The molecular mechanism of alpha-fetoprotein inhibiting cisplatin-induced apoptosis of hepatocellular carcinoma cells
10.3760/cma.j.cn311367-20190521-00223
- VernacularTitle:甲胎蛋白抑制顺铂诱导肝癌细胞凋亡的分子机制
- Author:
Chao ZHANG
1
;
Ying YAN
;
Haijian ZHAO
;
Gang LI
;
Chuanbao ZHANG
Author Information
1. 北京医院国家老年医学中心 国家卫生健康委临床检验中心 中国医学科学院老年医学研究院 100730
- From:
Chinese Journal of Digestion
2020;40(6):400-406
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the molecular mechanism of alpha-fetoprotein (AFP) inhibiting cisplatin-induced apoptosis of hepatocellular carcinoma cells.Methods:HepG2 (AFP positive) and QSG-7701 (AFP negative), two common hepatocyte carcinoma cell lines were selected. The expression of AFP was knockdown in HepG2 cells with RNA interference method and AFP expression plasmid was transfected in QSG-7701 cells. After the cells were cultured for 12, 24, 36 and 48 hours, the cell proliferation was detected by cell counting kit-8 (CCK-8) assay. After HepG2 and QSG-7701 cell lines were interfered or overexpressed AFP protein for 24 h, respectively, apoptotic inducer cisplatin (CDDP) was added. The effect of AFP on apoptosis induced by CDDP in hepatocellular carcinoma cells was determined by flow cytometry. The interaction between AFP and transcription factor retinoic acid receptor (RAR) was examined by protein coimmun oprecipitation (CoIP) technique. The effect of AFP expression level on the expression of DNA damage inducible transcript 1 ( DDIT1), and the effects of AFP and DDIT1 on apoptosis of hepatocellular carcinoma cells were tested by chromatin immunoprecipitation (ChIP) assay and Western blotting. T test was performed for statistical analysis. Results:The results of CCK-8 test showed that after plasmid transfected for 12, 24, 36 and 48 hours, the relative proliferation rates of QSG-7701 cells overexpressed AFP increased by 28.7%±2.7%, 49.8%±6.1%, 65.8%±3.0% and 79.3%±2.0%, respectively; however the relative proliferation rates of HepG2 cells after AFP knockdown decreased by 16.5%±6.1%, 28.5%±5.7%, 42.5%±1.7% and 57.6%±3.8%, respectively, when compared with the control group, and the differences were statistically significant ( t=3.978, 4.357, 3.461, 3.636, 2.858, 2.446, 3.233 and 4.492, all P<0.05). The results of flow cytometry indicated that after AFP overexpression for 24 and 48 hours, the apoptosis rate of QSG-7701 cells decreased by 46.3%±2.9% and 47.7%±7.4%, respectively, compared with cisplatin induced cells; however after AFP knockdown for 24 and 48 hours, the apoptosis rate of HepG2 cells increased by 86.7%±4.0% and 31.6%±10.5%, respectively, compared with cisplatin induced cells, and the differences were statistically significant ( t=3.543, 3.893, 2.336 and 2.561, all P<0.05). The results of CoIP experiment showed that AFP could interact with RAR. After AFP knockout in HepG2 cells, after nucleoprotein extracted, RAR entering the nucleus increased as compared with the control group. However, after overexpression of AFP in QSG-7701 cells, RAR entering the nucleus decreased compared with the control group. The results of ChIP experiments showed that AFP could regulate the expression of DDIT1. The expression of DDIT1 in AFP knockdown HepG2 cells was higher than that of control group, however the expression of DDIT1 in AFP overexpressed HepG2 cells was lower than that of control group. Compared with the control group, the apoptosis rate of HepG2 and QSG-7701 cells increased by 53.1%±4.0% and 73.3%±6.4% respectively after transfecting with DDIT1, and the differences were statistically significant ( t=3.462, 3.012, P=0.009, 0.017). In QSG-7701 cells, after AFP overexpression, the apoptosis rates decreased by 46.6%±4.8% compared with cisplatin added alone. After overexpression of AFP, cisplatin added and overexpression of DDIT1, the apoptosis rate increased by 43.6%±2.7% as compared with that of overexpression of AFP and cisplatin added, and the differences were statistically significant ( t=2.833 and 2.545, P=0.018 and 0.029). In HepG2 cells, after AFP knockdown the apoptosis rate increased by 73.3%±6.1% compared with cisplatin added alone; and after AFP knockdown, cisplatin added and DDIT1 knockdown the apoptosis rate decreased by 32.7%±3.7% as compared with AFP knockdown and cisplatin added, and the differences were statistically significant ( t=2.497 and 2.773, P=0.032 and 0.020). Conclusions:AFP can inhibit the expression of its downstream gene DDIT1 by interacting with the transcription factor RAR, which not only promotes the proliferation of hepatocellular carcinoma cells but also enhances the anti-apoptosis ability of hepatocellular carcinoma cells and weakens cisplatin induced apoptosis in hepatocellular carcinoma cells.
