Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

中国人群pitx3基因多态性与晚发散发性帕金森病的相关研究

目的分析pitx3基因多态性与晚发散发性帕金森病的遗传关联。方法使用连接酶检测反应(LDR)法,分析509例晚发散发性帕金森病(Parkinson's disease,PD)患者和494例正常对照者中pitx3基因单核苷酸多态性(SNPs)位点rs2281983、rs4919621和rs3758549。随机选取10%样品直接测序进行验证。结果 pitx3基因SNP位点rs2281983、rs4919621和rs3758549的基因频率在晚发散发性帕金森病患者和正常对照组间差异无统计学意义。3个SNP位点与晚发散发性PD无关联。结论中国人群中,pitx3不是晚发散发性帕金森病的易感因素。

中国人群PITX3基因多态性与帕金森病的相关性

目的分析中国人群PITX3基因多态性与帕金森病的相关性。方法使用连接酶检测反应（LDR）法，分析509例晚发性帕金森病（LOPD）患者、290例早发性帕金森病（EOPD）患者和494例正常对照中PITX3基因单核苷酸多态性（SNPs）位点rs2281983、rs4919621和rs3758549基因型。结果PITX3基因SNPs位点rs2281983、rs4919621和rs3758549的基因型和等位基因频率在799例帕金森病患者和494例正常对照组间（基因型频率P值分别为0．494，0．343，0．951；等位基因频率P值分别为0．369，0．297，0．823）、509例LOPD和494例正常对照组间（基因型频率P值分别为0．522，0．350，0．630；等位基因频率P值分别为0．413，0．328，0．571）、290例EOPD和494例正常对照组间（基因型频率P值分别为0．499，0．492，0．552；等位基因频率P值分别为0．321，0．301，0．931）、509例LOPD和290例EOPD间（基因型频率P值分别为0．577，0．710，0．127；等位基因频率P值分别为0．346，0．472，0．077）均差异无统计学意义。三个SNPs位点与PD无关联。结论中国人群中，PITX3基因多态性不是帕金森病的易感因素。