Heterochronic Genes Regulate The Lifespan Of Caenorhabditis Elegans And The Expression In The Development Of Mouse

Heterochronic genes control the temporal and spatial pattern of entire development process and specify the cell fate to ensure accurate position of the various organs and developmental time.In heterochronic genes mutation, stage-specific developmental events occur at earlier or later stages than they would normally.MicroRNAs and other heterochronic genes play key roles in regulating cell proliferation,cell development and apoptosis,as well as in controlling lifespan,tumor genesis and other diseases.In this paper,we used RNAi,lifespan analysis,western blot,immunoprecipitation and RT-PCR to study heterochronic genes in regulating C.elegans lifespan,and the expression pattern of the homologus genes lin-28 and lin-41 in vertebrate.First,the lifespan and temperature tolerance of different heterochronic genes mutantions were analysed and found that the lin-28 mutant lived longer than N2 wild type.The mutants of let-7 and lin-41 lived shorter than N2 wild type.There was no significant difference in the lifespan of lin-29 mutant compared with N2 wild type.The lifespan of daf-2(-) and daf-16(-) were analysed after lin-28 RNAi.The results showed that the lifespan of daf-2,lin-28 double mutant lived longer than a single mutant of daf-2,and there was no significant difference in daf-16(-),lin-28(-) double mutant compared with daf-16 single mutant,also the nuclear localization of DAF-16::GFP did not change significantly after lin-28 RNA interference.The expression of LIN-28 in short and long lifespan of worms was detected by western blot.The data showed that long lifespan of worms,including daf-2 mutant, the expression level of LIN-28 was low.In short lifespan of worms,including daf-16 mutant,expression level of LIN-28 was higher.In addition,Co-IP and RNAi were used to detect the interaction between daf-16 and lin-28.The results implied that lin-28 may perform a new function in regulating the lifespan of C.elegans by affecting daf-16.LIN-28 and DAF-16 could interact with each other.Over-expression of LIN-28 may inhibit the expression of DAF-16,and LIN-28 had abundance expression in daf-16(-) mutant.Immunoprecipitation and MALDI-TOF-MS were used to identify the proteins that interact with LIN-28.The results showed that the proteins may interact with LIN-28,including T22H9.3,a translation initiation factor 2C(eIF-2C),P granule protein PGL-2,hypothetical protein R13A1.10 and Aff_ZK484.8.The interaction between DAF-16 and LIN-14 which related to the lifespan regulation through the insulin/IGF-1 pathway was detected in the experiments.The study showed that in the background of daf-2 mutation,if lin-14 was loss of function, the expression of DAF-16 was increased;using immunoprecipitation and western blot, we found lin-14 could interact with daf-16;four proteins were identified which may interacted with LIN-14,including Y55-F3C.9,R12E2.1,C50C3.7 and mitochondrial ATP-inhibitor.In vertebrates,mouse lin-41 and lin-28 partial cDNA fragments were successfully amplified from the mouse embryonic day 9(E9.0) mRNA by RT-PCR. lin-28 and lin-41 cDNA were cloned into expression vector PET-32M for prokaryotic expression.The expression of LIN-41 and LIN-28 in mouse tissues and embryos from day 9 to 13 were detected by using RT-PCR and western blot.RT-PCR results showed that:lin-41mRNA was firstly detected in the embryo of day 9,then in day 10.5 and day 12 respectively,lin-28mRNA was firstly detected in the embryo of day 9 and day 10.5.Western blot results showed that the specific expression of LIN-41 were in the mouse heart,muscle,small intestine and day 9 to 12 embryo.LIN-28 was detected in the embryo of day 9,10.5 and 12,also in the adult mouse heart,kidney, liver,muscle,small intestine and brain.In addition,more abundant expression of LIN-28 was detected in mouse heart and small intestine than in other tissues.Finally, Co-Immunoprecipitation combine MALDI-TOF-MASS was used to identify the potential proteins interacting with LIN-41,the results showed that several proteins may interact with LIN-41 in mouse,including leucine zipper transcription factor, epidermal growth factor receptor,hemagglutinin-4(Galectin-4),mitochondria and Ras-related protein Rab- 13.