Investigation On Involvement Of Mir-51 In The Short-term Forgetting Via Regulating L-AChR In Caenorhabditis Elegans

In nature,animals retain useful environmental information to form short-term and/or long-term memory,which is considered a necessary condition for their survival;however,once the environmental stimulus changes and is inconsistent with the existing memory in the brain,the organism initiates forgetting,to prevent the interference of obsolete or irrelevant memory.Recent studies have found that forgetting can be activated by the learning process,and these two processes have similar signal transduction pathways,indicating that forgetting is an active process that occurs in organisms.Therefore,the molecular mechanism of forgetting has become increasingly concern by researchers.The functional genes and signaling pathways of Caenorhabditis elegans are homologous to higher mammals.Its nervous system has a simple structure and clear circuits.At the same time,many neurotransmitters and neuronal regulators such as acetylcholine,glutamate,dopamine,Serotonin,γ-aminobutyric acid and neuropeptides also have a high degree of homology with mammals.In recent years,C.elegans has become an important model for studying the complex neurobiological behaviors such as memory and forgetting.Micro RNAs(mi RNAs)are endogenouly expressed non-coding RNAs with the size of about 18～26 nucleotides.They have highly conservative and regulatory functions and widely found in eukaryotes.They participate in a variety of biological processes by promote the degradation of their target gene m RNA or inhibit the expression of its translation regulation target gene.Current studies have found that mi RNAs are highly expressed in the nervous system,especially in the brain,which affect learning and memory processes by regulating synaptic plasticity.However,the research about mi RNAs how regulate forgetting is very lacking.In addition,approximately 50% of the mi RNAs in C.elegans also share sequence homology with human mi RNAs.Therefore,using the C.elegans forgetting model,we discovered the mir-51 is the target mi RNA of egl-9,which effect the location and function of L-acetylcholine receptor(L-ACh R)on the synaptic though regulate LEV-10,and participates in the molecular mechanism of forgetting.This article mainly obtains the following research results:1.Using the short-term forgetting model of butanone and food associative memory,the synapse-related genes are screened by RNAi.We found that compared with wild-type nematode N2,lev-9 RNAi has a longer memory retention time for butanone,and LEV-9,LEV-10 and L-ACh R participate short-term forgetting process in the form of functional complexes in C.elegans.2.Bioinformatics analysis shows that EGL-9 and LEV-10 have potential interactions.Through behavioral experiment of egl-9 RNAi,egl-9 overexpression and egl-9 RNAi;lev-10(ok2111),we found that EGL-9 negatively regulates LEV-10 in the short-term forgetting.In addition,by comparing the changes in the transcription and post-transcriptional levels of egl-9 during the forgetting process,it is suggested that egl-9 participates the short-term forgetting process that occurs at the post-transcriptional level.3.Sequencing of the mi RNA transcriptome indicated 10 up-regulated mir RNAs and 5 down-regulated mi RNAs were involved in forgetting occurance.Bioinformatics analysis showed that the up-regulated mir-51 and mir-55 have the complementary pairing sites with the 3’ UTR region of egl-9.Phenotypic experiments suggest that both mir-51 and mir-55 can delay short-term forgetting;but pharmacological studies on levamisole sensitivity have shown that mir-51 can affect the function of L-ACh R and participate in the delay of short-term forgetting.Further the dual luciferase reporter system and vivo experiments in C.elegans proved that egl-9 is the target gene of mir-51.Finally,the phenotypic study of the double mutant nematode mir-51(n4473);lev-10(ok2111)and mir-51(n4473);egl-9RNAi,suggested that mir-51,egl-9and lev-10 may be in one pathway.4.In order to identify the transcription factors that regulate the expression of mir-51,the Transmi R v2.0 database was used to predict the transcription factors that regulate the expression of mir-51.Using the mir-51p::gfp reporter for experimental screening,it has been found that a few genes encoding the transcriptional factor,such as ceh-28,skn-1,daf-16 and hpl-2 are involved in regulating mir-51.But further behavioral experiments show that ceh-28 accelerates the process of short-term forgetting.The main innovations of this paper are as follows:1.In the short-term forgetting model of butanone and food associative memory in C.elegans,the LEV-9/LEV-10/L-ACh R functional complex regulates short-term forgetting.2.It is found that mir-51 targets egl-9 and then negatively regulates lev-10,which ultimately affects the signaling pathway of L-ACh R to delay forgetting.