RESUMEN
Objective To show evidences that autophagy is induced in human malignant glioma cell line U251 by enterovirus(EV71) and its effect on the expression of microtubule-associated protein 1 lightchain 3 (LC3) in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously.After 12 hours post infection of U251 cells with EV71,autophagic vacuoles of U251 cells were marked by monodansylcadaverine staining and observed under fluorescence microscope.After 24 hours post infection,expression and intracellular distribution of LC3 protein in U251 cells were observed under fluorescence microscope by immunofluorescence.Expressions of LC3-I and LC3-Ⅱ protein were measured by Western blot and analysis of LC3-Ⅱ protein expression was performed with semi-quantitative calculation at 2,4,8,12,24 and 48 hours post infection of U251 cells with EV71,respectively.Results Autophagic vacuoles stained by MDC in U251 cells appeared as distinct dot-like structures distributed under fluorescence microscope.The number of autophagic vacuoles were increased significantly at 12 hours post infection of U251 cells with EV71 when compared with control group.The morphological features of these cells became significantly shrunken,smaller and irregular shape at 24 hours post infection of U251 cells,and the expressions of LC3 protein were significantly higher in experimental group than those in control group.Under a fluorescence microscope,LC3 protein distributed within the cytoplasm or localizing in the perinuclear regions.At 4 hours post infection of U251 cells with EV71,the expression of LC3-Ⅱ protein started to increase,and was significantly higher than that in control group (P ＜ 0.01).Conclusion These results indicate that EV71 can effectively induce autophagy of human malignant glioma cell line U251,and play its oncolytic effect.
RESUMEN
Objective To investigate the effects of enterovirus 71 (EV71) on growth,apoptosis and human telomerase reverse transcriptase (hTERT) expression of human malignant glioma cell line U251 in vitro.Methods U251 cells were cultured in RPMI 1640 for 24 hours,then randomly divided into experimental group and control group.In the experimental group,EV71 were added in cell culture holes at multiplicity of infection (MOI) equal to 1,and cultured continuously for 72 hours with U251 cells,but in the control group,EV71 were not put.The morphological features and growth of cells were observed under inverted microscope at 0 hour,24 hours,48 hours and 72 hours of EV71 treatment,respectively.At the same time,cell apoptosis was measured by flow cytometry using Annexin V/PI double staining method.hTERT expression was detected with semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR).Results The growth of U251 cells was inhibited significantly at 24 h of EV71 treatment under inverted microscope in experimental group when compared with control group,and at 48 h and 72 h,the morphological features of these cells became significantly shrunken,smaller and irregular shape in experimental group,but which were uniform size in control group.At 24 h,48 h,72 h of EV71 treatment,flow cytometry showed the apoptosis rates were significantly higher in experimental group than that in control group [24 h:(12.55 ± 2.38) ％ vs (1.42 ± 0.21) ％,48 h:(65.60 ± 8.48)％ vs (1.42 ±0.17)％,72 h:(87.52 ±3.05)％ vs (1.41 ±0.16)％,all P =0.000],and there was upward trend day by day,but no change in control group.At the same time,hTERT mRNA expression levels of U251 cells were significantly lower in experimental group than control group [24 h:(0.58 ± 0.05) vs (0.89 ± 0.05),48 h:(0.23 ± 0.04) vs (0.89 ± 0.03),72 h:(0.10 ± 0.03) vs (0.90 ± 0.06),all P =0.000],and the downward trend was observed day by day,but there was no this trend in control group.Conclusions EV71 can effectively inhibit the growth of U251 cells,induce the cell apoptosis,and has the potential anti-tumor effect.Its mechanisms may be partly related to down regulation the hTERT expression of U251 cells.