The Role Of LncRNA-AK039862 In Paraquat Inhibiting Neurons Proliferation And Altering Pafah1b1/Foxa1 Expression

Objective: Dependent on lncRNA Microarray of substantia nigra in PD mice induced by paraquat(PQ),lncRNA-AK039862 was selected to investigate the temporal and spatial distribution.We investigated the role of AK039862 in the change of Pafah1b1/Foxa1 expression in neuronal microglia and/or dopaminergic neurons in vivo and in vitro.Methods:In vivo.(1)PD model mice were established with 0,5,10 mg/kg PQ and 30mg/kg MPTP.The expression levels of AK039862 and AK048895 in substantia nigra,hippocampus and cerebral cortex were detected by qRT-PCR(n=4).(2)Multiplex fluorescence in situ hybridization was used to investigate the spatial(cell,subcellular)distribution of AK039862 expression in the 10 mg/kg group.(3)The potential target genes of AK039862 were predicted and screened by CNC,GO,Pathway,RPISeq analysis;(4)qRT-PCR was used to detect the expression of target gene Pafah1b1/Foxa1 in substantia nigra in each group(n=4).In vitro.(1)bv2 cells were treated with 0,30,60,90,and 120 ?M PQ for 24,36,and 48 h respectively,CCK8 was used to detect cell proliferation(n=5);qRT-PCR and WB detected the mRNA expressions of lncRNA-AK039862,Pafah1b1,Foxa1 and Pafah1b1 in the 0,30,60 ?M PQ group(n=3);(2)Transfect with si-AK039862 or pcDNA3.1-AK039862 in bv2,CCK8 detected its proliferation activity(n=5);(3)After bv2 was transfected with si-AK039862 or pcDNA3.1-AK039862,bv2 cells were treated with 0,30,and 60 ?M PQ for 24 and 36 h respectively,CCK8 detected cell proliferation activity(n=5);qRT-PCR and WB detected lncRNA-AK039862,Pafah1b1,Foxa1 mRNA and Pafah1b1 protein(n = 3);(4)0,50,100,150,200 ?MPQ treatment of mn9 d cells,with normal medium and bv2 cells made conditioned medium(CM)for 24 and 36 h,CCK8 detected its proliferation activity(n=5);qRT-PCR and WB detected lncRNA-AK039862,Pafah1b1-mRNA,Foxa1-mRNA,and Pafah1b1 protein in 0,50,100?M CM-PQ group(n=3);(5)Transfect si-AK039862 or pcDNA3.1-AK039862 in mn9 d,CCK8 detected cell proliferation activity(n=5);(6)After mn9 d was transfected with si-AK039862 or pcDNA3.1-AK039862,mn9 d cells were treated with 0,50,and 100 ?M PQ for 24 and 36 h respectively.CCK8 detected cell proliferation activity(n=5);qRT-PCR and WB detected lncRNA-AK039862,Pafah1b1,Foxa1 mRNA and Pafah1b1 protein(n = 3);Result:(1)AK039862 was up-regulated in the substantia nigra(P<0.001),down-regulated in the hippocampus(P<0.001),and unchanged in the cerebral cortex of the 10 mg/kg PQ and 30 mg/kg MPTP group;AK048895 was up-regulated in substantia nigra(P<0.05),down-regulated in hippocampus and cerebral cortex of 10 mg/kg PQ,30mg/kg MPTP group(P<0.05).(2)AK039862 is widely distributed in the substantia nigra of mice in the 10 mg/kg PQ group and is distributed in the cytoplasm of dopaminergic neurons and other neurons.(3)Pafah1b1,Foxa1 and others gene are the potential target genes for AK039862.(4)Pafah1b1-mRNA expression was up-regulated in the substantia nigra of the 10mg/kg PQ and 30 mg/kg MPTP group(P<0.01);Foxa1-mRNA expression was up-regulated in the substantia nigra of the 5,10 mg/kg PQ and 30 mg/kg MPTP groups(P<0.01).Conclusion: Based on the above results,the following conclusions can be drawn:(1)LncRNA-AK039862 is widely distributed in the substantia nigra,hippocampus,and cerebral cortex of mice,and is expressed in the cytoplasm of PQ-treated dopaminergic neurons;AK039862 is up-regulated in the substantia nigra tissue of PQ-treated mice,and the target gene The mRNA of Pafah1b1 and Foxa1 was up-regulated and down-regulated in the substantia nigra of the PQ-induced PD mouse model.(2)Up-regulated AK039862 inhibited proliferation in PQ-treated bv2 cells;Down-regulated AK039862 promoted proliferation in CM-PQ-treated mn9 d cells.LncRNA-AK039862 plays a synergistic role in PQ inhibition of proliferation activity of neuronal microglia or/and dopaminergic cells.(3)LncRNA-AK039862 positively regulates transcription of Foxa1 gene in PQ-treated neuroglia and promotes transcription and translation of Pafah1b1;AK039862 reverses regulation of Foxa1 gene in CM-PQ-treated dopaminergic neurons.Transcription process and role in the transcription and posttranscriptional process of the Pafah1b1 gene.