The Mechanism Of Linc-133 Gain Of Function Mutation Regulating Survival Of Daf-18/PTEN Mutant Caenorhabditis Elegans

BackgroundPTEN is a tumor suppressor with double specific phosphatase activity.PTEN plays an important role in cell growth,apoptosis,adhesion,migration and infiltration.Caenorhabditis elegans(C.elegans)is a model organism with advantages in the research of basic medicine,genetics,cell biology and other fields.DAF-18 of C.elegans is highly homologous to the functional domains and active sites of human PTEN.It is found that human PTEN can completely replace DAF-18 in function and the missense mutation PTEN(Y138F),a protein phosphatase deficiency,is similar to the DAF-18(D137A)mutation in C.elegans.The life cycle of the worm changes from egg hatching to the first stage(L1)larval stage,where L1 larvae hatch without food and the subsequent developmental process stops,which is known as "L1 arrest".After daf-18/PTEN mutation,the survival rate of worms at L1 arrest stage was shortened sharply,and the life span of adult worms at L1 arrest stage was not as significant as that at L1 arrest stage.The phenotypic advantages of daf-18/PTEN mutant worms at L1 arrest stage were convenient for research and beneficial for rapid and accurate screening of new gene targets by forward genetics.daf-18/PTEN can control worm survival and postembryonic cell development during L1 arrest.Multiple gene targets and signaling pathways were found to be involved in the regulation of cell development.However,these known targets could not completely restore the survival of daf-18/PTEN mutant worm.This suggests that the mechanism of daf-18/PTEN regulating cell growth and individual survival may be independent of each other to a certain extent.To explore the mechanism of daf-18/PTEN regulating the survival of nematodes independent of cell proliferation at L1 arrest stage and to search for new targets,which may provide inspiration for improving the survival of human PTEN-related cancer patients.PurposeTo explore the mechanism of restoring survival time of daf-18 mutant nematode during L1 arrest.Method1.To explore whether the reported signal pathways regulating cell division can restore the survival time of daf-18 mutant nematodes by constructing double mutants and genetic epistasis detection.2.Overexpression of DAF-18 lipid phosphatase defective plasmid,DAF-18 protein phosphatase defective plasmid and DAF-18 diphosphatase defective plasmid were used to investigate whether the function of diphosphatase affected the survival of daf-18 mutant worm3.EMS mutagenesis and forward genetics were used to screen mutants,and sibling subtraction and whole genome sequencing were used to identify the specific information of SNP and In Del mutants.4.RNAi,microinjection transgenic technology,CRISPR/Cas9,construction of double mutants and other methods were used to identify the function of linc-133 mutation sites.Location of linc-133 RNA by single molecule fluorescence in situ hybridization.5.Transcriptome sequencing was used to evaluate the variation of differential gene expression in linc-133 gain of function mutant.KEGG is enriched into related signaling pathways.6.Survival detection,DAF-16 nuclear translocation detection,q RT-PCR and sod-3::GFP expression were used to detect whether linc-133 gain of function mutant activated transcription factor DAF-16.7.RNA pull down,RIP and Western bolt experiments were conducted to find the protein that binds to linc-133 gain of function mutant in daf-18 mutant worm,and to verify its function in regulating DAF-16.8.q RT-PCR,RNAi gene silencing,survival test and genetic epistasis of mutant were used to detect the expression of HSF-1-related heat shock protein regulated by linc-133 gain of function mutant.To explore the mechanism of linc-133 gain of function mutant and DAF18 protein phosphatase activity regulating protein homeostasis in daf-18 mutant worm.9.Western bolt ubiquitination protein detection and unfolded protein response demonstrated the role of linc-133 gain of function mutant in regulating the ubiquitination degradation pathway of unfolded proteins in endoplasmic reticulum in maintaining daf-18 mutant and DAF-18 protein phosphatase-deficient wormResult1.The genes or signaling pathways that regulate cell division in daf-18 mutant worm are not related to survival.2.The functions of DAF-18 lipid phosphatase and protein phosphatase are necessary to maintain the survival of worm during L1 arrest,and DAF-18 protein phosphatase may play a key role in the maintenance of survival.3.After the mutation sites were screened by EMS mutagenesis,it was proved that linc-133 mutation was a gain of function mutation,and linc-133 gain of function mutant restored the survival of daf-18 mutant worm during L1 arrest and sm FISH confirmed that linc-133 was cell-specific in AVK cells,and gain of function linc-133 played a role in cell non-autonomous regulation.4.The linc-133 gain of function mutant regulates L1 arrest survival by activating the DAF-16 transcription factor.Gain of function linc-133 competitively binds to 14-3-3 protein to block DAF-16 from exiting the nucleus in daf-18 worms,thereby regulating survival during L1 arrest through DAF-16.5.The linc-133 gain of function mutant prolongs the survival of daf-18 mutant worm during L1 arrest by degrading protein homeostasis through ubiquitination of endoplasmic reticulum unfolded proteins mediated by HSF-1 and P97.ConclusionBoth DAF-18 diphosphatase functions were involved in regulating the survival of worm during L1 arrest.linc-133 gain of function mutant to regulate the survival of daf-18 mutant worm by regulating the function of DAF-16 and activating the ubiquitination degradation pathway of unfolded proteins in the endoplasmic reticulum involving the molecular chaperones associated with HSF-1.