FAT10 Gene Promoting Aging Effect

BackgroundFAT10 gene is one member of ubiquitin protein family.It is highly expressed in the thymus,spleen,dendritic cells,lymph nodes and other tissues related to to immune system functions.Abnormal FAT10 expression affects cell cycle regulation,chromosome instability,DNA damage repair etc.But few studies have been done on the effect of FAT10 on aging.ObjectiveThis research is to study the effect of FAT10 on aging in vivo and in vitro,and to explore the possible mechanism of FAT10 affecting aging.MethodIn vitro experiments,we choosed CCN1 gene as a positive control.we constructed plasmids through gene amplification,vector extraction,restriction endonuclease digestion and connectivity of vectors and DNA fragments,transformation.Then,lentiviral vector was used to transfer the target genes into muscle fibroblasts.After that,?-galactosidase activity was used as a marker of senescence to study the effect of FAT10 overexpression on senescence.In addition,FAT10 gene was packaged into adeno-associated virus(AAV)and injected into C57BL/6J via tail vein.The serum levels of inflammatory factors IL-6,TNF-?,MCP-1,iNOS,IL-1 and IL-10 were detected by enzyme-linked immunosorbent assay.Result(1)FAT10 and CCN1 gene fragments were amplified by PCR and the products were examined on agarose gel.The results of gene sequencing showed that the inserted fragments in the recombinant plasmid were identical to FAT10 and CCN1.(2)Fluorescence could be observed after the infection of lentiviruses to imuscle cells and the flow cytometry analysis demonstrated that we successfully obtain lenti-virus/FAT10(LV-FAT10)and lenti-virus/CCN1(LV-CCN1),whose titer were3.85×105 p.f.u and 3.08×105 p.f.u,respectively.(3)SA-?-gal staining results showed that FAT10 could promote cell senescence,and was more likely to promote the senescence of C2Cl2 cells than CCN1.(4)The results of in vivo experiments showed that the overexpression of AAV9-FAT10 gene could accelerate the appearance of the senescent phenotype in C57BL/6J mice,such as bald,hair removal,white hair.(5)Overexpression of FAT10 could promote weakness.The frailty index of mice showed an increasing trend with age.twelve weeks of after tail vein injection,the score of the frailty index in AAV9-FAT10 group was higher than the PBS group.At the eighth week and twelfth week,the motor coordination of AAV9-eGFP group was higher than AAV9-FAT10 in C57BL/6J mice.(6)Overexpression of FAT10 can affect the metabolism of C57BL/6J mice.The weight of the experimental group was higher than that of AAV9-eGFP group at the second and twelfth week after tail vein injection.Glucose tolerance test showed that FAT10 could reduce glucose tolerance,and the area under the roc curve of glucose tolerance test was lower than that of group AAV9-eGFP.(7)At the twelfth week after injection,the study on serum inflammatory factors showed that AAV9-FAT10 could increase IL-6,TNF-alpha,MCP-1 and reduced the level of iNOS in serum.ConclusionPlasmids with FAT10 gene and CCN1 gene were successfully cloned and were used to generate LV-FAT10 and LV-CCN1.lenti-virus/ FAT10 infected C2Cl2 cells and promoted C2Cl2 cell senescence.Overexpression of FAT10 gene in vivo could promote the aging phenotype,such as bald,hair removal,white hair.At the same time it increased body weight,decreased forelimb grip,motor coordination and blood glucose tolerance test,and promote frailty.In addition,we found that FAT10 could increase the level of serum inflammation.So it was proposed that FAT10 could promote aging through regulating the levels of inflammatory cytokines in C57BL/6J mice.