Effects of Bcl-2 associated with athanogene-1 gene silencing on heat shock protein 70 expression and human neuroblastoma cell apoptosis induced by hypoxia
10.3969/j.issn.1008-9691.2017.01.019
- VernacularTitle:Bcl-2结合抗凋亡基因沉默对缺氧状态下人神经母细胞瘤细胞凋亡及热休克蛋白70表达的影响
- Author:
Yankun SONG
;
Zhi LI
;
Fengtao WANG
;
Haiyan LIU
;
Yan QU
;
Yun WANG
;
Chunyu XIE
;
Dan HU
- Keywords:
Bcl-2 associated with athanogene-1;
Heat-shock proteins;
Neural cells;
Hypoxia/reoxygenation;
Apoptosis;
RNA interference
- From:
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
2017;24(1):68-72
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the protective effects of Bcl-2 associated with athanogene-1 (BAG-1) gene on human neuroblastoma cells (SH-SY5Y) injury induced by hypoxia/reoxygenation,and its influence on heat shock protein 70 (HSP70) expression.Methods The SH-SYSY cells at logarithmic growth phase were collected.Lenti virus mediated RNA interference (RNAi) technology was used to suppress the BAG-1 expression.The cells screened out can be divided into four groups:the cell control group with no lentivirus infection,lentivirus control group (containing only fluorescein protein lentivirus infection),BAG-1 siRNA group (BAG-1 siRNA silencing group),including BAG-1 siRNA-α group and BAG-1 siRNA-β group with lentivirus containing fluorescein protein (GFP) but at different BAG-1 siRNA target sites of silencing.Western Blot was used to detect the protein expression of BAG-1 and HSP70 in target cells after infectious recombination lentivirus for 72 hours;the Cell Counting Kit-8 (CCK-8) was used to detect the activity of four different group cells after hypoxia;the flow cytometry was used to detect the cell apoptosis;the HSP70 mRNA transcription level were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) respectively in each group.Results After lentiviral infection for 72 hours,the Western Blot results showed that in the two BAG-1 siRNA silencing groups,the interference effect of BAG-1 siRNA-β group was superior to that of BAG-1 siRNA-α group.The cell viability of each group showed an increase followed by a decrease with the prolongation of hypoxia time,and reaching the peak at 8 hours.After hypoxia for 8 hours being given,the cell viability in BAG-1 siRNA-β group was significantly lower than that of the cell control group,lentivirus control group and BAG-1 siRNA-α group (A value:0.59 ±0.09 vs.0.94±0.12,0.90± 0.11,0.91± 0.14,P ＜ 0.01);the cell apoptosis rate was obviously higher in BAG-1 siRNA-β than that in the above three groups [(34.63 ± 3.46)％ vs.(14.83 ± 3.75)％,(19.93 ± 6.49)％,(16.40± 1.18)％,all P ＜ 0.01].There were no statistically significant differences in the HSP70 protein level and mRNA transcription level between BAG-1 siRNA-3 grroup,and the cell control group,lentivirus control group and BAG-1 siRNA-α group respectively (all P ＞ 0.05).Conclusion BAG-1 gene can play a role in protection of hypoxia nerve cells,reduce the apoptosis,and its protective effect can be independent of HSP70 gene.
