Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
, 35 (1), 11-22

MiR-34 Modulates Caenorhabditis Elegans Lifespan via Repressing the Autophagy Gene atg9

Affiliations
Comparative Study

MiR-34 Modulates Caenorhabditis Elegans Lifespan via Repressing the Autophagy Gene atg9

Jurong Yang et al. Age (Dordr).

Abstract

Evidence for a regulatory role of the miR-34 family in senescence is growing. However, the exact role of miR-34 in aging in vivo remains unclear. Here, we report that a mir-34 loss-of-function mutation in Caenorhabditis elegans markedly delays the age-related physiological decline, extends lifespan, and increases resistance to heat and oxidative stress. We also found that RNAi against autophagy-related genes, atg4, bec-1, or atg9, significantly reversed the lifespan-extending effect of the mir-34 mutants. Furthermore, miR-34a inhibits Atg9A expression at the post-transcriptional level in vitro, and the miR-34a binding sequences in the 3'-UTR of Atg9A contributes to the modulation of Atg9A expression by miR-34a. Our results demonstrate that the C. elegans mir-34 mutation extends lifespan by enhancing autophagic flux in C. elegans, and that miR-34 represses autophagy by directly inhibiting the expression of the autophagy-related proteins Atg9 in mammalian cells.

Figures

Fig. 1
Fig. 1
miR-34 expression increases with age in vivo. a Relative expression of miR-34a in heart, lung, brain, liver, thoracic aorta, and kidney from 3 and 24-month-old rats. Each estimate represents the mean of 3–5 quantitative RT-PCR reactions on independent RNA samples derived from five rats. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01. b The relative expression of miR-34 in wild-type N2 worms. Results presented are the average values obtained from three replicates. *P < 0.05, **P < 0.01 vs. 1 day adult worms. #P < 0.05 vs. 7-day adult worms
Fig. 2
Fig. 2
The mir-34 loss-of-function extends C. elegans lifespan. Survival curves of wild-type N2 and mir-34 mutant worms. Similar results were obtained in three identical experiments (the details are shown in Table S1)
Fig. 3
Fig. 3
Autophagy mediates mir-34 mutation-induced longevity in C. elegans. (ac) Effect of ad libitum (AL) and intermittent fasting (IF) on lifespan in N2 (a), mir-34(gk437) (b), and mir-34(n4276) (c) worms. Similar results were obtained in three identical experiments (the details are shown in Table S2). d, e Survival curves of wild-type (N2), mir-34(gk437), and mir-34(n4276) mutant animal populations subjected to heat (d) and oxidative stress (e). Similar results were obtained in three identical experiments (the details are shown in Table S3). fh Effect of N2 (f), mir-34(gk437) (g), and mir-34(n4276) (h) on lifespan in worms subjected to atg4.1, bec-1, or atg9 RNAi during adulthood. Similar results were obtained in three identical experiments (the details are shown in Table S4)
Fig. 4
Fig. 4
miR-34 inhibits autophagy in vitro. a Hela and HEK293 cells transfected with miR-34a mimics and inhibitors, respectively. SIRT1 protein expression level was detected by immunoblot 24 h later. b Hela cells were transfected with miR-34a mimics or miRNA control for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64d (both 10 mg/ml; Pep.A/E64d) or DMSO as a control, followed by immunoblot analysis of the LC3 proteins. c Diagram shows the autophagic flux of Hela cells with different miR-34a levels as described in b. Autophagic flux was determined by the strength of LC3-II accumulation in a 2-h treatment period with Pep.A/E64d. Therefore, normalized LC3-II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64d. Values are expressed as mean ± SEM. *P < 0.01 vs. mock, n = 3. d HEK293 cells were transfected with miR-34a inhibitors or siRNA control for 48 h, and the cells were treated as in b, followed by immunoblot analysis of the LC3 proteins. e Diagram shows the autophagic flux of HEK293 cells with different miR-34a levels as described in d. Values are expressed as mean ± SEM. *P < 0.01 vs. mock, n = 3
Fig. 5
Fig. 5
Atg9A is a direct target of miR-34a. a Hela and HEK293 cells transfected with miR-34a mimics and inhibitors, respectively. Atg9A proteins expression level was detected by immunoblot 48 h later. b Quantitative RT-PCR assays for Atg9A. Expression values were normalized to the GAPDH housekeeping gene, and expression in untreated cells was set to 1. Values shown represent means ± SEM of three independent experiments. c Reporter plasmids in which the luciferase coding sequence was fused to the 3′-UTR of Atg9A were transfected into Hela cells in conjunction with miR-34a mimics or HEK293 cells in conjunction with miR-34a inhibitors. Renilla luciferase activity was normalized to firefly luciferase activity. Results shown are the mean ± SEM of triplicate determinations from one of three identical experiments. *P < 0.01

Similar articles

See all similar articles

Cited by 53 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback