Prediction And Experimental Validation Of MicroRNAs That Regulate The Expression Of GSK3β In Mouse

microRNAs (miRNAs) constitute a family of small endogenous non-coding RNAs which can regulate gene expression at post-transcriptional level through recognition of specific binding sites located mainly in the 3'-untranslated region (3'UTR) of target gene and play important roles in many biological progress in vivo. Recent evidence shows that miRNAs are also involved in the pathological progress of AD. Glycogen synthase kinase 3β(GSK3β) is the main kinase that phosphorylates tau protein which is the core component of neurofibrillary tangles (NFT) in AD patient. Here, we investigated whether there were miRNAs that could regulate GSK3βexpression.1. Computational identification of miRNAs that can target to GSK3βin miceFirstly, four online prediction softwares----TargetScan, Pictar, DIANA-micro and miRanda were used to predict miRNAs which may target to GSK3β3'UTR. Then candidate miRNAs were selected based on the conservation level of their binding sites in human and mouse and the free energy between the binding sites and miRNAs. Using this approach, we identified miR-128a, miR-26a, miR-29c and miR-23a that might regulate GSK3βexpression.2. Analysis of luciferase reporter gene assayLuciferase reporter gene assay were performed to confirm the binding of miRNA to GSK3β3'UTR. GSK3β3'UTR luciferase vectors were constructed by inserting partial 3'UTR of GSK3βthat contains the potential miRNA binding sites to downstream of luciferase gene. MiR128a, miR-26a, miR-23a, miR-29c and vector were co-transfected into 293T cell with this reporter vector, respectively. The results showed that over- expression of miR128a, miR-26a, miR-23a and miR-29c could significantly reduce luciferase activity. To further confirm the binding of miRNA to the possible binding site in GSK3β3'UTR, luciferase reporter vectors containing single wild type binding site or mutant binding site were established. The constructs and corresponding miRNA-expressing vector were co-transfected into 293T cells and the luciferase activities were examined. The results showed that miR-128a, miR-26a and miR-29c could bind to wild type binding sites, but not mutant binding site to decrease luciferase expression. These results suggested that miR-128a, miR-26a and miR-29c could bind to GSK3β3'UTR.3. Effect of miRNAs over-expression on GSK3βmRNA and protein expressionMiRNAs can inhibit the expression of their target genes at post-transcription level, In order to further validate whether GSK3βis target genes for miR128a, miR-26a, miR-23a and miR-29c, these miRNAs were over-expressioned in N2a cells. Protein and mRNA were extracted to perform western blot and RT-PCR experiments, respectively. The results have shown that these four miRNAs had no significant effects on GSK3βmRNA level. However, miR-128a and miR-26a overexpression can decrease GSK3βprotein expression levels in N2a cells compared with cells without miRNA overexpression. Over-expression of miR-29c and miR-23a has no obvious effect on GSK3(3 protein level. These results indicated that miR-128a and miR-26a could negatively regulate GSK3P expression.In conclusion, our study provides the first evidence that GSK3βmay be under the regulation of miR-128a and miR-26a. This will do benefits to further investigation on the role of miRNAs in AD.