MicroRNA-mediate Regulation Of Differentiation Of Dopaminergie Neurons

Parkinson’s disease (Parkinson’s diseases, PD) is caused by the selective death of the midbrain substantia nigra dopaminergic neurons or significant reduction of striatum dopamine level when pathological changes take place in the cells of midbrain substantia nigra. Owing to the root cause of PD is that the release of DA neurotransmitter is not enough because of the significant reduction of dopaminergic neurons. Thus, the research of DA neurons induced differentiation has potential medical significance and clinical value.Nr4a2/Nurrl is one of the members of super-family transcription factors, which is highly expressed in the ventral midbrain and the midbrain dopamine neurons. Studies have reported that Nr4a2/Nurrl (nuclear receptor-related factor I) is a nuclear receptor associated factor1, which is closely related with the incidence of dopaminergic neurons, which is one of the most important transcription factors that decide the development and differentiation of dopamine neurons. So far, microRNAs (miRNAs) have been reported to have some mediation function to Nr4a2/Nurrl which is the transcript of dopaminergic neuron differentiation. As is known, microRNAs (miRNAs) are a class of single endogenous non-coding RNA with20to25nt’s Length, which is mainly involved in gene regulation at the transcription level, and play an important role in various life activities. Thus, Systematic studies the regulation function of transcription factors of Nr4a2/Nurrl caused by miRNAs, which could have a thorough understanding of dopamine neurons differentiation mechanism, exploring miRNAs as nucleic acid molecular in its role in the development of Parkinson’s disease.Using Dual-luciferase assays system In this paper and preliminary screening the miRNAs reduced Nr4a2/Nurrl expression significantly. We test the miRNAs including miR-17、miR-183、miR-211、miR-106b、miR-20a、miR-19a and miR-206b. The results show that the expression quantity of target genes NR4A2by miR-17、 miR-183、miR-211,miR-106b and miR-20a reduced significantly (0.01<P<0.05). According to the testing results of the Dual-luciferase assays, we clone mi RNAs with regulatory role into lenti-virus carrier. In vitro packaging lenti-virus of the plasmid into293t cells by calcium phosphate trans-fection method, whi ch get the virus expression of miR-17、miR-183、miR-211、miR-106b、miR-19a、miR-20a and the contrast miRNA.Western Blotting detection show that compare with and positive control, o ver-expressing miR-17, miR-183, miR-106b in neural stem cell, which reduce t he expression quantity of target genes NR4A2significantly, while no significan t differences with miR-19a, miR-20a and miR-211.Real-time-PCR detection show that, over-expressing miR-17, miR-183in the neural stem cell, the expression of target genes NR4A2reduce more significantly at the transcription level (p<0.01),and miR-106b reduced significantly (p<0.05), while miR-211raised the expression significantly (p<0.01).Using real-time-PCR and detecting the expression level of target genes of differentiation from embryonic stem cell to dopaminergic neurons. The target genes including NR4A2、TH、β-Tubulin. The result shows that miR-17and miR-183affect the expression of target gene TH and β-Tubulin through down-regulated the expression of target gene NR4A2.Through the experiments above, we found that miR-17and miR-183participate in the regulation of transcription factor Nr4a2/Nurrl in order to mediate the generation of dopamine neurons, which confirmed that miRNAs has great connection with the occurrence of Parkinson’s disease, which has provided a new way of thinking for the cure for Parkinson’s disease and new drugs development.