RESUMEN
Objective:To investigate the mechanism of microRNA-4298 (miR-4298) targeting inhibition of peptidylarginine deiminase 4 (PADI4) expression in U251 cells apoptosis induced by histone deacetylase inhibitor trichostatin A (TSA).Methods:The experiment was divided into TSA group (0.2 μmol/L TSA) and contral group. CCK8 method was used to detect the effect of TSA on the proliferation of U251 cells. Flow cytometry was used to detect the apoptosis level of U251 cells after drug action. Reverse transcription PCR and Western blotting experiments were used to determine the changes of PADI4 gene and protein expression after miR-4298 interference. Luciferase assay was used to determine the effects of miR-4298 on targeted binding and luciferase activity in the 3′UTR region of PADI4. U251 cells were transfected with PADI4 to observe the rescue effect of miR-4298 on apoptosis. Each experiment was divided into 3 groups, NC group, miR-4298 mimic group, TSA+ miR-4298 mimic group and NC group, miR-4298 inhibitor group, TSA+ miR-4298 inhibitor group.Results:U251 cells were treated with 0.2 μmol/L TSA for 4 days, the Absorbancy ( A) values of the control group was 1.168±0.148, which was higher than those of the TSA group (0.737±0.007), with statistically significant difference ( t=4.948, P=0.008). Compared with the control group, after treatment with 0.2 μmol/L TSA for 48 h, U251 cells showed obvious apoptosis (27.62%±3.49% vs. 4.99%±0.13%, t=11.190, P<0.001). Compared with those before the drug treatment, the PADI4 gene expression (0.386±0.020 vs. 0.903±0.021) and protein expression (0.276±0.041 vs. 0.777±0.031) after TSA treatment for 48 h both were decreased, with statistically significant differences ( t=30.400, P<0.001; t=16.770, P<0.001). The expression of miR-4298 during the process of TSA-induced U251 cell apoptosis increased (2.573±0.289 vs. 1.003±0.136; t=8.487, P=0.001). There was a negative correlation between the miR-4298 expression and PADI4 expression ( r=-0.877, P=0.002). Luciferase experiment confirmed that miR-4298 has targeted binding and luciferase inhibitory effects on the 3′UTR region of wild-type PADI4. Compared with NC group (0.920±0.026), the relative expression levels of PADI4 gene of miR-4298 mimic group (0.413±0.049) and TSA+ miR-4298 mimic group (0.213±0.035) were decreased, with statistically significant differences (all P<0.001). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (3.78%±0.68%), the apoptosis levels of miR-4298 mimic group (7.96%±1.10%) and TSA+ miR-4298 mimic group (13.74%±1.26%) were increased, with statistically significant differences ( P=0.005; P<0.001). Compared with NC group (0.183±0.025), the PADI4 gene expression of miR-4298 inhibitor group (0.483±0.032) and TSA+ miR-4298 inhibitor group (0.386±0.025) were increased, with statistically significant differences ( P<0.001; P=0.015). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (4.96%±0.59%), the apoptosis levels of miR-4298 inhibitor group (23.83%±2.20%) and TSA+ miR-4298 inhibitor group (9.55%±1.49%) were increased, with statistically significant differences (all P<0.001). In the rescue experiment, the expression level of PADI4 in miR-4298 mimic group was significantly increased ( P<0.001), while the expression level of PADI4 in miR-4298 mimic+ PADI4 group was relatively reversed ( P=0.002). Conclusion:miR-4298 can participate in the process of U251 cell apoptosis by targeting the expression of PADI4 gene, and miR-4298 may be the target of targeted intervention therapy for glioma.
RESUMEN
Objective To investigate the effect of euphorbiasteroid on inducing the apoptosis of HL-60 cells and demonstrate whether the Fas/FasL signaling pathway is involved in the induction of apoptosis. Methods HL-60 cells were treated with dose of 2.5,10,40 μg/ml of euphorbiasteroid in vitro for 24 h respectively.After that,cell counting Kit-8 was used to detect cell proliferation.The morphology of HL-60 cells were observed under light and fluorescent microscopy.The early cell apoptosis was detected by using flow cytometry with Annexin Ⅴ-FITC /PI double staining.The expressions of Fas,FasL,caspase-8 and caspase-3 mRNA were analyzed by the method of RT-PCR.The activities of caspase-8 and caspase-3 were examined by chromatometry.Results Compared with 1640 control group,HL-60 cell proliferation was inhibited significant-ly by euphorbiasteroid.The inhibition rates were (34.9 ±3.7)%,(54.6 ±5.2)% and (61.3 ±4.3)%respectively.Moreover,HL-60 cells exhibited typical morphological features.Early cell apoptosis rates of HL-60 cells were (23.4 ±3.1)%,(35.7 ±4.3)% and (53.2 ±3.9)% respectively.Furthermore,the expressions of Fas,FasL,caspase-3 and caspase-8 mRNA were up-regulated significantly after euphorbiasteroid administration in a dose-dependent manner (P<0.01 ).After treated with euphorbiasteroid,the activities of caspase-8 and caspase-3 were significantly enhanced (P<0.01 ).Conclusion The up-regulation effect of euphorbiasteroid on Fas/FasL signaling pathway might contribute to the apoptosis of HL-60 cells.