Fmr1敲除小鼠海马组织microRNA的差异表达及其功能研究

发布时间：2018-09-03 17:13

【摘要】：【研究背景】 脆性X综合征（Fragile X syndrome，FXS）是常见的遗传性智力低下疾病，是由位于Xq27.3的脆性X智力低下基因1（fragile X mental retardation1，FMR1）突变引起脆性X智力低下蛋白（fragile X mental retardation protein，，FMRP）表达下降或缺失所致。大部分FXS患者为5′非翻译区（CGG）n三核苷酸重复序列的动态突变，导致其上游CpG岛异常甲基化，引起其编码的FMRP表达下调或缺失。极少数则是由于FMR1有义突变或缺失所致。FMRP通过作用于mRNA和microRNA（miRNA）在转录水平选择性地调控基因表达来参与脑功能的调控。MiRNAs通过与mRNAs3′非翻译区（3′UTR）以反义序列不完全配对的形式，抑制mRNA翻译或促进mRNA降解影响蛋白表达，参与细胞分化、增殖、凋亡以及各个组织器官的正常发育。近年来有研究证实，miRNA在中枢神经系统高表达，并且某些miRNA已被证实参与神经发生及脑的发育。有文献报道，在FXS动物模型中发现miRNA表达异常，异常表达的miRNA通过形成RNA-诱导的沉默复合体（RISC）或其他途径作用于靶基因mRNA，影响靶基因mRNA的稳定性，导致树突棘的发育异常，参与FXS的发病机制。已有研究证实，FMRP在生物化学以及遗传学上与miRNA相关通路密切联系。FMRP，miRNA都具有选择性的抑制蛋白翻译的作用，并且FMRP可以与miRNA成熟过程中的关键Dicer酶相互作用，参与miRNA在胞浆中的成熟过程。但是，FMRP作为选择性的RNA结合蛋白是否在胞核内对miRNA译的作用，并且FMRP可以与miRNA成熟过程中的关键Dicer酶相互作用，参与miRNA在胞浆中的成熟过程。但是，FMRP作为选择性的RNA结合蛋白是否在胞核内对miRNA的生成产生影响，异常表达的miRNA是否参与FXS的发病，目前尚无报道。 【研究目的】 通过分析脆性X综合征动物模型-Fmr1敲除（KO）小鼠海马组织miRNA表达情况、成熟过程中pri-miRNA及pre-miRNA的改变，初步探究FMRP对miRNA成熟过程可能的影响；并进一步分析差异表达的miRNA对潜在的靶基因表达调控的影响，在基因表达调控水平上进一步揭示该疾病的发病机制。 【方法】 本研究通过Q-PCR探究Fmr1转录本在不同发育时期小鼠海马组织中的动态表达情况，找到合适研究天数的小鼠，采用miRNA芯片技术分析FVB品系野生型小鼠与Fmr1敲除小鼠海马组织中miRNA表达谱系在该时间点的改变情况，对芯片结果中差异表达的miRNA采用荧光定量PCR进行验证，选取改变幅度大于100倍的miRNA，进一步观察其pri-miRNA和pre-miRNA的表达情况。采用生物信息学软件对上述明显上调的miRNA进行靶基因的预测，采用Gene Ontology分析预测到的靶基因，找到与神经系统相关的基因。采用半定量PCR和荧光定量PCR验证与神经系统相关的靶基因在WT与KO鼠中转录本含量有无差异。构建Met3′UTR双荧光素酶真核报告基因表达质粒和相关miRNA真核表达质粒，将构建的Met3′UTR的质粒分别与其相关的miRNA质粒瞬时共转染小鼠神经源性N1E115细胞及Neuro2A细胞，采用双荧光素酶基因报告系统对共转染质粒双荧光素酶活性变化进行检测，分析miRNA对靶基因3′UTR活性的影响。 【结果】 1. Fmr1在野生型小鼠海马组织中的动态表达 通过qRT-PCR检测不同发育时期（从胚胎期18天到出生后60天）小鼠海马组织中Fmr1的表达水平。在P1、P7和P15Fmr1mRNA表达水平分别为E18天的0.1、0.6和0.2倍。E18和P60之间表达无差异(P0.05)。毗邻两个发育时期之间Fmr1表达水平有显著差异（P 0.001）。 2. Fmr1敲除小鼠海马组织中miRNA的表达 通过全基因组miRNA表达谱系分析显示，与野生型小鼠相比，P7KO小鼠在1080种miRNA中有38个表达上调（P 0.001），其中33个上调高达15~250倍；有26个miRNA表达下调约2~4倍（P 0.001）。通过qRT-PCR验证表达上调大于100倍的9个miRNA，上述miRNA均显著上调，与芯片结果相符。 3. Fmr1敲除小鼠海马组织中，上调大于100倍miRNA的pri-miRNA及pre-miRNA的表达 通过qRT-PCR检测9个上调miRNA的pri-miRNA和pre-miRNA的表达水平。与野生型小鼠相比，9个pre-miRNA在KO鼠海马组织中表达上调约5~10倍。而9个pri-miRNA在KO鼠海马组织中表达与野生型小鼠相比未见明显变化。 4.在Fmr1敲除小鼠海马组织中miRNA靶基因的表达 通过生物信息学软件预测以及分析上述9个表达上调miRNA的靶基因，有约5000个潜在的靶基因，其中392个与神经系统功能相关。392个靶基因中的绝大多数只受9个miRNA中的1个调控，小部分靶基因受9个miRNA中的多个调控。对受其中5~6个miRNA共同调控的10个靶基因，应用RT-PCR检测mRNA水平，发现与野生型小鼠相比，上述10个靶基因mRNA水平在敲除小鼠海马组织中的表达均明显下调。 5. Met3′UTR及其相关miRNA构建质粒共转染细胞后活性的检测 已预测Met3′UTR及调控其表达miRNA的相关信息，构建Met3′UTR及其相关miRNA（miR-34b， miR-340，miR-148a和miR-101a）的质粒，通过双荧光素酶报告系统检测预测的miRNA是否可以通过作用于Met3′UTR的序列下调报告基因的表达。将psi-CHECK2-Met3′UTR质粒以及相关的pCMV-EGFP-pre-miRNA质粒共转染小鼠神经源性N1E-115和Neuro-2A细胞，发现miR-34b，miR-340和miR-148a可以通过与Met3′UTR的序列相互作用下调报告基因的表达，活性约为阳性对照组的75%-50%。 【结论】 FMRP可能参与miRNA由pri-miRNA转变为pre-miRNA的转录后加工成熟过程，选择性调控miRNA表达。KO鼠表达上调的miRNA通过降低靶基因mRNAs的表达，可能参与FXS的发病机制。
