MiR-125b Manipulates Neural Cell Adhesion Molecule Involved In Mice Dementia

microRNA(miRNA),a family of small noncoding RNA,target mRNA by complementary pairing,play a crucial role in post-transcriptional regulation by either degrading mRNA or repressing its translation.miRNAs participate in a variety of physiological processes including cell growth,differentiation and etc.In most cases,cause and progression of disease are always accompanied with abnormal miRNAs expression.Investigations on miRNAs and their target genes contribute to understanding the physiological mechanism,pathogenesis and pathological process;also they may help to find out the therapeutic strategies and early diagnosis as biomarkers.In this study,miRNAs expressed in Presenilin 1/2 conditional double knock out(DKO)mice were screened by miRNA array.We then focused on the abnormal expressed miRNAs as well as their target genes.Furthermore,we investigated the influenced physiological function resulted from disrupted miRNA regulation.Quantitative real-time PCR and Western Blotting were performed to analyze the expression of our interested miRNAs and their predicted targeting genes in DKO mice.miR-125 b showed an significant up-regulation in prefrontal cortex(PFC)of DKO mice.Repression of miR-125 b to its predicted target neural cell adhesion molecule(NCAM)was confirmed on both N2 a and PC12 cell lines.In addition,we observed that expression of miR-125 b leaded to the increase of glycogen synthase kinase-3β(GSK3β)activity both in vitro and in vivo.Moreover,phosphorylation of tau protein was affected as well.Conclusions are following:1.Array based analysis of miRNAs in PFC of DKO miceAccording to miRNA array data,we verified the up-and down-regulated miRNAs in DKO mice,quantitative real-time PCR approach showed the significant up-regulation of miR-125 b in PFC of 12 M DKO mice than their littermate controls.We aslo observed an age-dependent increase of miR-125 b in DKO mice compared to control.2.miR-125 b regulated expression and function of NCAMUsing available databases and bioinformatics methods we evaluated three potential target genes of miR-125 b.BDNF and ASIC1 a showed no difference on transcripts in PFC of DKO and control mice,yet NCAM mRNA was significantly decreased in 12 M DKO mice.Dual Luciferase Reporter Assay System was used to validate the suppression of miR-125 b to the 3’-UTR of NCAM in N2 a cells.Consistently,protein synthesis of NCAM was interfered in PC12 cells when exogenous miR-125 b was expressed steadily.Over-expressing miR-125 b in PC12 cells resulted in restricted differentiation due to the participation of NCAM in neurite outgrowth,performed as the limited length and extension of neurites.Moreover,up-regulating miR-125 b in PFC of C57B/6 mice decreased the density of dendritc spines.3.miR-125 b affected GSK3β activity and tau protein phosphorylation via regulating NCAM expressionThe relationship between NCAM and GSK3β was rarely investigated.In this study,we observed that miR-125 b could regulate GSK3β activity positively.The elevated activity might caused by the decreased phosphorylation of GSK3β on Ser9 site which was related with NCAM activity.In cell differential stage,NCAM synthesis was inhibited;phosphorylation of GSK3β on Ser9 site was decreased as well.Meanwhile,increased phosphorylation of tau protein was observed.During apoptosis procedures,high activity of GSK3β was observed in PC12 cells that expressed exogenous miR-125 b than in control cells,activity of caspase-3 and phosphorylated tau protein were increased as well.In vivo test showed similar elevated activity of GSK3β and phosphorylation of tau protein.Taken together,miR-125 b regulated expression of NCAM that resulted in altered synaptic structures and dendritc spine density.Moreover,inhibition of NCAM to GSK3β was also suppressed by miR-125 b,which would induce the incease of GSK3β activity and tau phosphorylation.These regulations might participate in the pathological process of dementia in DKO mice.These results provided some hints for investigation on pathology of dementia,including the formation of neuronal fibrillary tangles and neuronal apoptosis.