MicroRNA-mediate Regulation Of Differentiation-Related Genes Of Dopaminergic Neurons

Parkinson's disease is caused by the slowing selective death of dopaminergic neurons in the substantia nigra, and the significant reduction of dopamine levels in the striatum of PD patients.The incidence of PD had become the second hightest neurodegenerative diseases in the world.Obviously,insufficient release of dopamine transimitter caused by the major decrease of dopaminergic neurons resulting in the motor ataxia,is a radical reason of PD. Consequently,find a proper way to regulate the number of dopaminergic neurons and to restore the neurotransmitter levels in vivo is the key to cure PD.microRNAs (miRNAs) are 20-25 nt, endogenous non-coding small molecule RNA family that posttranscriptionally regulate gene expression through base paring to the 3'UTR of target gene. Many studies have focused on the fundamental role of these small molecules during the development and treatment of diseases.Based on the published researchs, we found three key genes—Lmxla, Msxl and Nr4a2/Nurrl that are highly associated with differentiation of dopaminergic neurons. Therfore, we utilized Microcosm Targets, MicroRNA.org, TargetScan. and miRDB four miRNA on-line analysis softwares for bioinformatics assay and predicted miRNAs that could regulate the three target genes. Finally, we validated the role of these miRNAs by Dual-luciferase assays system and Western Blotting. The results are summarized as follows:1 We chose Lmxla, Msxl, Nr4a2/Nurrl three genes, which are functional during differentiation of dopaminergic neurons, as the objects of our study. The potential miRNAs that could regulate these genes were predicted by four miRNA on-line analysis softwares. miRNAs could be predicted in at least three or four softwares are selected as candidate miRNAs.Actually,there are 3 miRNAs target Lmxla,4 miRNAs target Msx 1,13 miRNAs target Nr4a2/Nurr1.2 The 3'UTR sequence of three target genes were cloned into psiCHECK-2 vectors,and totally 17 miRNAs, miR-9, miR-19b, miR-19a, miR-17, miR-20a, miR-106, miR-211, miR-206, miR-183, miR-34a, miR-467b, miR-144, miR-374, miR-29a, miR-30e, miR-30b and miR-384-5p were cloned into T-Vectors, respectively.3 The candidate miRNAs and 3'UTR sequence of the target genes which had been cloned into dual-reporter luciferase vectors are co-transfected into mouse embryonic neural stem cells. Luciferase activity was measured using a GloMaxTM-Multi+ Mircroplate Luminometer and the Renilla luciferase counts were then normalized to Firefly luciferase counts.We find that among all the predicted miRNAs,only miR-206/Nr4a2, miR-467b/Lmxla,miR-374/Msxl show no significant differences, while others are consistent with the prediction.4 After transfecting mouse embryonic neural stem cells by the candidate miRNAs, the expression of Nr4a2, Lmxla and Msxl were detected by Western Blotting. We find that all the miRNAs of Nr4a2 group can significantly reduce the protein levels, but the inhibitory effect of Lmxla and Msxl groups did not show obvious effect compared to Nr4a2 group.Our work verify miRNAs' fundamental role in the regulation of differentiation-related genes of dopaminergic neurons, which not only confirmed the great link between miRNA and the incidence of Parkinson's disease, but also provides a new idea for the treatment of Parkinson's disease and the development of new drugs.