Spatiotemporal Expression Pattern F MiR-34c In Mouse Central Ervous System

microRNA (miRNA) is a class of endogenous small single-stranded non-coding RNA,about21to24nucleotides, derived from a pre-miRNA with a hairpin structure(length about70~80nt). Mature miRNA work as post-transcriptional regulator in the cytoplasm, which iscomplementary pair with3’-untranslated region nucleotide sequence of target gene mRNA.Thereby inhibit post-transcriptional translation or induce degradation or silencing of thetarget gene. miR-34c is the member of the miR-34family, which plays an important role inlots of processes within the cells, such as proliferation, differentiation and apoptosis. Inaddition, it is widely involved in learning and memory, anti-anxiety, germatogenesis andother major life process of mice. Olfactory bulb, neocortex, cerebellar cortex andhippocampus are important parts of the central nervous system, which is closely related to thesmelling, cognition, learning and memory，movement and other physiological activities. Inthe present study, we established a stable and efficient detection system of real-timequantitative PCR to detect the expression of miR-34c in these four tissues at differentdevelopmental stages, and also utilized FISH technology to examine the distribution ofmiR-34c in these tissues. Furthermore, to explore the effects of miR-34c on learning andmemory, we established different mouse models, in which miR-34c in hippocampus wasexamined by real-time quantitative PCR. Our results indicated that miR-34c may be involvedin the developmental process of mouse central nervous system, such as olfactory bulb,neocortex, cerebellar cortex and hippocampus and their functions, for example, learning,memory and cognition.1. Establishment of quantitative real-time PCR system for miR-34cWe used stem-loop structure method to design the reverse transcription primers, andthen utilized concentration gradient and temperature gradient PCR method to identify the bestextend annealing temperature and template concentration of miR-34c. Finally, wesuccessfully established a real-time quantitative detecting system with the best annealingtemperature of62.4℃and the appropriate RNA quantity is50ng/μL, which turn into cDNAthrough inverse transcription in each reaction. By examing the expression of miR-34c inmouse brain and lung, we confirmed the high repeatability and specificity of this system.2. Expression profiling of miRNA-34c at different developmental stages of male mice brain tissuesUsing0,3,7,14,21,28,60and90d postnatal male mice, we performed florescentreal-time quantitative PCR to examine the relative expression levels of miR-34c in olfactorybulb, neocortex, cerebellar cortex and hippocampus at eight different developmental stages ofmouse. We also compared the relative expression levels of miR-34c in above four differenttissues of adult male mice. After statistic analysis, the results showed that expression levels ofmiR-34c in olfactory bulb, neocortex, cerebellar cortex and hippocampus gradually increasedwith the development of the mice. Hippocampus showed the highest expression level ofmiR-34c in the four in adult animals.3. Cellular distribution of miR-34c in olfactory bulb, neocortex, cerebellar cortex andhippocampus of adult male miceAfter perfusion with4%PFA, the olfactory bulb, neocortex, cerebellar cortex andhippocampus tissue were prepared from the adult male mice and embedded in paraffin. ThenFISH experiment was performed to examine the distribution of miR-34c. Our results showedthat miR-34c was mainly detected in mitral cells of olfactory bulb, Purkinje’s cells ofcerebellar cortex and CA3pyramidal cells of hippocampus, indicating that miR-34c is likelyinvolved in the regulation of physiological and behavioral activities, such as: smelling,movement, learning and memory of mouse.4. Expression comparision of miR-34c in hippocampus of different mouse modelsWe established mouse models with rich environment, stress, epilepsy and Alzheimerdiesease (AD). The expression levels of miR-34c in hippocampus of these models wereexamined and compared using real-time PCR. Our results showed that expression of miR-34cin AD model is higher than that in control group (P <0.05), while miR-34c in other micemodels is lower than that in control group. We predicted the target genes of miR-34c usingsoftware in websites, and achieved a series target genes. Then choose GABRA3gene, whichone is closely related to learning and memory of mouse. Furthermore, we examined theexpression of miR-34c and GABRA3gene in control mice, AD and enriched environmentmodel mice by real-time PCR, and initially confirmed that the expression levels of miR-34cand GABRA3were negatively related, suggesting that miR-34c may be involved in learningand memory through targeting GABRA3.