Identification Of Differently Expressed MiRNA And Its Target Gene In The Hippocampus Tissues Of Fmr1 Gene Knockout Mice

MicroRNAs (miRNAs) are short (～22 nucleotide) noncoding RNAs that mediate posttranscriptional gene silencing. miRNAs usually bind their target mRNAs through imperfect base pairing in the 3'untranslated region (UTR) and impact protein expression by inhibiting mRNA translation or by promoting mRNA decay,which participate in cell differentiation, proliferation, apoptosis and all normal development process of the tissues and organs.In mammals, several hundred distinct miRNAs have been discovered, including those selectively expressed in the brain, yet only a small number of miRNA targets have been validated and shown to be functionally important in vivo.MiRNAs present study shows that plays an important role in early development and diseases such as cancer and neurodegenerative.In mammals, miR-132 regulates dendrite development by targeting p250GAP and miR-134 and 138 have been implicated in dendritic spine development through repression of LIMK1 and APT1.Fragile X syndrome (fragile X syndrome, FXS) is one of the most common forms of inherited mental retardation, which usually results from a trinucleotide repeat (CGG) expansion in the 5-untranslated region of the gene FMR1 and subsequent hypermethylation which lead to transcriptional silencing of FMR1 and loss of its encoded protein, the fragile X mental retardation protein (FMRP). FMRP is biochemically and genetically linked to the miRNA pathway. FMRP interacts with proteins (e.g., Argonaute and Dicer) in the RNA interference silencing complex (RISC) and with miRNAs, but FMRP itself is not essential for RNAi-mediated mRNA cleavage. The well-established immature dendritic spines was found in Fmr1 knockout (KO) mouse, which are also central to the pathogenesis of FXS in humans. The discovery of miRNA provides a new direction for FXS study, due to both FMRP and miRNA have inhibitory of translation activity, fragile X syndrome become the first human disease of RNAi study.We used miRNA microarray comparison of normal mice and Fmr1 knockout mouse miRNA expression profiling of hippocampus tissues, access to a number of differentially expressed miRNAs, and bioinformatics software predict miRNAs target gene validated by Real-time PCR, which to explore these miRNAs role in the pathogenesis of FXS and dendritic spines morphology and synaptic function.This study of the mechanism of FMRP-miRNA interaction is expected to help to elucidate the pathogenesis of FXS provides a new thinking. Materials and MethodsThe expression of microRNAs and mRNAs were detected in hippocampus from 1 week male Fmr1 knockout mice(KO1W) of FVB strain and age-matched wildtype mice(WT1W)as control (n=3 for each group).Tail DNA were extracted before the experiment, and PCR identify for genetype.Intraperitoneal injection of 1% sodium pentobarbital (sodium pentobarbital, 0.1-0.2 ml) anesthetized animals, which brains were removed quickly and hippocampal tissue was isolated from the brain in the ice.Hippocampus tissues were saved with 1ml RNALater.Each mouse need to do a technology repeat of miRNA chip, a total of 12 miRNA chip. Differently expressed miRNAs screening criteria: q-value (%)≤5, while Fold Change more than 1.5 times and the number of samples≥3, Ratio = KO / WT.RESULT:1. The expression of miRNAs between Fmr1 knockout mice and wildtype mice in hippocampal tissue8 of the upregulated miRNA (see Excel 1) were obtained from the number of samples (n=3 for each group, 12 chips cluster analysis) and no downregulated miRNA;56 of the upregulated miRNA(see Excel 2) and 20 of the downregulated miRNA(see Excel 2) were obtained from the number of samples (n=2 for each group,8 chips cluster analysis).2. Differently expressed miRNAs were verified by Real-time PCR Differently expressed miRNAs were verified by Real-time PCR, the results showed that miR-449a (P = 0.014) was upregulated in Fmr1 knockout mice.miR-714 (P = 0.035), miR-720 (P = 0.017) were downregulated in the chip results, but were upregulated in the Real-time PCR results.3. The target genes of differently expressed miRNAs prediction Predicted target genes of 3 miRNAs were validated by Real-time PCR, the results showed that:Target genes of neurons/dendritic spines development related (miR-714: Ephb4/Ephb2,Ret,Cdkn1c; miR-720: Ndel1,Fyn,Cdk5,Relb; miR-449a: MAP1A,ARHGAP1).Target genes of synaptic plasticity related (miR-714: Dlgap2,Ephb4/Ephb2,Pxk,Cpne6,Snta1; miR-720: Dlgap2,Ephb4/Ephb2,Pxk,Cpne6,Snta1; miR-449a:SYT1).4. The target genes of miRNAs were verified by Real-time PCR The target genes of miRNAs prediction were verified by Real-time PCR, the results showed that Cdk5 (P = 0.045) of neurons/dendritic spines development related was downregulated in Fmr1 knockout mice. Ubb(P= 0.014 ),SYT1(P= 0.003 ),Efna1(P= 0.019 )of synaptic plasticity related were downregulated in Fmr1 knockout mice.ConclusionA variety of differently expressed miRNAs were obtained by miRNAs chip in Fmr1 knockout mouse brain, suggest abnormal expression of miRNA may be involved in the pathogenesis of FXS.The target genes of differentially expressed miRNAs were predicted by bioinformatics and verified by Real-time PCR. We found that the target genes of development of dendritic spines related and synaptic plasticity related in hippocampus tissue of Fmr1 knockout mice, suggesting that miRNAs may be involved in the pathogenesis of FXS by regulation development of dendritic spines and synaptic plasticity.