| Objective:hepatoblastoma(HB)is a common malignant tumor in pediatrics,and its incidence is increasing year by year.The onset of hepatoblastoma is more hidden,and the clinical harm is great,and the treatment is difficult.Chemotherapy is one of the treatment methods of hepatoblastoma,but at present,there are few chemotherapy drugs suitable for children.Therefore,it is necessary to seek further chemotherapy drugs or targeted drugs to treat hepatoblastoma.Everolimus is a new generation of mammalian target of rapamycin(m TOR)inhibitors.It is initially used for immune rejection after organ transplantation,and later studies have found that it also has anti-tumor activity and has been approved for the treatment of a variety of tumors.Everolimus is also an effective autophagy activator,which can activate autophagy and induce apoptosis,and can be used as an effective tumor inhibition mode.Currently,everolimus has been verified to inhibit tumor proliferation in vitro and in vivo experiments.However,its role in tumor inhibition(especially in hepatoblastoma)is still largely undetermined.It may be a new way to treat hepatoblastoma.The objective of this study was to investigate the effects of everolimus on the proliferation,migration,apoptosis,and autophagy of hepatoblastoma cells(Hep G-2,Huh-6),and to initially explore the mechanism of action,and to establish patient-derived xenograft,patient-derived xenograft.PDX)model to evaluate the therapeutic effect of everolimus and provide experimental basis for the treatment of hepatoblastoma with everolimus.Method:1.Experimental grouping: hepatoblastoma cells were divided into control group,low,medium and high concentration everolimus treatment group;2.The effect of everolimus on hepatoblastoma cell activity was detected by MTT colorimetry.3.Transwell was used to detect the effect of everolimus on the migration ability of hepatoblastoma cells.4.Flow cytometry was used to detect the effect of everolimus on apoptosis of hepatoblastoma cells.5.The expression of hepatoblastoma cell proliferation indexes(nuclear antigen(PCNA)),apoptosis indexes(Bax,Bcl-2),autophagy indexes(LC3B,ATG16L1)and protein EVA1 A were detected by Western Blot.6.Hepatoblastoma PDX model was established as the evaluation platform of everolimus.Control group,positive control group(cisplatin group),paclitaxel group and everolimus treatment group were set up to compare the therapeutic effects of hepatoblastoma.7.Statistical methods: SPSS14.0 statistical analysis software was used to analyze the experimental data,and the description of experimental data was in the form of mean ± standard error(X±SEM).For those obeying normal distribution,one-way analysis of variance is used,while non-parametric test is used for those disobeying normal distribution.Results:1.Effects of everolimus on proliferation and migration of hepatoblastoma cells(1)hepatoblastoma cells were treated with everolimus,and the cell activity of hepatoblastoma cells was affected to different degrees,and the difference was statistically significant compared with the control group.(2)Compared with the control group,the expression level of PCNA in hepatoblastoma cells treated with everolimus decreased with increasing concentration,and the difference was statistically significant.(3)Compared with the control group,everolimus had an inhibitory effect on the migration ability of hepatoblastoma cells,and the inhibitory effect increased with the increase of everolimus concentration,the difference was statistically significant.2.Effect of everolimus on apoptosis of hepatoblastoma cells(1)Compared with the control group,the proportion of apoptosis of hepatoblastoma cells treated with everolimus increased with the increase of everolimus concentration,and the difference was statistically significant.(2)Compared with the control group,the expression level of Bax/Bcl-2 in hepatoblastoma cells treated with everolimus increased with the increase of everolimus concentration,the difference was statistically significant;3.Effect of everolimus on autophagy of hepatoblastoma cells Compared with the control group,the expressions of autophagy related proteins LC3 B and ATG16L1 in hepatoblastoma cells treated with different concentrations of everolimus were up-regulated in a dose-dependent manner,and the high concentration group had the most obvious effect on hepatoblastoma cells,with statistically significant differences.4.Effects of everolimus on autophagy mechanism related protein EVA1 A in hepatoblastoma cells Compared with the control group,the expression level of EVA1 A protein in hepatoblastoma cells treated with different concentrations of everolimus was up-regulated in a dose-dependent manner,and the difference was statistically significant.5.The PDX model of hepatoblastoma was established.Compared with the traditional chemotherapy drugs cisplatin and paclitaxel,the mean tumor volume measured in the everolimus treatment group was significantly lower than that in the cisplatin and paclitaxel groups,showing the optimal therapeutic effect,and the differences in measurement data between groups were statistically significant.Conclusion:(1)Everolimus inhibited the proliferation and migration of Hep G-2 and Huh-6cells in vitro and promoted apoptosis,suggesting that everolimus could induce apoptosis of hepatoblastoma cells.(2)Everolimus can induce autophagy in hepatoblastoma cells and increase the expression of autophagy related proteins,and the expression of EVA1 A protein is up-regulated.It is speculated that everolimus can induce autophagy in hepatoblastoma cells by regulating EVA1 A.(3)Efficacy evaluation was conducted on PDX model of hepatoblastoma,and it was found that the efficacy of everolimus in the treatment of hepatoblastoma was better than that of traditional chemotherapy drugs cisplatin and paclitaxel,suggesting that everolimus could be a potential drug for the treatment of hepatoblastoma.In conclusion,in cell experiments,it was found that everolimus could inhibit the proliferation and migration of hepatoblastoma cells,promote autophagy and induce apoptosis.In addition,it was verified in the hepatoblastoma PDX model that the tumor inhibition effect of everolimus was better than that of traditional chemotherapy drugs,which may be used as a new drug for the treatment of hepatoblastoma. |
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