The Protective Effect Of Cold-Shock Protein RBM3 Against NO-induced Neuronal Apoptosis And Its Molecular Mechanism

BackgroundAs an important messenger molecule in the nervous system,nitric oxide(NO)has many physiological functions.But excessive NO is believed to be a mediator of neurotoxicity and is an important cause of neurodegenerative diseases in human.Recent studies have shown that cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults.However,whether RBM3 can protect neural cells from NO-induced apoptosis is unclear.ObjectivesTo explore the role of RBM3 in NO-induced human SH-SY5 Y neuroblastoma cells apoptosis and the possible mechanisms.Methods1.Exploring the role of mild hypothermia(32 ℃)in NO-induced SH-SY5 Y cells apoptosisSH-SY5 Y cells were pre-cultured at 37(nomothermia)or 32(mild hypothermia)℃ ℃for 1 d,and then treated with SNP at 37 ℃for 16 h,and the apoptosis effect of NO was detected by MTT,DAPI staining and Western blotting assay.2.Exploring whether the protective effect of mild hypothermia depends on RBM3,using RBM3-specific siRNA2 d post transfection of RBM3 siRNA or scramble siRNA,cells were pre-cultured for 1 d at 37 ℃or 32 ℃then treated with SNP for 16 h,and the apoptosis effect of NO was detected by MTT,DAPI staining and Western blotting assay.3.Upregulating RBM3 expression to further confirm its effect on NO-induced apoptosis in SH-SY5 Y cellsSH-SY5 Y cells were transfected with plasmid pXJ40-myc or pXJ40-myc-RBM3 for 2 d and then insulted with 1mM SNP for 16 h,the apoptosis effect of NO was detected by MTT,FACS and Western blotting assay.4.The signal pathways that RBM3 was able to control were detected using Western BlottingSH-SY5 Y cells transfected with plasmid pXJ40-myc or pXJ40-myc-RBM3 were treated with 1 mM SNP for 16 h,and Western blotting was performed to dected total and phosphorylated AMPKα,AKT,p38α,JNK1/2,and ERK1/2.5.The specific inhibitors of the kinase were used to determine the results of the previous stepIn the presence of various inhibitors,SH-SY5 Y cells were treated with 1 mM SNP for 16 h,and cell viability was assessed by MTT,DAPI staining and Western blotting assay.6.Determiningwhether RBM3 confers protection against NO via regulation on miR-143qPCR was performed to detect miRNA-143 expression for all the following samples.(1)SH-SY5 Y cells were transfected with plasmid pXJ40-myc or pXJ40-myc-RBM3 for 2 d,and treated with 1 mM SNP for 16 h.(2)Cells were per-cultured at 37 or 32 for 1 ℃ ℃d and treated with 1 mM SNP for 16 h.(3)2 d post transfection of RBM3 siRNA or scramble siRNA,cells were treated as shown in panel 1.(4)In the presence of p38 inhibitor,cells were treated with 1mM SNP for 16 h.7.Exploring the role of miR-143 in NO-induced SH-SY5 Y cells apoptosisSH-SY5 Y cells were transfected with miR-143 inhibitor or negative control inhibitor for 2 d,and treated with 1 mM SNP for 16 h.The apoptosis effect of NO was detected by MTT,FACS and Western blotting assay.Results:1.Mild hypothermia(32 ℃)protects SH-SY5 Y neuroblastoma cells from NO-induced apoptosis;2.RBM3 silencing mediated by siRNA impedes the neuroprotective effect of mild hypothermia;3.Overpression of RBM3 mimics neuroprotective effect of mild hypothermia;4.RBM3 potently inhibits NO-induced p38 activation;5.p38 signaling is required for NO-induced cytotoxicity;6.RBM3 downregulates NO-induced mi R-143 in p38-dependent manner;7.MiR-143 mediates NO-induced apoptosis.Conclusion:RBM3 prevents NO-induced apoptosis in human neuroblamtoma cells by modulating p38 signaling and miR-143.