The Effects Of Cold Shock Protein RBM3 On Retinoic Acid Induced Apoptosis In Neuroblastoma Cells In Vitro

BackgroundAll-trans retinoic acid(RA),the active metabolite of vitamin A,is the main mediator of the biological function of vitamin A.RA is widely distributed in immature and mature tissues,which is a key regulator of cell differentiation,cell proliferation and programmed cell death.However,excessive use of vitamin A has toxic and adverse effects on the nervous system.The cold-stress protein RBM3 is a very important cold shock protein,which is widely involved in a variety of physiological processes including hypoxia-ischemia stress,ultraviolet radiation stress,cell proliferation,skeletal muscle regulation,growth and development,and has protective effects on nerve cells.The purpose of this study is to investigate the effects of RBM3 on RA-induced neuronal apoptosis and to elucidate its potential molecular mechanisms.Objectives1.To explore the protective effects of mild hypothermia on RA-induced apoptosis in neuroblastoma cells;2.To elaborate the protective effects and molecular mechanisms of RBM3 on RA-induced apoptosis in neuroblastoma cells.Methods1.SH-SY5 Y cells,a model of neural cell apoptosis established in our laboratory,were used as objects of the study.MTT assay and flow cytometry were both employed to determine the optimum concentration of RA on cell survival.2.The cells were pretreated by mild hypothermia(32?)for 24 h,and the apoptosis of SH-SY5 Y cells was induced by RA.The expression of cleaved PARP and cleaved caspase 3 were measured by Western blotting,and cell viability was detected by MTT assay.Meanwhile,TUNEL staining was also used to detect the levels of apoptosis.The results were analyzed whether mild hypothermia made contributions to reduce the toxicity of RA and increase the rate of cell survival.3.The results showed that the expression of cold shock protein RBM3 was increased significantly after treatement of mild hypothermia,therefore,we speculated that the neuroprotective effects of mild hypothermia were relevant to RBM3.Then,the gene expression of RBM3 was silenced by small-interfered-RNA under the condition of mild hypothermia.After treatment with RA for another 24 h,the expression level of RBM3 and the apoptotic hallmarks were analyzed by Western blotting.It was analyzed that RBM3-silenced affects on the neuroprotective effects of mild hypothermia.4.After RBM3 was transfected for 48 h,cells were stimulated by RA for 24 h.Cell viability and apoptotic markers were examined by MTT assay and Western blotting,respectively.And DAPI staining was adopted to analyze apoptosis in SH-SY5 Ycells.All the measures were used to analyze whether the expression of RBM3 had protective effects on RA-induced apoptosis.At the same time,we analyzed the changes of phosphorylation of key kinases in multiple stress-induced signaling pathways and we will elucidate which signaling pathways mediate the neuroprotective effects of RBM3.5.Using specific inhibitor or activator of the key kinase of these signal pathways blocked or activated the signal pathway at the earlier step screened.MTT assay and Western blotting were employed into our further study and the mechanisms of the protective effects of RBM3 on the apoptosis in SH-SY5 Y cells were elucidated.Results1.The results showed that mild hypothermia could protect cells from RA-induced apoptosis,accompanied by up-regulation of cold shock protein RBM3.In addition,RBM3-silenced reduced the protective effects of mild hypothermia,obviously.The level of the hallmark of apoptosis,cleaved PARP,was also significantly increased.2.Studies have shown that RBM3 played a critical role in the protection of mild hypothermia(32?)against apoptosis.In the process of RA-induced apoptosis in SH-SY5 Y cells,RBM3-overexpressed can significantly reduce the damage caused by RA.Moreover,RBM3 could evidently inhibit the activation of MAPK/JNK and MAPK/p38 signaling pathways and significantly activate the AMPK signaling pathway.Compared with RA treated group,the corresponding specific blockers(SP600125,SB203580)and AMPK activator(AICAR),the rates of cell survival were all significantly increased.Conclusion1.Mild hypothermia can protect SH-SY5 Y cells from overdose of RA induced apoptosis.2.The protective effects of mild hypothermia on cells mainly rely on the induction of the expression of RBM3,that is to say that RBM3 is a key mediator of mild hypothermia protection.3.RBM3 could modulate the apoptosis in SH-SY5 Y cells via inhibiting JNK and p38 signaling pathways as well as activating AMPK signaling pathway.This study can provide a meaningful reference for vitamin A abuse induced diseases.