The Sir-2.1 Gene RNA Interference In Carenorhabditis Elegans And The Preliminary Study On The Molecular Mechanisms Of DhHP-6 On Extending Life Span In Caenorhabditis Elegans

Peroxidases are an important class of iron porphyrin-containing enzymes, they catalyze substrate oxidations using hydrogen peroxide, but the therapeutical potential of natural peroxidases is limited by their scarce solubility, instability, cost and other factors. In our previous work, we designed a novel peroxidase mimics Deuterohaemin- Ala-His-Thr-Val-Glu-Lys (DhHP-6) and investigated its peroxidase activity and anti-cataract activity in vitro. The peroxidase activity of DhHP-6 is very similar to that of MP-11, but DhHP-6 has very good water solubility, stability and its chemical synthesis is much more simple.In the present study, we used the model organism Caenorhabditis elegans (C.elegans) to evaluate the pharmacological effect of DhHP-6 on aging. C. elegans is a popular model for studying aging and longevity due to its short lifespan and the well defined genetics and environmental factors affecting lifespan. Treatment of wild-type C. elegans by DhHP-6 increased its lifespan and delayed the age-related decline of fast body movement in our laboratory culture condition at 20℃. DhHP-6 treatment could also increase the expression of sir-2.1,ctl-1,ctl-2 and sod-3. The results indicate that DhHP-6 had direct or indirect effects on these gene expressions.Sir2 gene family is a conservative NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases of ancient bacteria to mammals. The C. elegans homolog gene sir-2.1 plays important roles in multiple cellular processes including aging, apoptosis and stress response. It has also increasingly been widespread concerned in recent years.RNA interference technology can separate special gene silence in Caenorhabditis elegans in vivo and in vitrowhich result in function lose or reduce the mutation. The phenotypes of inhibition of the gene are similar to that by direct deletion of the same gene. RNA interference technology is a good means of research gene function.Here we use the nematode Caenorhabditis elegans as a model system to investigate the function of the sir-2.1 gene.Based on the technique of double-stranded RNA interference , we established the model in C.elegans by controlling the expression of sir-2.1 gene. We observed the change of mRNA expression of sir-2.1 in C.elegans after RNA interference and the effect of gene silence. To further determine the relationship between the sir-2.1 gene RNA interference in Carenorhabditis elegans and the effect of DhHP-6 on extending life span, we tested the effect of DhHP-6 upon longevity and aging using RNA interference type C. elegans.Based on the technique of gene engineering technology, we successfully established the L4440- sir-2.1 vector and E.coli HT115(DE3),that can produced dsRNA by IPTG induced. We explored feeding as a means of delivering dsRNA for RNAi. We observed the change of expression of sir-2.1 in C.elegans after RNA interference and the effect of gene silence and tested the effect of 100μM DhHP-6 upon longevity and aging using wildness type C. elegans, sir-2.1 RNA interference type C. elegans and sir-2.1 (ok434) null mutation strains of C.elegans.Then,we gained some results and conclutions:We examined the effect of DhHP-6 on some aging-related genes. DhHP-6 treatment had no effect on the expression of pha-4 and ftt-2. It blocked the increase of the expression of the hsp16.1, which suggests that DhHP-6 has the anti- oxidative damage activity in vivo and this activity may promote the life span of C.elegans. It could also increase the expression of ctl-1,cep-1 and sir-2.1 on the 8th day. The result indicates that DhHP-6 had direct or indirect effects on these gene expressions.In this paper,we choose sir-2.1 as the target gene to preliminary study on the molecular mechanisms of DhHP-6 on extending life span in Caenorhabditis elegans.In this assay, we survey sir-2.1 mRNA expression in 100μM IPTG concentration. Empty vector group has no effect on the mRNA expression of sir-2.1. However, without IPTG group C. elegans sir-2.1 mRNA decreased 95% compare with the wildness type. The result proves that the links between the effect of IPTG concentration and the interference are not that necessary, C. elegans in the course of their metabolism may product IPTG or its analogues.In RNA interference type C. elegans sir-2.1 mRNA decreased 96% compare with the wildness type. The result also confirms the conclusion that IPTG induce dsRNA expression is successful.Though C. elegans life span determination experiments, we found that treatment with 100μM DhHP-6 can extend the lifespan of wildness type C. elegans,but could not prolong lifespan of sir-2.1 RNA interference type C. elegans and sir-2.1 (ok434) null mutation strains of C.elegans.Prove that the effect of DhHP-6 on extending life span needs the existence of sir-2.1 gene. One possible explanation of this founding is that the effects of DhHP-6 treatment are mediated, at least in part, through sir-2.1 gene.Though all the experiments, this study successfully established the model of sir-2.1 RNA interference type C. elegans and proved that the effect of DhHP-6 on extending life span needs the existence of sir-2.1 gene. To further determine the relationship between the sir-2.1 gene RNA interference in Carenorhabditis elegans and the effect of DhHP-6 on extending life span, the SIR-2.1 protein and sir -2.1 gene-related signaling pathway in C. elegans under the treatment of DhHP-6 need to be futher studied.In conclusion, we believe that RNAi C.elegans could be used as a model system to investigate the effect and the aging mechanism of anti-oxidative drug. Data presented in this paper have provided a framework and a basis for further investigation towards the molecular mechanisms of the effect of DhHP-6 upon aging and longevity.