Abstract: Objective To analyze the mechanism of formononetin on inhibiting the proliferation, migration and invasion of esophageal carcinoma (EC) cells by hepatocyte nuclear factor 4α(HNF4A). Methods It was concluded by network pharmacology analysis that HNF4A might beone of the targets of formononetin. The expression level of HNF4A was closely related to clinical phenotypes of EC. EC109 and KYSE510 were treated with different concentrations of formononetin. The relative survival rate of cells was detected by CCK8. The cells proliferation was detected by CCK-8. The cancer cells were cultured by cells cloning assay. The number of colony cells before and afterformononetin treatment was detected. The migration and invasion of EC109 and KYSE510 cells before and after formononetin treatment were detected by transwell assay. Nude mice were subcutaneously injected with tumor cells and treated with formononetin to observe the changes in volume andweight of xenograft tumors. The expression level of Ki67 protein in xenograft tissues was detected byimmunohistochemistry. The effect of formononetin on the expression of HNF4A gene were detected byWestern blotting. EC109 and KYSE510 cell lines with stable overexpression of HNF4A were constructed and treated with formononetin to observe the effect on the expression of HNF4A. Results After formononetin treatment, the relative survival rates of EC109 and KYSE510 cells were significantly decreased (P< 0. 05). Compared with that of cells without formononetin treatment, the relative survival rate of cells was significantly decreased after treated with formononetin (≥150 μmol / Lfor EC109 cell line, and ≥ 120 μmol / L for KYSE510 cell line) ( P < 0. 05). The proliferation,migration and invasion of EC109 and KYSE510 cells in the formononetin group was significantly decreased when compared with those in the control group (P< 0. 05). The volume and weight of xenograft tumors were significantly decreased in the formononetin group (P< 0. 05), and the expression level of Ki67 protein was significantly lower than that in the control group (P< 0. 05). The effect offormononetin on the down-regulation of HNF4A protein was dependent on the concentration and time of the treatment. HNF4A overexpression could promote cells proliferation, invasion and migrationwhich were inhibited by formononetin (P< 0. 05). Conclusion formononetin can inhibit the proliferation, migration and invasion of EC cells by regulation of HNF4A.

Key words: formononetin, esophageal carcinoma cell, proliferation, invasion, migration