[Abstract]:[background]
Fragile X syndrome (FXS) is a common inherited mental retardation disorder. It is caused by a mutation in the fragile X mental retardation gene 1 (FMR1) located in Xq27.3, resulting in a decrease or deletion of the expression of fragile X mental retardation protein (FMRP). Dynamic mutations in the n-trinucleotide repeats of the translation region (CGG) result in abnormal methylation of the upstream CpG islands, resulting in the down-regulation or deletion of the FMRP expression encoded by the CpG islands. Few are due to meaningful mutations or deletions in FMR1. FMRP participates in the regulation of brain function by selectively regulating gene expression at the transcriptional level by acting on mRNA and microRNA (microRNA). Control. MiRNAs inhibit mRNA translation or promote mRNA degradation affecting protein expression, participate in cell differentiation, proliferation, apoptosis and normal development of various tissues and organs by incompletely matching with the 3'-untranslated region of mRNAs (3'-UTR). Recent studies have confirmed that microRNAs are highly expressed in the central nervous system, and some microRNAs have been identified. Abnormal expression of microRNAs has been found in FXS animal models. Abnormal expression of microRNAs affects the stability of target gene mRNA through the formation of RNA-induced silencing complexes (RISC) or other pathways, resulting in abnormal development of dendritic spines, and participates in the pathogenesis of FXS. Studies have shown that FMRP is biochemically and genetically closely related to the microRNA-related pathways. FMRP and microNA both selectively inhibit protein translation, and FMRP can interact with key Dicerase during the maturation of microRNAs and participate in the maturation of microRNAs in the cytoplasm. However, whether FMRP, as a selective RNA binding protein, affects the production of microRNAs in the nucleus and whether abnormally expressed microRNAs are involved in the pathogenesis of FXS has not been reported.
