microRNA论文：microRNA对多巴胺能神经元分析化基因的调节作用.doc

microRNA论文：microRNA对多巴胺能神经元分析化基因的调节作用【中文摘要】帕金森氏病(Parkinsons disease, PD)是由于缓慢发生的中脑黑质多巴胺能神经元选择性死亡和纹状体多巴胺含量显著减少所导致,PD已成为发病率高居第二的神经系统退行性疾病。显而易见,多巴胺能神经元(dopaminergic neuron, DA neuron)数量的锐减导致其所释放的DA递质含量不足,进而引起运动失调,是PD产生的根本原因。所以,通过合适的途径调控体内DA神经元的数量,从而恢复神经递质平衡是治疗PD的关键所在。microRNAs(miRNAs)是一类长度约为20-25nt的内源性非编码小分子RNA,可通过与靶mRNA的3UTR区互补配对而在转录后水平负调控基因表达。近期的众多研究都着眼于此类小分子RNA家族在疾病产生及治疗方面的重要作用。本文基于现有的研究基础,将三个与多巴胺能神经元分化相关的关键基因Lmxla、Msx1和Nr4a2/Nurrl作为研究对象。利用Microcosm Targets、MicroRNA.org、TargetScan和miRDB等四款miRNA在线分析软件进行生物信息学预测,搜索筛选出对三个靶基因具有调控作用的miRNAs,再经过萤光素酶双报告基因检测(Dual-luciferase assays system)和Western Blotting对软件预测结果进行实验验证,结果小结如下：1、通过文献查找,选定Lmxla、Msx1和Nr4a2/Nurr1三个与多巴胺能神经元分化密切相关的基因作为研究对象。以这三个基因的3UTR为靶序列,在四款miRNA数据库中进行生物信息学预测,搜索出可能调控这三个靶序列的miRNAs。选取四款软件或至少三款软件均能预测到的miRNAs作为候选miRNAs。其中,预测对Lmx1a有作用的miRNAs有3个；预测对Msx1有作用的miRNAs有4个；预测对Nr4a2/Nurr1有作用的miRNAs有13个。2、克隆Lmxla、Msx1和Nr4a2/Nurr1三个靶基因3UTR序列到psiCHECK-2载体中,克隆miR-9、miR-19b、miR-19a、miR-17、miR-20a、miR-106b、miR-211、miR-206、miR-183、miR-34a、miR-467b、miR-144、miR-374、miR-29a、miR-30e、miR-30b、miR-384-5p,17个miRNAs到T载体中。3、利用小鼠的胚胎神经干细胞作为转染对象,共转染已构建的候选miRNAs及携带靶基因3UTR序列的萤光素酶双报告基因载体。利用Dual-luciferase assays system,分析海肾萤光素酶(Rluc)报告基因的表达水平,发现预测的miRNAs中,除miR-206/Nr4a2、miR-467b/Lmxla、miR-374/Msx1三个实验组无差异显著的结果外,其他miRNAs均与预测结果一致,对靶基因3UTR具有明显抑制作用。4、利用小鼠的胚胎神经干细胞作为转染对象,单独转染已构建的候选miRNAs载体,提取蛋白进行Western Blotting检测。发现Nr4a2组的各miRNAs都能够显著降低靶基因的蛋白水平,进一步证实了预测结果。而Lmxla和Msx1纽的抑制效果没有Nr4a2组明显。经过上述验证,我们可以肯定miRNA参与调控多巴胺能神经元分化相关基因的表达,并通过靶向这些基因而间接调节多巴胺能神经元的分化,从而参与帕金森氏病的发生。本研究不但证实了miRNA与帕金森氏病病的发生之间存在莫大的联系,还为帕金森氏病病的治愈及新药的研发提供了一个新思路。【英文摘要】Parkinsons 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 3UTR 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 genesLmxla, 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 3UTR 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 3UTR 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 Parkinsons disease, but also provides a new idea for the treatment of Parkinsons disease and the development of new drugs.【关键词】microRNA 多巴胺能神经元 生物信息学预测 萤光素双报告基因检测 Western Blotting【英文关键词】microRNA dopaminergic neurons bioinformatics predicted Dual Luciferase Reporter Gene Assay Western Blotting【目录】microRNA对多巴胺能神经元分析化基因的调节作用摘要3-5Abstract5-6第1章 引言11-211.1 立题的背景和意义11-121.2 帕金森氏病的介绍12-141.2.1 帕金森氏病的发病机制及病理特征121.2.2 多巴胺及多巴胺能神经元12-141.2.3 多巴胺能神经元相关的分化基因141.3 microRNA的介绍14-211.3.1 microRNA的发现151.3.2 microRNA的生物合成机制15-161.3.3 microRNA的特征16-171.3.4 microRNA的作用机制及靶基因预测17- microRNA的作用机制17- microRNA靶基因预测18-191.3.5 microRNA对帕金森氏病的影响19-21第2章 材料与方法21-292.1 材料与试剂21-272.1.1 仪器和设备21-222.1.2 试剂和耗材22-232.1.3 引物和测序23-252.1.4 动物、细胞和质粒25-272.2 计算机分析27-29第3章 实验方法29-493.1 实验技术路线293.2 靶基因的确定及对其具有调控作用的miRNA预测293.3 靶基因克隆29-373.3.1 小鼠神经干细胞(NPC)的培养29-313.3.2 PCR扩增靶基因及琼脂糖凝胶电泳检测31-323.3.3 靶基因割胶回收32-333.3.4 靶基因与载体PsiCHECK-2的酶切和酶连33-353.3.5 转化353.3.6 单克隆菌落培养及测序353.3.7 克隆菌落测序结果比对及质粒提取、鉴定35-373.4 miRNA克隆37-413.4.1 lenti-137质粒提取(BIOMIGA质粒小提试剂盒)37-383.4.2 PCR扩增miRNA及琼脂糖凝胶电泳检测38-393.4.3 纯化产物加A尾393.4.4 miRNA的TA克隆及重组质粒转化大肠杆菌39-403.4.5 单克隆菌落培养及测序403.4.6 miRNA单克隆菌落测序结果比对及质粒提取、鉴定40-413.5 细胞转染41-433.5.1 小鼠神经干细胞(NPC)的培养413.5.2 小鼠神经干细胞(NPC)的贴壁培养41-423.5.3 靶基因和miRNA共转染NPC42-433.5.4 luciferase检测(Promega)433.6 Western Blotting检测靶基因目的蛋白表达43-493.6.1 小鼠神经干细胞(NPC)的培养433.6.2