A Study Of YAP1 Expression In Hepatoblastomas And The Effect Of YAP1 Targeted Inhibition

ObjectiveTo investigate the expression of Yes-associated protein 1(Yesassociated protein-1,YAP1)in hepatoblastoma of children and the effect of chemotherapy on the expression of YAP1 in hepatoblastoma cells,and to detect the amplification of YAP1 gene in the tumor cells before and after chemotherapy.To statistically analyze the correlation between YAP1 nuclear expression in tumor cells and the prognosis of the patients.To observe the effect of YAP1 siRNA and Verteporfin on the proliferation and apoptosis of hepatoblastoma cell line HepG2 in vitro.MethodThis experiment includes two parts:1.The expression of YAP1 in hepatoblastoma and its correlation with the patients’ prognosis.The expression of YAP1 and β-catenin in 38 cases of hepatoblastoma and the paratumor tissue,and YAP1 expression in 48 cases of chemotherapied hepatoblastoma were detected by immunohistochemical staining.The expression of YAP1 protein in 11 cases of tumor biopsy tissue and the chemotherapied hepatoblastoma was compared.FISH was used to detect YAP1 gene amplification.The correlation between YAP1 nuclear expression in hepatoblastoma and the prognosis of patients was analyzed statistically.2.The effect of YAP1 siRNA and vitipofen on proliferation and apoptosis of hepatoblastoma HepG2 cells.In vitro,YAP1 siRNA was transfected into HepG2 cells for 48 h,the YAP1 mRNA and YAP1 protein were detected by real-time fluorescence quantitative PCR(real-time quantitative PCR,RT-qPCR)and Western blot(western blot).After down-regulation of YAP1 expression by siRNA transfection or co-culture vitipofen with HepG2 cells,their effect on proliferation and apoptosis rate of HepG2 cells were detected by flow cytometry.Results1.The positive nuclear expression rate of YAP1 was 50.00%(19/38)in non-chemotherapy hepatoblastoma and 0.00%(0/21)in paratumor liver tissue(χ2=15.49,P<0.01),and the expression level was negatively correlated with the prognosis of the patients.The positive nuclear expression rate of YAP1 in hepatoblastoma after chemotherapy was 70.83%(34/ 48),which was significantly higher than that in non-chemotherapy hepatoblastoma(χ2=3.893,P<0.05).In the 11 cases of hepatoblastoma,the nuclear YAP1 expression in tumor cells was significantly increased after chemotherapy(t=12.251,P<0.01);however,no amplification of YAP1 was found in hepatoblastoma before and after chemotherapy by FISH.The positive expression rate of β-catenin in hepatoblastoma was 52.63%(20/38),but there was no β-catenin expression in paratumor liver tissue 0.00%(0/21)(χ2=16.72,P<0.01).The statistical analysis showed that the nuclear expression of YAP1 in tumor cells was positively correlated with the expression of β-catenin.2.After transfected YAP1 siRNA for 48 h,the mRNA and protein expression of YAP1 in HepG2 cells was significantly lower than that in the blank control HepG2 cells.The proliferation rate of HepG2 cells decreased and the apoptotic rate of HepG2 cells increased.After co-cultured with Verteporfin for 48 h,the proliferation rate of HepG2 cells was significantly lower than that of the control group(P<0.01),and the apoptosis rate of HepG2 cells was higher than that of the control group(P<0.01).ConclusionYAP1 expressed in 50% of hepatoblastoma,and the nuclear expression of YAP1 in hepatoblastoma was significantly increased after chemotherapy,but no amplification of YAP1 gene was found in hepatoblastoma cells before and after chemotherapy.The nuclear expression of YAP1 in hepatoblastoma was negatively correlated with the prognosis of the patients.YAP1 might cooperate with β-catenin to promote the growth of hepatoblastoma.Both down-regulation of YAP1 expression by siRNA and the targeted inhibition of YAP1 by vitipofen decreased the proliferative rate and increased the apoptosis rate of hepatoblastoma HepG2 cells.Therefore,YAP1 may be used as a new target for the treatment of hepatoblastoma,and Vitipofen may be used as a potential alternative drug.