A MicroRNA Profile In Fmr1Knockout Mice Reveals MicroRNA Expression Alterations With Possible Rolesin Fragile X Syndrome

IntroductionFragile X syndrome (FXS), a common form of inherited mental retardation, isresulted from a deficiency of the fragile X mental retardation protein (FMRP). FMRP isinvolved in brain functions by interacting with mRNAs and microRNAs (miRNAs) thatselectively control gene expression at translational level. However, little is known aboutthe role of FMRP in regulating miRNA expression. Most FXS cases are caused byunstable expansion of the CGG trinucleotide repeat in the promoter region of the FMR1gene that leads to a deficiency of FMRP by translational suppression. A few cases arecaused by deleting part or all of the FMR1gene, or by point mutations that lead to achange in one of the amino acids in the gene. FMRP is involved in brain functions byinteracting with mRNAs and miRNAs that selectively control gene expression attranslational level. MiRNAs can supress the translation of specific target mRNAs bycomplementing with antisense sequences in the3′untranslated region (3′UTR) of thesemessages, modulating cell differentiation, proliferation, apoptosis and the organdevelopment. Recent studies confirmed that miRNA is highly expressed in the centralnervous system, and some have been determined to participate in the neurogenesis andbrain development. It has been reported that miRNA is abnormal expressed in the brain of both human patient with fragile X syndrome and Fmr1knockout mice, which formsRNA induced silencing complex (RISC) or takes part in other pathways to inhibittranslation, affect the stability or destruct the target mRNAs.FMRP has been verified tointeract with miRNA pathway chemically and genetically. Both FMRP and miRNAsinhibit translation, of them, FMRP can interact with proteins that participate in miRNApathway. The hypothesis in the present study is FMRP can modulate the genesis ofmiRNA.ObjectiveTo confirm whether the expression of miRNAs in the hippocampus of Fmr1knockoutmice is changed by the deficiency of FMRP and the altered miRNAs due to the lackingof FMRP should participateinthe pathogenesis ofFXS.MethodsTo find the proper development stage for study, we explored the dynamic expressionof Fmr1gene in the hippocampus of wildtype mice by Q-PCR. The expressions ofmiRNAs in the hippocampus of Fmr1KO and WT mice at P7weredetermined usingmiRNA microarray. The expressions of the alteredmiRNAs werethen validated byQ-PCR.9miRNAs were increased more than100folds in the hippocampus of Fmr1KO mice, and the levels of pri-miRNA and pre-miRNA of those increased miRNAswere determined by Q-PCR.The target genes of the increased miRNAs were predicted by three bioinformaticsoftwares. Gene Ontology analysis（GO）was used to predictthe biological functions ofthose potential target genes. Semi-PCR and Q-PCR were used to validate the differentexpression of potential target genes in the hippocampus of Fmr1KO and WT mice.Met3′UTR luciferase reporter gene expression plasmids and related miRNAexpression vectors were constructed. These recombinant constructs were thencotransfected into mouse N1E115and Neuro2A cells and detected by dual luciferaseassay toexplore whether those miRNAs can affect the expression of the reporter geneby interacting with theMet3′UTR. Results1. Dynamic transcription of Fmr1in the mouse hippocampusTo determine the temporal expression pattern of Fmr1in the mouse hippocampus,qRT-PCR experiment was done to detect Fmr1mRNA transcripts at different stagesfrom embryonic day18(E18) to postnatal day60(P60). The results showed that therelative mRNA expression levels (Fmr1/β-actin) at P1, P7and P15were about0.1,0.6and0.2fold of that at E18, respectively. No significance was found between E18andP60(P>0.05). Significant alterations (P <0.001) in the relative mRNA expressionlevels were observed between two adjacent stages.2. Altered miRNA expression in the hippocampus of Fmr1KO miceA genome-wide miRNA profile experiment was carried out to determine the miRNAexpression levels in the hippocampus of Fmr1KO andwildtype mice at P7. Comparedwith the wildtype mice,38of the1080miRNAs in the KO mice showed up-regulatedexpression with P value <0.01. Of them,33miRNAs had an increase with about15~250folds. Twenty-six miRNAs in the KO mice showed down-regulated expressionwith a decrease of only about2~4folds. Nine of the most up-regulated miRNAs withchanges>100-fold were confirmed by qRT-PCR analysis，which were increased about40~70folds in the KO mice.3. The up-regulated miRNAs in the hippocampus of Fmr1KO mice are resultedfrommiRNA processing of pri-miRNA into pre-miRNAThe qRT-PCR was used to quantify the levels of the pri-miRNA and the pre-miRNAof9up-regulated miRNAs. The relative levels (pre-miRNA/U6) of the9pre-miRNAsin the KO mice were about5~10folds of that in the wildtype mice. No significantdifference in the relative levels (pri-miRNA/U6) of the9pri-miRNAs was observedbetween the KO mice and the wildtype mice.4. Down-regulation of miRNA target genes in the hippocampus of Fmr1KO miceBy bioinformatic analysis，about5000genes were predicted as the targets, of them,392genes were associated with neuronal function. Most of the392genes had only onepotential target for the9increased miRNAs and6genes had up to6targets. Using qRT-PCR analysis, the mRNA transcripts of10target genes which are highlyexpressed in mouse hippocampus and have5or6potential miRNA targets in their3′UTRs were detected. And the relative mRNA levels of the10genes in thehippocampus of the KO mice were all significantly lower than those in the wildtypemice.5. Overexpression of miRNA down-regulate the reporter gene expression throughinteracting with the Met3′UTRA serial of recombinant constructs of Met3′UTR and related miRNAs (miR-34b, miR-340,miR-148a and miR-101a) were produced to examine whether those miRNAs regulate thereporter gene expression by dual luciferase assay.The constructs of psi-CHECK2-Met3′UTRs cotransfected with related pre-miRNA constructs pCMV-EGFP-pre-miRNA inN1E-115cells and Neuro-2A cells were75%to50%of that cotransfected withpCMV-EGFP. MiR-34b, miR-340and miR-148a could down-regulate the reporter geneexpression by interacting with the Met3′UTR.ConclusionThis study demonstrates that the expression of more than50miRNAs are alteredinthe hippocampus of P7Fmr1KOmice. Our results also suggest that FMRP might beinvolved in the miRNA processing of pri-miRNA into pre-miRNA. The alteredmiRNAs due to the lacking of FMRP shouldparticipateinthe pathogenesis ofFXS.