Hep1 cells with/without IFN-γ stimulation were observed under transmission electron microscope(TEM). In experiment 4, SK-Hep1 cells were grouped into control group, IFN-γ group, Z-VAD-FMK group and Z-VAD-FMK+IFN-γ group. Proteins related to autophagy were tested by Western Blot analysis. In experiment 5, SK-Hep1 cells were grouped into control group, negative control(si RNANC) group,(IRF-1) si RNA1 group, si RNA2 group, si RNA3 group, si RNANC+IFN-γ group and si RNA1+IFN-γ group. The levels of proteins related to autophagy were tested by Western Blot analysis.Results: The results of Western Blot analysis showed that the levels of IRF-1, p STAT1 and cleaved Caspase-3 were increased, while the levels of Ki-67, LC3-II were decreased after IFN-γ stimulation. PI staining and flow cytometry showed an increase in the proportion of death cell after IFN-γ stimulation. Fluorescent spots of LC3 were reduced after IFN-γ stimulation. The number of autophagosomes was also decreased in ultrastructure imagines of SK-Hep1 cells stimulated with IFN-γ. Compared with cells treated with Z-VAD-FMK(Caspase inhibitor), no significant decrease in the levels of Beclin1, LC3-II, Atg5 and Atg7 was found in SK-Hep1 cells treated with IFN-γ and Z-VAD-FMK together. After SK-Hep1 cells was transfected with IRF-1 si RNA, LC3-II and LC3-II/LC3-I ratio decreased significantly. When si RNA transfected SK-Hep1 cells were stimulated with IFN-γ, the level of LC3-II was increased and the level of IRF-1 was decreased in comparison with si RNANC+IFN-γ group.Conclusion: In the SK-Hep1 cell line, IRF-1 generates a positive feedback to IFN-γ signal by activating STAT1; IFN-γ promotes apoptosis and suppresses autophagy via IRF-1 expression and Caspase activation; the basic expression of IRF-1 is important to the autophagy. |
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