Functional And Mechanical Study Of Long Non-coding RNA-NEAT1 In SUDEP Via Regulating The Distribution Of KCNAB2 In The Brainstem

Background:Sudden unexpected death in epilepsy(SUDEP)is one of the leading causes of death in patients with epilepsy.Brainstem is the key structure related to SUDEP.On the one hand,the structural abnormalities of brainstem caused by repeated seizures lead to the susceptibility of SUDEP;On the other hand,the functional abnormalities of brainstem(i.e.,brainstem spreading depolarization)after specific seizures were associated with the occurrence of SUDEP.However,the underlying molecular mechanisms in these two processes are still unclear.Considering the role of seizures in SUDEP susceptibility,it is speculated that epigenetic mechanisms,such as long noncoding RNA(lnc RNA)may be involved in SUDEP.NEAT1 is a highly conserved mammalian lnc RNA.Vitro studies indicated that NEAT1 was associated with neuronal excitability,which could regulate the expression of various ion channels and was related to the distribution of some ion channel-interacting proteins.The expression of NEAT1 in the brain increased after seizures,which suggested that NEAT1 has the potential to participate in the occurrence of SUDEP.In addition,the potassium channel family was highly related to SUDEP.Functional loss and deletion mutation of Kv1.1 channel were highly related to SUDEP.Animal studies confirmed that the number and function of voltage-gated potassium channels were crucial for SUDEP,which is worthy of further study.KCNAB 2(Kvβ2)was a regulatory molecule of Kv1.1 、 Kv1.4 and Kv4.2,which could stabilize the structure of potassium channels and decrease the excitability of neurons via interacting with NEAT1.Therefore,NEAT1 might play a vital role in SUDEP via influencing KCNAB2.Objective:We investigated lnc RNAs and m RNAs expression profile in the brainstem of SUDEP model to explore the relationship between lnc RNA and SUDEP.Furthermore,performing brainstem tissue and cell experiments reveal the role of lnc RNA-NEAT1 in regulating KCNAB2 in SUDEP.Materials and Methods:It has been widely accepted that DBA/1 is a SUDEP model.We compared the expression profile of lnc RNAs and m RNAs through Arraystar mouse lnc RNA Microarray V3.0(Arraystar,Rockville,MD)between DBA/1 and C57BL/6 mice.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were determined to sreen target lnc RNA.q RT-PCR and Western blot were also carried out to study the expression of NEAT1 and KCNAB2.In HT22 cells,Neat1 was knocked down by antisense oligonucleotide(ASO)and overexpressed by lentivirus.Then we used Western blot,immunofluorescence(IF)and RNAscope to explore the effect of NEAT1 on KCNAB2 in vitro based on HT22 cell.Patch-clamp technique was conducted to assess the potassium channel current of HT22 cell after ASO targeting NEAT1.Furthermore,RIP experiment was conducted to verify the binding of NEAT1 and KCNAB2.Finally,the findings in vitro experiments were examined by RNAscope technique in brainstem tissue sections of DBA/1 mice.Results:A total of 1072 lnc RNAs and 438 m RNAs were differentially expressed between the brainstem of DBA/1 and C57BL/6 mice,including 257 up-regulated,815 down-regulated lnc RNAs,and 138 up-regulated,300 down-regulated m RNAs.The expression of lnc RNA-NEAT1 was significantly different between DBA/1 and C57BL/6 mice.q RT-PCR showed that the expression of NEAT1 m RNA was gradually rised during the induction of SUDEP sensitivity in DBA/1 mice.Westernblot showed that the expression of KCNAB2 decreased with the high expression of NEAT1.In HT22 cells,knockdown of NEAT1 could significantly promote the expression of KCNAB2.IF showed that knockdown of NEAT1 increased the cytoplasmic distribution of KCNAB2,while overexpression of NEAT1 could decrease the cytoplasmic distribution of KCNAB2.In situ hybridization(ISH)/IF co-detection suggested with the increase of fluorescence intensity of NEAT1 in nucleus,the expression of KCNAB2 in cytoplasm gradually decreased within 24 hours after KCl stimulation which mimic the occurrence of seizure.The results of patch-clamp showed that knockdown of NEAT1 could result in a significant increase of outward potassium current.RIP experiment confirmed that NEAT1 could directly bind to KCNAB2.In tissue,ISH/IF co-detection also showed that the expression of NEAT1 which located in PAG was negatively correlated with the fluorescence intensity of KCNAB2.Conclusion:We identified thousands of differently expressed lnc RNAs,including NEAT1 in the brainstems of DBA/1 and C57BL/6 mice,which might be related to the susceptibility of SUDEP.NEAT1 is up-regulated in the process of SUDEP susceptibility acquisition,which may be involved in the occurrence of SUDEP.NEAT1 could negatively regulate expression and distribution of KCNAB2 in vivo and vitro by directly binding to KCNAB2.NEAT1 could also induce decreasing outward potassium current and increasing the excitability of neurons,thus facilitating the spread of abnormal discharges to the brainstem and participating in the occurrence of SUDEP.