Effect Of HSP70 Gene Silence On Apoptosis Of SH-SY5Y Induced By Hypoxia And Its Mechanism

ObjectiveUsing RNA interference technology to silence the expression of heat shock protein 70(HSP70),observation the effect of HSP70 silencing on hypoxia neuroblastoma cells(SH-SY5Y)apoptosis and the expression of BAG-1,NF-?B and caspase-3.MethodsLogarithmic growth phase cells,using RNA interference technology to silence the expression of heat shock protein 70(HSP70),the SH-SY5 Y cells were divided into 4 groups,the cell control group(normal SH-SY5 Y cells),the lentiviral control group(infected with GFP but not containing siRNA lentivirus),the lentiviral infection group?(infected with GFP and siRNA-?lentiviral),the lentiviral infection group?(infected with GFP and siRNA-?lentiviral).HSP70 protein expression of SH-SY5 Y cells were detected by Protein immunoblotting test(Western blotting),HSP70 m RNA expression of SH-SY5 Y cells were detected by real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR).Treatment with hypoxia for 4h,8h,12 h,respectively,and re-oxygenation 24 h,CCK-8 assays were performed and cell viability were illustrated according to OD values.The apoptosis rate and cell cycle distribution of SH-SY5 Y cells were detected by flow cytometry;the m RNA transcription level of HSP70 and BAG-1 of SH-SY5 Y cells were detected by q RT-PCR;Western blotting was used to detect the expression level of BAG-1,NF-?B and caspase-3 protein of SH-SY5 Y cells.Results1.After transfected with lentiviral,the expression of HSP70 m RNA were decreased significant than the cell control group and the lentiviral control group(2-??ct were 1.00 ± 0.04,0.98 ± 0.12,0.22 ± 0.03,0.15 ± 0.01,P<0.05),the expression of HSP70 protein were decreased significant than the cell control group and the lentiviral control group(the gray values of the four groups were 1.31 ± 0.23,1.26 ± 0.17,0.77 ± 0.10,0.80 ± 0.17,P<0.05).2.With the prolongation of time of hypoxia,the cell viability in all groups appeared peak at 8 hours,follow by a declining tendency.The cells of the lentiviral infection group?and lentiviral infection group? were grow more slowly than the cell control group and the lentiviral control group(OD value were 0.85 ± 0.02,0.84 ± 0.03,0.23 ± 0.03,0.25 ± 0.02,P<0.05).3.The apoptosis rate were significantly increased(apoptosis rate(%)were 11.17 ± 1.12,11.40 ± 0.40,68.97 ± 6.82,58.10 ± 3.16,P<0.05)in lentiviral infection group?and lentiviral infection group?,compared with the cell control group and the lentiviral control group.4.Treatment of SH-SY5 Y cells with HSP70 silence had no effect on BAG-1 m RNA(2-??ct were 1.00 ± 0.06,0.94 ± 0.07,0.92 ± 0.05,0.91 ± 0.09,P>0.05),compared with the cell control group and the lentiviral control group;there were no significant difference in the expression level of BAG-1 protein(gray values of BAG-1L were 0.96 ± 0.10,0.99 ± 0.03,1.03 ± 0.02,1.02 ± 0.07,P>0.05;gray values of BAG-1M were 0.95 ± 0.03,0.98 ± 0.05,1.02 ± 0.09,1.00 ± 0.01,P>0.05;gray values of BAG-1S were 1.02 ± 0.04,1.00 ± 0.07,1.03 ± 0.07,0.96 ± 0.04,P>0.05),compared with the cell control group and the lentiviral control group;down-regulation of HSP70 led to increased expression of NF-?B(gray values were 1.00 ± 0.00,1.01 ± 0.08,1.95 ± 0.22,2.03 ± 0.16,P<0.05),compared with the cell control group and the lentiviral control group;down-regulation of HSP70 led to increased expression of caspase-3(gray values were 0.82 ± 0.04,0.83 ± 0.03,1.19 ± 0.04,1.21 ± 0.04,P<0.05),compared with the cell control group and the lentiviral control group.ConclusionsHSP70 silencing promoted the apoptosis of SH-SY5 Y cells,decreased the viability of SH-SY5 Y cells,increased the protein expression level of NF-?B and caspase-3,but had no effect on the expression of protein and the level of m RNA transcription of BAG-1.Therefore,HSP70 sh RNA could increase the sensitivity of SH-SY5 Y cells to hypoxia,probably by up-regulating the NF-?B and caspase-3 expression.