Fmr1敲除小鼠海馬組織microRNA的差異表達及其功能研究
發(fā)布時間：2018-09-03 17:13
【摘要】：【研究背景】 脆性X綜合征（Fragile X syndrome，F(xiàn)XS）是常見的遺傳性智力低下疾病，是由位于Xq27.3的脆性X智力低下基因1（fragile X mental retardation1，F(xiàn)MR1）突變引起脆性X智力低下蛋白（fragile X mental retardation protein，，F(xiàn)MRP）表達下降或缺失所致。大部分FXS患者為5′非翻譯區(qū)（CGG）n三核苷酸重復序列的動態(tài)突變，導致其上游CpG島異常甲基化，引起其編碼的FMRP表達下調(diào)或缺失。極少數(shù)則是由于FMR1有義突變或缺失所致。FMRP通過作用于mRNA和microRNA（miRNA）在轉(zhuǎn)錄水平選擇性地調(diào)控基因表達來參與腦功能的調(diào)控。MiRNAs通過與mRNAs3′非翻譯區(qū)（3′UTR）以反義序列不完全配對的形式，抑制mRNA翻譯或促進mRNA降解影響蛋白表達，參與細胞分化、增殖、凋亡以及各個組織器官的正常發(fā)育。近年來有研究證實，miRNA在中樞神經(jīng)系統(tǒng)高表達，并且某些miRNA已被證實參與神經(jīng)發(fā)生及腦的發(fā)育。有文獻報道，在FXS動物模型中發(fā)現(xiàn)miRNA表達異常，異常表達的miRNA通過形成RNA-誘導的沉默復合體（RISC）或其他途徑作用于靶基因mRNA，影響靶基因mRNA的穩(wěn)定性，導致樹突棘的發(fā)育異常，參與FXS的發(fā)病機制。已有研究證實，F(xiàn)MRP在生物化學以及遺傳學上與miRNA相關(guān)通路密切聯(lián)系。FMRP，miRNA都具有選擇性的抑制蛋白翻譯的作用，并且FMRP可以與miRNA成熟過程中的關(guān)鍵Dicer酶相互作用，參與miRNA在胞漿中的成熟過程。但是，F(xiàn)MRP作為選擇性的RNA結(jié)合蛋白是否在胞核內(nèi)對miRNA譯的作用，并且FMRP可以與miRNA成熟過程中的關(guān)鍵Dicer酶相互作用，參與miRNA在胞漿中的成熟過程。但是，F(xiàn)MRP作為選擇性的RNA結(jié)合蛋白是否在胞核內(nèi)對miRNA的生成產(chǎn)生影響，異常表達的miRNA是否參與FXS的發(fā)病，目前尚無報道。 【研究目的】 通過分析脆性X綜合征動物模型-Fmr1敲除（KO）小鼠海馬組織miRNA表達情況、成熟過程中pri-miRNA及pre-miRNA的改變，初步探究FMRP對miRNA成熟過程可能的影響；并進一步分析差異表達的miRNA對潛在的靶基因表達調(diào)控的影響，在基因表達調(diào)控水平上進一步揭示該疾病的發(fā)病機制。 【方法】 本研究通過Q-PCR探究Fmr1轉(zhuǎn)錄本在不同發(fā)育時期小鼠海馬組織中的動態(tài)表達情況，找到合適研究天數(shù)的小鼠，采用miRNA芯片技術(shù)分析FVB品系野生型小鼠與Fmr1敲除小鼠海馬組織中miRNA表達譜系在該時間點的改變情況，對芯片結(jié)果中差異表達的miRNA采用熒光定量PCR進行驗證，選取改變幅度大于100倍的miRNA，進一步觀察其pri-miRNA和pre-miRNA的表達情況。采用生物信息學軟件對上述明顯上調(diào)的miRNA進行靶基因的預測，采用Gene Ontology分析預測到的靶基因，找到與神經(jīng)系統(tǒng)相關(guān)的基因。采用半定量PCR和熒光定量PCR驗證與神經(jīng)系統(tǒng)相關(guān)的靶基因在WT與KO鼠中轉(zhuǎn)錄本含量有無差異。構(gòu)建Met3′UTR雙熒光素酶真核報告基因表達質(zhì)粒和相關(guān)miRNA真核表達質(zhì)粒，將構(gòu)建的Met3′UTR的質(zhì)粒分別與其相關(guān)的miRNA質(zhì)粒瞬時共轉(zhuǎn)染小鼠神經(jīng)源性N1E115細胞及Neuro2A細胞，采用雙熒光素酶基因報告系統(tǒng)對共轉(zhuǎn)染質(zhì)粒雙熒光素酶活性變化進行檢測，分析miRNA對靶基因3′UTR活性的影響。 【結(jié)果】 1. Fmr1在野生型小鼠海馬組織中的動態(tài)表達 通過qRT-PCR檢測不同發(fā)育時期（從胚胎期18天到出生后60天）小鼠海馬組織中Fmr1的表達水平。在P1、P7和P15Fmr1mRNA表達水平分別為E18天的0.1、0.6和0.2倍。E18和P60之間表達無差異(P0.05)。毗鄰兩個發(fā)育時期之間Fmr1表達水平有顯著差異（P 0.001）。 