[research purposes]
By analyzing the expression of microRNAs in hippocampus of Fmr1 knockout (KO) mice and the changes of pri-microRNAs and pre-microRNAs during maturation, the possible effects of FMRP on the maturation of microRNAs were preliminarily explored, and the effects of differentially expressed microRNAs on the regulation of potential target gene expression were further analyzed. One step forward is to reveal the pathogenesis of the disease.
[method]
In this study, the dynamic expression of Fmr1 transcripts in the hippocampus of mice at different developmental stages was investigated by Q-PCR. The suitable number of mice for study was found. The changes of the expression of microRNAs in the hippocampus of FVB strain wild type mice and Fmr1 knockout mice were analyzed by using microarray technology. The differences between the results of microarray were shown. The expression of pri-microRNAs and pre-microRNAs was further observed. The target genes were predicted by bioinformatics software. Gene Ontology was used to analyze the predicted target genes and find the genes related to the nervous system. Genes. Semi-quantitative PCR and fluorescence quantitative PCR were used to verify the difference of transcripts between WT and KO mice. Met3'UTR double luciferase eukaryotic reporter gene expression plasmid and related microRNA eukaryotic expression plasmid were constructed, and the constructed Met3'UTR plasmid was transfected with its related microRNA plasmid. Mice N1E115 cells and Neuro2A cells were co-transfected with two luciferase gene reporter systems to detect the activity of double luciferase and analyze the effect of microRNAs on the activity of 3'-UTR.
[results]
Dynamic expression of 1. Fmr1 in hippocampus of wild type mice
The expression levels of Fmr1 in hippocampus of mice at different developmental stages (from 18 days of embryo to 60 days after birth) were detected by qRT-PCR. The expression levels of Fmr1 mRNA in P1, P7 and P15Fmr1 were 0.1, 0.6 and 0.2 times higher than those in E18 days, respectively. There was no difference between E18 and P 60 (P 0.05).
Expression of miRNA in hippocampal tissue of 2. Fmr1 knockout mice
Compared with wild type mice, 38 of the 1080 microRNAs in P7KO mice were up-regulated (P 0.001), 33 of which were up-regulated as high as 15-250 times, and 26 of them were down-regulated by 2-4 times (P 0.001). Nine microRNAs were up-regulated by qRT-PCR, and all of them were up-regulated significantly. The result is consistent.
The expression of pri-miRNA and pre-miRNA in hippocampus of 3. Fmr1 knockout mice was upregulated more than 100 times miRNA.
Nine of the up-regulated pri-microRNAs and pre-microRNAs in the hippocampus of KO mice were detected by qRT-PCR. Compared with wild-type mice, nine pre-microRNAs were up-regulated about 5-10 times in the hippocampus of KO mice.
4. expression of miRNA target genes in hippocampus of Fmr1 knockout mice
About 5000 potential target genes were predicted and analyzed by bioinformatics software, of which 392 were related to nervous system function. Most of the 392 target genes were regulated by only one of nine microRNAs, and a few were regulated by multiple of nine microRNAs. RT-PCR was used to detect the mRNA levels of the 10 target genes in the hippocampus of knockout mice. The results showed that the mRNA levels of the 10 target genes were significantly down-regulated compared with those of wild-type mice.
Detection of the activity of 5. Met3 'UTR and its associated miRNA construction plasmid co transfected cells
Met3'UTR has been predicted and information related to the regulation of its expression of microRNAs has been obtained. Plasmids of Met3'UTR and its related microRNAs (microRNAs - 34b, microRNAs - 340, microRNAs - 148A and microRNAs - 101a) have been constructed to detect whether the predicted microRNAs can down-regulate the expression of the reporter gene through the sequences that act on the Met3'UTR. Mice neurogenic N1E-115 and Neuro-2A cells were co-transfected with pCMV-EGFP-pre-microRNA plasmids. It was found that microRNAs-34b, microRNAs-340 and microRNAs-148a could down-regulate the expression of reporter genes by interacting with the sequence of Met3'UTR, and the activity was about 75-50% of the positive control group.
[Conclusion]
FMRP may be involved in the post-transcriptional maturation of microRNAs from pri-microNA to pre-microNA and selectively regulate the expression of microRNAs.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.93

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