MiR-34a Reduces Degradation Of A? By Suppressing Autolysosome Pathway

Alzheimer's Disease is one of the neurodegenerative diseases,which occurs in aged people mostly over 65-year old.Patients with AD generally show memory deficiency,language disability,cognitive difficulties,executive dysfunction,etc.The injured area focuses on cerebral cortex and hippocampus.The pathogenesis of AD is still unclear but two hypotheses are recognized,tau hyperphosphorylation and deposition of A?.Research suggested that A? was degradation by microglia via autophagy pathway.Autophagy is an important activity by which cells eliminate dysfunctional organelles or proteins in lysosome and recycle materials and energy to ensure cellular homeostasis and execution of various physiological process.Autophagy is involved in all kinds of functions such as cell proliferaton,differentiation,aging and metabolism,as well as various diseases such as cancer,atherosclerosis,neurodegenerative disease.miRNAs are small non-coding ribonucleic acid consisting of 22 to 24 nucleotides.miRNA is encoded by RNA polymerase III followed by cleavage under various enzymes into mature miRNA.miRNA takes post-transcriptional regulation of the particular gene by target binding the 3'untranslated region of mRNA in a complex containing RISC.miR-34 a is a famous tumor suppressor which promotes cell apoptosis and inhibits cell proliferation while conserved in mammalian.There were reports that miR-34 a is up-regulated in a double transgenic mouse model of Alzheimer's disease.In our research,we first veryfied the up-regulation of miR-34 a in hippocampus of our mice of AD model.We found miR-34 a inhibited degradation of A?42 in U251 with FACS.To study the relationship between miR-34 a and autophagy,we used Western Blotting to find out the increase of ratio between LC3-II and LC3-I as well as P62 in U251 both transfected with and stable expressing miR-34 a.Under confocal fluorescent microscopy we observed that with the effects of miR-34 a autophagosome was increased and so was autolysosome which indicated that miR-34 a could inhibit autophagy substrate degradation in autolysosome.With the help of bioinformatics softwares we found two potential target genes,lysosome enzymes Cathepsin B and Cathepsin D.Results of Q-PCR and Western Blotting showed the decrease of Cathepsin B and Cathepsin D in U251 transfected with miR-34 a,which suggested the confirmation of the lysosome enzymes as targets of miR-34 a.We also demonstrated the target sequence of miR-34 a in mRNA of the two lysosome enzymes by constructing the dual-luciferase reporters and the mutant ones containing the target sequence in 3'UTR.Consequently,miR-34 a reduced degradation of A?42 by inhibited the lysosome enzymes Cathepsin B and Cathepsin D to suppress autolysosome pathway.