芒柄花黄素通过HNF4A抑制食管癌细胞增殖、迁移和侵袭

Formononetin Inhibits Proliferation,Migration and Invasion of Esophageal Carcinoma Cells by HNF4A

目的分析芒柄花黄素(formononetin,FORM)通过肝细胞核因子4α(hepatocyte nuclear factor 4α,HNF4A)抑制食管癌细胞增殖、迁移和侵袭的机制。方法使用不同浓度的芒柄花黄素处理EC109和KYSE510,利用CCK8检测细胞相对存活率和细胞的增殖,利用细胞克隆实验培养癌细胞,并检测加入芒柄花黄素前后细胞克隆数量,利用Transwell检测加入芒柄花黄素前后EC109和KYSE510细胞的迁移和侵袭。裸鼠皮下注射肿瘤细胞,加入芒柄花黄素,观察移植瘤体积和重量变化,并利用免疫组化检测移植瘤组织中Ki67蛋白表达。采用蛋白质印迹法检测芒柄花黄素对HNF4A基因表达的影响。构建稳定过表达HNF4A的EC109和KYSE510细胞系,加入芒柄花黄素,观察HNF4A的表达情况。结果加入芒柄花黄素后,EC109和KYSE510的细胞相对存活率明显降低(P<0.05),与0μmol/L芒柄花黄素作用比较,EC109细胞系在芒柄花黄素浓度≥150μmol/L时,细胞相对存活率降低显著(P<0.05),KYSE510细胞系在芒柄花黄素浓度≥120μmol/L时,细胞相对存活率降低显著(P<0.05);芒柄花黄素组EC109和KYSE510增殖、侵袭、迁移的细胞数量均明显低于对照组(P<0.05)。移植瘤体积和重量明显低于对照组(P<0.05),Ki67蛋白表达明显低于对照组(P<0.05)。随着芒柄花黄素浓度越高、作用时间越久,HNF4A蛋白表达下调更为明显。HNF4A过表达可以恢复芒柄花黄素抑制的细胞增殖、侵袭和迁移(P<0.05)。结论芒柄花黄素通过HNF4A抑制食管癌细胞增殖、迁移和侵袭。

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 be one 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 after formononetin 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 and weight of xenograft tumors.The expression level of Ki67 protein in xenograft tissues was detected by immunohistochemistry.The effect of formononetin on the expression of HNF4A gene were detected by Western 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(≥150μmol/L for EC109 cell line,and≥120μmol/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 of formononetin 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 migration which were inhibited by formononetin(P<0.05).Conclusion formononetin can inhibit the proliferation,migration and invasion of EC cells by regulation of HNF4A.