| BackgroundAging is a deleterious and complex process characterized by the progressive decline of cellular and organismal homeostasis and represents the primary risk factor in major human chronic pathologies,including diabetes,cardiovascular disorders,and neurodegenerative diseases.Therefore,researches on the delay of the process of aging and elimination of the potential risk factors for the age-related diseases seem to be urgently required.The short life cycle and tractable genetics of C.elegans made this animal model as an attractive subject which are widely referred to investigate the mechanisms of regulations in aging and lifespan.C.elegans dve-1 is an ortholog of human satb1/sotb2,which is mainly involved in Colorectal Cancer(CRC).satb1/dve-1 was also reported to regulate the lifespan by dietary restriction.Howerver,the mechanism remains further uncovered.Previously studied on dve-1 regulation in lifespan was based on RNAi,which was predicted that the mitochondrial UPR is correlated with dve-1-mediated longevity in C.elegans.However,We found that dve-1(tm4803)mutant shows decreased lifespan and it has nothing to do with the mitochondrial UPR.In the present study,we intend to further explore how dve-1 regulates lifespan and aging using the dve-1(tm4803)mutant,and the involvement of UPRmt,the classic insulin/IGF-1 signaling pathway and peroxisome dysfunction is mainly discussed.Our findings provide a new direction for explaining the function of dve-1 in regulating lifespan.ObjectiveIn this work,we are trying to uncover the mechanism of dve-1 in lifespan by using the C.elegans tm4803 mutant.Methods1.Analyze the phenotypes of dve-1(tm4803):to collect the data of the lifespan,average egg laying amount,reproductive cycle and and length of N2wild type and dve-1(tm4803).2.Test the stress tolerance of dve-1 during its lifespan controlling process:to select adult elegans of N2 wild type and dve-1(tm4803)on Dayl and Day 4 to have heat-shock experiment at 35℃ for 10h.The amount of N2 wild type and dve-1(tm4803)mutants survived at different time were collected and compared.3.Analyze the reason why the lifespan of mitochondria UPR and dve-1(tm4803)are shorten:to hybridize two genetically modified moleculars of UPRm1 marked with hsp-6p::gfp and hsp-60p::gfp with dve-1(tm4803),then to deal with their progenies with spg-7 RNAi.Finally,to reveal the relationship between mitochondria UPR and dve-1(tm4803)with fluorescence result.4.The genetic relationship between dve-1 and the main genes of Insulin/IGF-1 classical regulatory pathway was revealed.daf-2(e1370),daf-18(ok480)and daf-16(mu86)were selected to construct double mutants with dve-1(tm4803)for genetic analysis.These constructed double mutants were compared with their single mutants,as well as N2 wild type and dve-1(tm4803)mutants,in order to analyze the genetic relationship of these genes with the mechanism of dve-1 regulating lifespan.5.The regulatory relationship between dve-1 and the important transcription factor DAF-16 was analysis.The strains of daf-16::gfp and dve-1(tm4803);daf-16::gfp on the Day 1 and Day 4 were selected and divided into two groups for cultivation at 20℃ and heat-shock experiment at 35℃.The fluorescence localization of DAF-16::GFP was observed after culture(cultivation)at room temperature and heat-shock experiment,and the ratio of DAF-16::GFP entering N2 wild type and dve-1(tm4803)mutants under normal conditions and heat-shock conditions.6.Identify the regulatory relationship between dve-1 and daf-2 in dauer formation:Culture the eggs of daf-2(e1370),dve-1(tm4803),daf-2(e1370);dve-1(tm4803)at different temperatures(15℃,20℃,25℃)for 48h,and calculate the ratio of their entering dauer.At the same time,select daf-16 to treat the above three mutants with RNAi at 20℃ to observe whether the regulatory relationship between dve-1 and daf-2 in dauer formation depends on daf-16.7.Detect the role of peroxisome function in dve-1-mediated life span regulation:Hybridize molecular markers of PTS1 peroxisome with GFP::PTS1 into dve-1(tm4803)mutant.Observe the changes of fluorescence volume on Day 1,Day 4 and Day 7 after L4,and count the fluorescence expression volume and compare their differences.Results1.The lifespan and egg laying amount results indicated that the average lifespan of dve-1(tm4803)mutants was shorter than that of N2 wild type;the average egg laying amount of dve-1(tm4803)was lower than that of N2 wild type.The length measurement results indicated that the length of dve-1(tm4803)was slightly longer than that of N2.2.The number of surviving dve-1(tm4803)mutant C.elegans on the 1st and 4th day following heat stress at 35℃ for 10 h was lower compared with wild-type N2 strains,which was much more pronounced on the 4th day.3.spg-7 RNAi results indicated that the fluorescence intensity of the control group was significantly enhanced after treatment,while the fluorescence intensity of the dve-1(tm4803)group was very close to that of the control group.4.The genetic analysis results indicated that the neither the lifespan nor the egg-laying amount of the double mutant constructed by dve-1(tm4803)with the mutants of the three different genes of insulin/IGFP-1 was consistent with the phenotype of any of the three mutants,suggesting that the way that dve-1 regulates lifespan is parallel to the IIS signaling pathway.5.DAF-16::GFP was not imported into nuclei in daf-16::gfp and dve-1(tm4803);daf-16::gfp strains,but stimulated by heat stress at 35℃.6.Eggs of daf-2(e1370),dve-1(tm4803),daf-2(e1370);dve-1(tm4803)double mutant C.elegans were cultured at 25℃ for 48 h were stimulated to dauer formation.The rate of dauer formation of daf-2(e1370)at 20℃ was significantly higher than that of daf 2(e1370);dve-1(tm4803).Dauer formation of dve-1(tm4803)mutant strains failed under the three different temperatures,neither as process by daf-16 RNAi at 20℃.7.Fluorescence signal of GFP::PTS1 in dve-1(tm4803)mutant C.elegans on the 1st,4th and 7th day of L4 larval stages was higher than that of wild-type N2 strains,which was the most pronounced on the 4th day.Conclusion:1.dve-1(tm4803)mutant shows decreased lifespan,and its survival percentage also displays lower than that in wild type at the same time point after heat shock.2.The mechanism of dve-1 in the regulation of lifepan may be not related to the mitochondrial UPR.3.dve-1 may function in parallel with the Insulin/IGF-1 signaling pathway,and it did not affect the expression and localization of DAF-16::GFP.4.when it was at 20℃,the dauer formation of daf-2(e1370)became lower because of the loss of dve-1.5.The decreased lifespan of dve-1(tm4803)mutant might be related to the abnormal function of peroxisomes. |
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