The Role Of Foxd3 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is famous for its high incidence and poor prognosis.Studies have shown that epigenetic abnormalities of tumor suppressor genes,especially the methylation of DNA,play a key role in HCC development.Purpose We found that the promoter of Foxd3 gene was highly methylated in primary hepatocellular carcinoma by gene methylation microarray,suggesting that the expression of Foxd3 protein was regulated by epigenetics.Therefore,we intend to further explore the function and mechanism of the gene in the occurrence and development of primary hepatocellular carcinoma in this study.Methods We evaluated themethylation status of Foxd3 promoter by methylation-specific-PCR and bisulfite genome sequencing.Foxd3 mRNA and protein expression level was examined by semi-quantitative RT-PCR and immunohistochemistry.Finally,we tested effects of re-express Foxd3 protein on the migration,invasion,apoptosis and autophagy of tumor cells.ResultsDNA promoter methylation was detected in hepatocellular carcinoma cell lines and primary liver cancer tissues,and DNA promoter methylation was detected in both tumor cells and tumor tissues with abnormal expression.We found that the expression of Foxd3 gene was positive in 44.8%(47 cases)of HCC tissues and 91.4%(96 cases)para-normal tissues.The difference was statistically significant(p=0.04).Negative Foxd3 protein expression was significantly correlated higher HBV infection(p=0.016),higher AFP level(p=0.038),bigger tumor size(p=0.007)and worse tumor TNM stage(p=0.033).We also found that the 5-year overall survival rate and tumor-free survival rate of patients with positive expression of Foxd3 in cancer tissues were significantly higher than those of patients with negative expression(p=0.032,p=0.027,respectively).The model of hepatoma cell lines stably expressing Foxd3 was established.In vitro and in vivo experiments,it was found that Foxd3 could significantly inhibit tumor growth.Furthermore,it was found that Foxd3 gene could promote apoptosis of hepatoma cells by promoting the expression of cleaved-caspase3,caspase7,and caspase9,and enhance the apoptosis of hepatoma cells induced by starvation by inhibiting the AMPK-Akt mTOR pathway.ConclusionsFoxd3 is a tumor suppressor gene whose expression is regulated by DNA methylation.The reexpression of Foxd3 protein can promote the starvation induced autophagy of hepatoma cells,inhibit tumor cell migration,tumor formation or growth in vivo.