Study on the mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 and miRNA-1236 / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the molecular mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 (miR-329) and miRNA-1236 (miR-1236).Methods:Hepatoma cell line HepG2 was treated with icaritin at different concentrations (2.5, 5.0, 10.0, 20.0, 40.0 μmol/L), and the control group only added dimethyl sulfoxide (DMSO). The half inhibitory concentration ( IC50) of icaritin on HepG2 cells and cell proliferation rate were detected by CCK-8 after cultured for 36 h. HepG2 cells were treated with 400 μg/L alpha fetoprotein (AFP). After cultured for 0, 12, 24, 36, 48 and 60 h, the effect of AFP on the proliferation of HepG2 cells was detected by CCK-8 method. AFP-3'UTR reporter plasmid pmirGLO-AFP-3'UTR plasmid was constructed, pmirGLO blank vector plasmid, pmirGLO-AFP-3'UTR plasmid, miR-329 or miR-1236 mimics or inhibitors, control plasmid of mimics (NC), control plasmid of inhibitors (INC) were respectively co-transfected with HepG2 cells, and the effect of miR-329 and miR-1236 on the luciferase activity was detected by dual-luciferase reporter assay after cultured for 24 h. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the effects of icaritin on the expressions of AFP, miR-329 and miR-1236 in HepG2 cells. HepG2 cells were respectively transfected with mimics and inhibitors of miR-329 and miR-1236 to detect the effects of miR-329 and miR-1236 on the expression of AFP. Results:The cell proliferation rates of 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were (80.4±2.3)%, (73.2±1.6)%, (51.7±3.3)%, (38.2±4.6)%, (29.5±4.3)%, and (94.0±2.9)%, respectively, and the difference was statistically significant ( F = 75.65, P < 0.01); the differences in the proliferation rate of HepG2 cells between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). The IC50 of icaritin on HepG2 cells was 10 μmol/L. The relative expressions of AFP mRNA in 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were 0.83±0.06, 0.69±0.02, 0.53±0.07, 0.45±0.01, 0.33±0.07, and 1.00±0.01, respectively, and the difference was statistically significant ( F = 42.67, P < 0.01); the differences in the relative expressions of AFP mRNA between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). HepG2 cells were treated by 400 μg /L AFP for 0, 12, 24, 36, 48 and 60 h, and the cell proliferation rates were (102±5)%, (138±13)%, (186±24)%, (260±12)%, (311±15)%, and (348±25)%, respectively, and the difference was statistically significant ( F = 27.483, P < 0.01); the differences in the cell proliferation rate between different time of AFP treatment and 0 h were statistically significant (all P < 0.01). Compared with the control group, different concentrations of icaritin can promote the expression of miR-329 and miR-1236 (all P < 0.01). After co-transfection of miR-329 and miR-1236 mimics and AFP-3'UTR, the luciferase activity decreased by about 40%; after co-transfection of miR-329 and miR-1236 inhibitors and AFP-3'UTR, the luciferase activity increased about 1.5 times. Both miR-329 and miR-1236 can reduce the expression levels of AFP protein and mRNA (both P < 0.05). Conclusion:Icaritin can promote the binding of miR-219, miR-1236 and AFP-3'UTR by promoting the expression of miR-329 and miR-1236, inhibit the stability and translation activity of AFP mRNA, inhibit the expression of AFP, and thus inhibit the proliferation of hepatoma cells in vitro.