title: [Inhibition of Beclin 1 enhances apoptosis by H2O2 in glioma U251 cells].
abstract: Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1-siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptotic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.
authors.affiliation: Wenzhou Medical College, Wenzhou, China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Kong
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Zhang
authors.forename: Zhao-Qin
authors.initials: ZQ
authors.lastname: Chen
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Fan
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: Gong
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology*; D000071186:Beclin-1; D001932:Brain Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D005910:Glioma / Q000473:pathology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D006861:Hydrogen Peroxide