| Objective:To insight into the regulatory mechanism of endocrine factors expression in regional cerebral source, the differences of GH, IGF-1, CREB1and microRNAs expression in hippocampus were compared in newborn, adult and aged Lewis dwarf and wild type rats, and the relationship of GH/IGF-1/CREB1with growth and development of hippocampal neurons was studied during aging process. Furthermore, the regulatory mechanism of miRNAs on key signaling molecule CREB1expression was revealed.Methods:1. Comparing the ability of Learning and memory, and expression level of GH, IGF-1, CREB1in hippocampus of different months old dwarf and wild type rats.Morris water maze was used to test the differences of learning and memory ability between dwarf and wild-type rats of10months and20months old. Further, the expression level of GH, IGF-1, CREB1were detected in hippocampus of newborn,10months,20months and30months old dwarf and wild type rats by Western blot.2. Analyzing the miRNAs expression differences in hippocampus of different months old dwarf and wild type rats.microRNA chips (V16.0, Exiqon) were used to detect the miRNAs expression level in hippocampus of newborn,10months and20months old Lewis wild type and dwarf rats, and miRanda, DAVID and KEGG were used to analyze the results from the microRNA chips according to the function classification, such as growth and development, ageing degradation and so on for bioinformatics comparison. It is involved in the down-regulated miRNAs with the body development and up-regulated miRNAs with ageing degeneration out of all over1.5-fold-changed miRNAs. the target genes of these differentially expressed miRNAs were enriched (≥3miRNAs’ predicted targets) for DAVID function annotation clustering analysis, and then the important entries associated with physiology function of hippocampus were searched signal pathways using KEGG databank.3. Investigating the regulation mechanism of related miRNA on CREB1expression. Based on the analysis of bioinformatics and related articles, it speculate the key signaling molecule of IGF-1pathways, CREB1is the target gene of miR-181a; Farther the expression vector pGL3C-CREB1UTR and pGL3-CREB1UTR mutant contain CREB13’UTR sequence were constructed, then the mechanism of miR-181a acts on CREB1was analyzed by dual luciferase report system, and the regulatory mechanism was conformed with a series of cell experiments.Results:1. Learning and memory ability and expression level of GH, IGF-1, CREB1in hippocampus in the same age of dwarf and wild type rats.The results of learn training and probe test showed there was no difference of learning and memory ability between dwarf and wild-type in20months old rats. The results of Western blot indicated that the expression level of GH, IGF-1and CREB1in hippocampus were increased remarkably from newborn to adult, but the expression of GH, IGF-1were gradually reduced in aging; Noteworthily, GH and IGF-1expression in hippocampus reduced very slowly in aged dwarf rats, so that no difference was observed between20months and30months old dwarf rats. However, GH and IGF-1expression decreased rapidly in the homologous wild type rats, so that both of them were significantly lower than old dwarf rats.2. miRNA expression profiles and the function analysis for the differentially expressed miRNAs in hippocampus.The reportorial680rat source miRNAs were detected in different ages Lewis dwarf and wild type rats by microRNA chips. The differentially expressed miRNAs between dwarf and wild type rats were analyzed by bioinformatics. The results showed:miR-165, miR-15b, miR-195, miR-10-5p may target BDNF mRNA, miR-21, miR-365, miR-125a-5p, miR-125b-5p, miR-351, miR-23b, miR-23a, miR-34c, miR-15b, miR-195may target Bc12mRNA, miR-125a-5p, miR-125b-5p, miR-351may target Grin2mRNA etc.(Table5), participate in regulation of neurotrophin signal pathway, apoptosis signal pathway and long term potentiation related signal pathway etc, and then involved in development, degeneration and learning and memory function of hippocampus. In addition, on the basis of the differentially expressed miRNAs (>1.5-fold changes) of above mentioned, combined with bioinformatics analysis,45miRNAs were chose to analyze their expression level, which have predicted binding sites on mRNA of GH/GHR, IGF-1/IGF1R, STAT3, STAT5, CREB1etc. Several of them, such as miR-181a, miR-27b, let-7f, miR-153may be selected to investigate the regulation mechanism of miRNAs act on the key factors in GH/IGF-1axis.3. miR-181a is involved in IGF-1mediated regulation of CREB1expression.Western blot analysis revealed that IGF-I, total CREB1and p-CREB1protein levels were significantly higher in hippocampus of adult Lewis rats than that in the newborns.The results of microarray and QPCR illustrated that miR-181a expression was much lower in hippocampus of adult Lewis rats than that in the newborns. The luciferase activity was significantly repressed when miR-181a mimic was co-transfected with pGL3C-CREB13’UTR. The luciferase activity did not reduce notably when miR-181a mimic co-transfection with pGL3C-CREB13’UTR mutant. miR-181a mimic was transfected into PC12cells, the CREB1expression of the cells was significantly repressed (P<0.05), while, the transfection of the cells with miR-181a inhibitor significantly upregulated the expression of CREB1product (P<0.005). PC12cells were treated with different concentrations of IGF-1(25ng/ml,50ng/ml,100ng/ml and200ng/ml), IGF-1remarkably upregulated CREB1protein expression with50ng/ml being most effective (P<0.001), but IGF-1(50ng/ml) caused a significant decrease in miR-181a expression to about45%of the control level, the primary-cultured hippocampal neurons, transfection with miR-181a mimic decreased total dendritic length by about60%and branching by about70%in comparison to scramble miRNA, while transfection with miR-181a inhibitor increased the total dendritic length by about65%and branching by about60%. These results demonstrated that miR-181a is involved in IGF-1mediated regulation of CREB1expression through an interaction with CREB1mRNA’s3’UTR, and then regulate the growth and development of neuron cells.Conclusion:This study suggested that GH, IGF-1, CREB1in hippocampus of Lewis dwarf rat are not lower than that in the homologous Lewis wild type rat, as well as there was no difference of learning and memory ability between them. With miRNA microarray and bioinformatics analysis, this study found69miRNAs, which might involve in regulation of development, degeneration, learning and memory functions of hippocampus. Furthermore, the present study demonstrated for the first time that miR-181a is involved in IGF-1mediated regulation of CREB1expression by targeting its mRNA’s3’UTR, and effects on the growth and development of hippocampal neurons. |
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