The Role And Mechanism Of Zfp326 Regulating LncRNA NR₀30777 In Neurodegenerative Damage By Paraquat

Objective: Based on the in vitro cell level,we investigated the role of Zfp326 in the expression of NR₀30777/Cpne5 in PQ-induced neuronal damage and the mechanism of Zfp326/NR₀30777/Cpne5 in paraquat-induced neuronal damage.Methods:?1?After N2 a and MN9 D was transfected with si-Zfp326,N2 a and MN9 D were treated with 0,100 and 300 ?M PQ for 24 and 36 hours respectively,q RT-PCR and WB detected Zfp326,NR₀30777,Cpne5 m RNA,and ZFP326 and CPNE5 protein?n=3?;?2?After N2 a was transfected with si-Zfp326,N2 a were treated with actinomycin D?Act D?,q RT-PCR detected NR₀30777 and Cpne5 m RNA at different time points?n=3?;?3?After N2 a and MN9 D was transfected with si-Zfp326,N2 a and MN9 D were treated with 0,100 and 300 ?M PQ for 24 and 36 hours respectively,CCK8 detected cell proliferation activity?n=6?;?4?After N2 a was transfected with si-Zfp326,N2 a were treated with 0,100 and 300 ?M PQ for 24 hours,Flow cytometry detected cell apoptosis;WB detected SEPTIN2 and DBC-1 protein?n=3?;?5?After NR₀30777 was pull down by TRAP technology,MS,NGS detected proteins?RBPs?and RNA that combined with NR₀30777;GO and Pathway analyze these molecules bound to NR₀30777;?6?After N2 a was transfected with si-Zfp326,N2 a were treated with 0,100 and 300 ?M PQ for 24 hours,WB detected SF3B3 protein?n = 3?;?7?After N2 a was transfected with si-Sf3b3,N2 a were treated with 0,100 and 300 ?M PQ for 24 hours,q RT-PCR detected Sf3b3,NR₀30777 m RNA?n=3?.Result:?1?After transfected with si-Zfp326,Zfp326 m RNA and protein was down-regulated?P <0.01?;NR₀30777 and the stability of NR₀30777 was down-regulated?P <0.01?;Cpne5 m RNA and protein was no change.?2?PQ inhibits neuronal proliferation and promotes apoptosis,that has a dose-response relationship;knockdown of Zfp326 can increases cell proliferation activity and inhibits apoptosis?P <0.01?;SEPTIN2 and DBC-1 protein was no change.?3?The proteins and RNAs binding to NR₀30777 are widely involved in various life activities of N2 a cells,mainly involved in neurodegenerative diseases,such as Huntington's disease and Parkinson's disease,as well as cellular oxidative stress processes.?4?PQ promotes the expression of SF3B3 protein;knockdown of Zfp326 can down-regulation of SF3B3 protein expression?P <0.05?;knockdown of Sf3b3 can down-regulation of NR₀30777?P <0.05?.Conclusion: Based on the above results,the following conclusions can be drawn:?1?In PQ-induced neuronal damage,knockdown of Zfp326 regulated NR₀30777 by down-regulating SF3B3 splicing factor expression and decreasing NR₀30777 stability.?2?In PQ-induced neuronal damage,knockdown of Zfp326 reverses neuronal cell proliferation declining and reduce neuronal apoptosis.?3?The proteins and RNAs binding to NR₀30777 are mainly involved in neurodegenerative diseases,such as Huntington's disease and Parkinson's disease,as well as cellular oxidative stress processes.NR₀30777 is essential for PQ-induced neuronal damage.