MiR-52 Extends Lifespan By Inhibiting Non-canonical Wnt Signaling Pathway Component VANG-1/Van Gogh In Caenorhabditis Elegans

Wnt signaling pathway,including canonical and non-canonical Wnt pathway,is highly conserved in various animals.Wnt signaling pathway regulates many biological events during embryonic development,including cell apoptosis,asymmetric cell division,cell migration and synaptogenesis.Recent evidence shows that Wnt pathway regulates not only embryonic development,but also lifespan and human aging-related diseases,such as diabetes and cancer.Caenorhabditis elegans(C.elegans)is a classic model for aging study,because of its short life cycle and highly conserved pathway of aging control.Previous studies have shown that mutation of Wnt signaling pathway genes extends or shortens the worm lifespan,indicating Wnt signaling pathway genes play key roles in aging regulation.In this study,we found that the expression levels of several Wnt pathway genes significantly decreased during aging process in C.elegans.However,the molecular factors that inhibit Wnt pathway gene expressions to affect aging remain unclear.Gene expression is well-known regulated in transcriptional,post-transcriptional,translational and post-translational ways.microRNA(miRNA)is a class of short-chain non-coding RNA,which inhibits gene expression by binding the 3'UTR region oftarget genes.Previous studies have reported that miRNAs are critical for longevity of mammals and C.elegans,as well as human aging-related diseases.Therefore,we propose that Wnt pathway genes are suppressed by microRNAs,which results in the change of lifespan in worms.Using “Targetscan” algorithm,miR-51 family,which a highly conserved withmiR-100 family in other organisms,was predicted as a candidate to target six components of Wnt pathway(egl-20,cwn-2,lin-44,mom-2,vang-1 and bar-1)that have been reported to regulate the longevity of C.elegans.There are six members in miR-51 family,including miR-51,miR-52,miR-53,miR-54,miR-55 and miR-56.Utilizing lifespan assays and rescue experiments,we found miR-52 significantly extended the lifespan.Moreover,the expression of miR-52 was observed gradually increased in aging process.Screening by luciferase reporter assays,VANG-1/Van Gogh,a component of non-canonical Wnt pathway,was identified as a direct target of miR-52.Then,Pvang-1:: gfp::vang-13'UTR plasmidwas constructed.We obtained a stable expression strain of the plasmid by CRISPR-Cas9 and microinjection.By comparing the fluorescence intensity of worms at L4,Young Adult,Day 1,Day 3,Day 7 and DAY 11 stages,we found that the expression level of VANG-1/Van Gogh decreased significantly after entering the adult stage,and decreased continuously during the aging process.Genetic analysis verified that miR-52 acted in the same pathway as VANG-1,and promoted longevity dependent on germline signaling.DAF-16/FOXO is akey transcriptional factor in germline signaling pathway.By analyzing the localization of DAF-16/FOXO in intestinal nucleus and the expression levels of its target genes,we found that DAF-16/FOXO activity was required for the function of miR-52 in promoting longevity via inhibition of VANG-1/Van Gogh.These data indicate that miR-52-mediatedrepression of VANG-1 extends the lifespan by DAF-16/FOXO activity in germline of C.elegans.These findings have key implications for exploring the role of miRNAs in repressing Wnt pathway gene expressions during post-developmental stage,which is crucial to control natural aging and human aging-related diseases.