Molecular Mechanism Of Cold Shock Protein Rbm3 Gene Transcription Under Mild Hypothermia

BackgroundMild hypothermia is a clinically recognized treatment that is commonly used to relieve nerve damage caused by ischemia and hypoxia.Mild hypothermia can also cause various complications,such as electrolyte imbalance and hypoxia,which limits the clinical application of hypothermia.A large number of studies have shown that RNA-binding motif protein 3(RBM3),as a cold stress protein,mediates the neuroprotective effect of mild hypothermia to a large extent.Therefore,inducing the specific expression of RBM3 in nerve cells is expected to become a new clinical treatment strategy.However,there are still unclear that how RBM3 is induced in nerve cells under mild hypothermia condition and the molecular mechanism of its gene transcription.Objectives1.To explore the effects of mild hypothermia on various signal pathways in nerve cells;2.To clarify the molecular mechanism of RBM3 gene transcription regulation during mild hypothermia;3.To understand the neuroprotective effects of signaling molecules involved in the regulation of RBM3 gene expression.Methods1.Effects of mild hypothermia(32?)on various signaling pathways in SH-SY5 Y cellsThe SH-SY5 Y cells were cultured in mild hypothermia(32?)environment for a different time course,and Western blot was used to detect the level of RBM3 protein expression and the phosphorylation level of MAPK,FAK,PLC,AMPK,AKT and other pathways.2.Effects of pathway-specific inhibitors on RBM3 transcription and expression induced by mild hypothermiaThe cells were pre-treated with specific inhibitors of the various pathways for 1 h,and then the cells were cultured at 32°C for 24 h.The m RNA and protein levels of RBM3 were detected by q PCR and Western blot to determine which signal pathways that affect RBM3 gene expression.3.Effects of key kinases-specific si RNA on the transcription and expression of RBM3 induced by mild hypothermiaAfter transfecting with kinase-specific si RNA for 24 hours,the cells were cultured at32°C for 24 hours,and the m RNA and protein levels of RBM3 were detected by q PCR and Western blot.4.Roles of the selected signal pathways in the hypothermia neuroprotectionAfter the kinase-specific inhibitors were used to treat the cells,the cells were treated with Rotenone(ROT)to construct a cell apoptosis model in vitro.The CCK-8 method was used to detect the cell viability,and Western blot was used to detect the levels of the apoptotic protein cleaved PARP.Analyzed the roles of the selected signal pathways in the mild hypothermia neuroprotection against apoptosis.5.Determine the transcription factors downstream of selected signaling pathways that affect RBM3 gene expressionTreated cells with specific inhibitors of transcription factors,and used q PCR and Western blot to detect its effects on the transcription and expression of RBM3 induced by mild hypothermia,and determined which transcription factors affect RBM3 gene expression;Treated cells with the above kinase-specific inhibitors and si RNA,and the activity of transcription factor was detected by Western blot.Results1.MAPK,PLC,AMPK and AKT pathway inhibitors had no significant effect on RBM3 gene expression induced by mild hypothermia;2.FAK specific inhibitors and si RNA inhibited RBM3 transcription and expression induced by mild hypothermia;3.Src acted as a downstream activating kinase of FAK,inhibiting its activity significantly reduced the transcription and expression of RBM3 induced by mild hypothermia;4.Inhibition of FAK/Src pathway significantly impaired the neuroprotective effect of mild hypothermia on ROT;5.After the FAK/Src signaling axis was activated by mild hypothermia,it further activated the transcription factor NF-?B p65.At the same time,NF-?B specific inhibitors significantly inhibited the induction of RBM3 by mild hypothermia.ConclusionThe FAK/Src signaling axis and the transcription factor NF-?B p65 jointly constitute a new hypothermia response pathway and participate in the transcriptional regulation of RBM3 under mild hypothermia.In addition,the FAK/Src signaling axis also mediates the neuroprotection of mild hypothermia.This suggests that FAK-Src-RBM3 may play an important role in the treatment of neurodegenerative diseases,providing a new approach for the development of specific small molecule drugs that mimic hypothermia therapy.