2. Fmr1敲除小鼠海馬組織中miRNA的表達 通過全基因組miRNA表達譜系分析顯示，與野生型小鼠相比，P7KO小鼠在1080種miRNA中有38個表達上調(diào)（P 0.001），其中33個上調(diào)高達15~250倍；有26個miRNA表達下調(diào)約2~4倍（P 0.001）。通過qRT-PCR驗證表達上調(diào)大于100倍的9個miRNA，上述miRNA均顯著上調(diào)，與芯片結(jié)果相符。 3. Fmr1敲除小鼠海馬組織中，上調(diào)大于100倍miRNA的pri-miRNA及pre-miRNA的表達 通過qRT-PCR檢測9個上調(diào)miRNA的pri-miRNA和pre-miRNA的表達水平。與野生型小鼠相比，9個pre-miRNA在KO鼠海馬組織中表達上調(diào)約5~10倍。而9個pri-miRNA在KO鼠海馬組織中表達與野生型小鼠相比未見明顯變化。 4.在Fmr1敲除小鼠海馬組織中miRNA靶基因的表達 通過生物信息學軟件預測以及分析上述9個表達上調(diào)miRNA的靶基因，有約5000個潛在的靶基因，其中392個與神經(jīng)系統(tǒng)功能相關(guān)。392個靶基因中的絕大多數(shù)只受9個miRNA中的1個調(diào)控，小部分靶基因受9個miRNA中的多個調(diào)控。對受其中5~6個miRNA共同調(diào)控的10個靶基因，應用RT-PCR檢測mRNA水平，發(fā)現(xiàn)與野生型小鼠相比，上述10個靶基因mRNA水平在敲除小鼠海馬組織中的表達均明顯下調(diào)。 5. Met3′UTR及其相關(guān)miRNA構(gòu)建質(zhì)粒共轉(zhuǎn)染細胞后活性的檢測 已預測Met3′UTR及調(diào)控其表達miRNA的相關(guān)信息，構(gòu)建Met3′UTR及其相關(guān)miRNA（miR-34b， miR-340，miR-148a和miR-101a）的質(zhì)粒，通過雙熒光素酶報告系統(tǒng)檢測預測的miRNA是否可以通過作用于Met3′UTR的序列下調(diào)報告基因的表達。將psi-CHECK2-Met3′UTR質(zhì)粒以及相關(guān)的pCMV-EGFP-pre-miRNA質(zhì)粒共轉(zhuǎn)染小鼠神經(jīng)源性N1E-115和Neuro-2A細胞，發(fā)現(xiàn)miR-34b，miR-340和miR-148a可以通過與Met3′UTR的序列相互作用下調(diào)報告基因的表達，活性約為陽性對照組的75%-50%。 【結(jié)論】 FMRP可能參與miRNA由pri-miRNA轉(zhuǎn)變?yōu)閜re-miRNA的轉(zhuǎn)錄后加工成熟過程，選擇性調(diào)控miRNA表達。KO鼠表達上調(diào)的miRNA通過降低靶基因mRNAs的表達，可能參與FXS的發(fā)病機制。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R749.93
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R749.93
【相似文獻】
相關(guān)期刊論文 前10條
1 孫素菊,尹桂山,馮忠軍,趙建宏,朱惠民;過量碘對仔鼠海馬組織烯醇化酶活性的影響[J];中華預防醫(yī)學雜志;2000年01期
2 陸彩玲;王金子;唐深;胡峰;付強;李習藝;郭松超;;海馬組織蛋白質(zhì)雙向電泳條件實驗研究[J];中國職業(yè)醫(yī)學;2012年03期
3 ;健康速遞[J];醫(yī)療保健器具;2001年10期
4 王布爾,程介士;海馬內(nèi)強啡肽和電針抗癇作用的關(guān)系[J];科學通報;1992年14期
5 魏小龍,張永祥,魏婉麗,徐海,程軍平,聶偉,楊勝,馬淵,周金黃;用mRNA差異顯示技術(shù)篩選和克隆小鼠海馬有關(guān)基因[J];中國藥理學通報;2000年05期
6 楊學森,索玉蘭,張廣斌,余爭平;電磁輻射后大鼠血清和海馬組織兒茶酚胺的變化及防護部位的選擇[J];中華勞動衛(wèi)生職業(yè)病雜志;2002年04期
7 田美玲;金國華;譚雪鋒;朱惠霞;秦建兵;施金洪;趙荷艷;;切割穹窿海馬傘海馬提取液和銀杏葉提取物聯(lián)合誘導海馬NSCs向膽堿能神經(jīng)元的分化[J];神經(jīng)解剖學雜志;2009年05期
8 李昱，呂長虹;海馬中γ-氨基丁酸與癲癇的關(guān)系[J];重慶醫(yī)科大學學報;1998年02期
9 劉明月;董憲U
本文編號：2220640
本文鏈接：http://sikaile.net/yixuelunwen/jsb/2220640.html
最近更新
教材專著
