The Alteration Of LncRNA Expression In The Brain Of Mice Induced By Paraquat : The Role Of Transcription Factor Nrf2

Objective: Previous studies suggest that Long non-coding RNA（LncRNA）and Nrf2 play a key role in neurodegenerative diseases. However, little is known about what the relationship between LncRNAs and Nrf2 contribute to dopaminergic neuronal damaged by paraquat（PQ） and 1-methyl-4-phenyl-1, 2, 3, 6- tetrahydropyridine（MPTP）. In the present study, we used microarray analysis to show the differential expression of LncRNA induced by PQ and MPTP between Nrf2（+/+） and Nrf2（-/-） mouse model of Parkinson’s disease. And compared the change patterns of LncRNA expression which are caused by these two nerve toxicants. Applied the Nrf2（-/-） mouse to investigate what is the possible role of Nrf2 in the substantia nigra,when paraquat and MPTP mice induced the altevation of LncRNA expression.Methods:（1） Saline, 10 mg/kg PQ, 30 mg/kg MPTP treated Nrf2（+/+） and Nrf2（-/-） ICR mice. Then applied PD model, established by PQ, to analyse the changes of LncRNA and mRNA expression patterns（n=3） used LncRNA and mRNA gene microarray in the tissue of substantia nigra. We used the Volcano Plot filtering to identify differentially expressed LncRNA and mRNA（fold change,FC） >1.5 and p < 0.05), and gene ontology（GO） analysis and KEGG pathway analysis are applied to analyse the differential expression of mRNA.（2） After Nrf2（+/+） and Nrf2（-/-） ICR mice treated with saline, 5 mg/kg, 10 mg/kg PQ, 30 mg/kg MPTP（n=4）,we save the substantia nigra, hippocampus, cerebral tissue. Then,we selected 6 LncRNA with the biggest chang fold in defferential LncRNA expression: NR₀30777、NR₀27648、AK078503、Ak047865、NR₀24257 and uc007 nsi.1. RT-PCR verify whether the expression levels of LncRNA is in accordance with gene microarray results in the mice substantia nigra,and explore the levels of them in the hippocampus,cerebral cortex. At the same time,we play fluorescence in situ hybridization to explore NR₀30777、NR-027648 distribution at the different organizations and different subcellulars（n=3）.（3） Applied coding-non-coding（CNC）relation analysis to predict potential target genes of 6 LncRNAs,then combine with differently expression mRNA profles、GO analysis、KEGG pathway analysis to affirm downstream target genes.（4） QRT-PCR and Western blot verify part of the target gene expression level of NR₀30777 and NR₀27648.Results: In vivo, PQ and MPTP exposure can change LncRNA expression profiles, which might be their neurotoxic mechanism. The changes of LncRNAs expression between them are distinguishing but related. MPTP might change LncRNA expression profiling in substantia nigra through the interaction with Nrf2, and then result in the change of NR₀30777/Cpne5/Zfp326 and NR₀27648/Cybb mRNA pathway, which may be the mechanism of MPTP-induced neurotoxincity. Similarly, PQ change LncRNA expression profiling in substantia nigra through the interaction with Nrf2. However, this change showed difference with PQ dose. The result of target gene prediction analysis and mRNA microarray analysis suggested that Cybb mRNA may be the downstream target gene of NR₀27648, Cpne5 mRNA may be the downstream target gene of NR₀30777. Taken together, this study demonstrated that transcription factor Nrf2 plays a role through LncRNA expression alteration in mice substantia nigra induced by PQ and/or MPTP.Conclusions:（1） For the first time, PQ exposure can change the LncRNA expression profile in vivo, and this is likely to be involved in the mechanisms of neurotoxicity induced by PQ.（2） For the first time, MPTP exposure can change the LncRNA expression in vivo, which may be involved in the mechanism of neurotoxicity induced by MPTP, which is different from PQ, but there are related.（3） There may be a potential basis physiological regulation relationship between transcription factor Nrf2 and LncRNAs. Nrf2 plays a role in LncRNA expression changes induced by PQ or MPTP.（4） MPTP might change LncRNA expression profiling in substantia nigra through the interaction with Nrf2, and then result in the change of NR₀30777/Cpne5/Zfp326 and NR₀27648/Cybb mRNA pathway, which may be the mechanism of MPTP-induced neurotoxincity. Similarly, PQ might changes LncRNA expression profiling in substantia nigra through the interaction with Nrf2. However, this change show difference with different PQ dose. The result of target gene prediction analysis and mRNA microarray analysis suggests that Cybb mRNA may be the downstream target genes of NR₀27648 and Cpne5 mRNA may be the downstream target genes of NR₀30777.（5） The different expressed LncRNA and their downstream target genes, which is related to functional disorder induced by paraquat and MPTP in nerve cells. Experimental research on gene function need further experimental study. Especially, related to Nrf2, the regulation of the downstream target genes of NR₀27648（Cybb, Zc3h14, Duox1 and Duoxa2） NR₀30777（zfp326 and cpne5）, need to further study.