Mechanism Of MiR-34 Regulating Senescence Via Autophagy

MicroRNA (miRNA) are endogenously encoded single-stranded RNAs of about 22 nt in length that are highly conserved and regulate protein expression through interactions with the 3'untranslated region (UTR) of mRNA. The ability of miRNAs to regulate a variety of target genes allows them to induce changes in multiple pathways and processes such as development, senescence, immune system, cancer and other physiological contexts. The evidence for a regulatory role of the miR-34 family in senescence is growing. Recent studies have shown that the expression of miR-34 increases with aging. Overexpression of miR-34 in many cell lines leads to cell cycle arrest, increases the expression of senescence marker, SA-β-Gal (senescence-associatedβ-galactosidase). Autophagy, a highly conserved mechanism of quality control inside cells, plays a critical role in the regulation of aging and age-related degenerative diseases. Recent studies have revealed that the SIRT1 and p53 signaling pathways regulate lifespan via autophagy. miR-34s are direct transcriptional targets of p53. Furthermore, miR-34a can represses SIRT1 translation through a miR-34a-binding site within the 3'-UTR of SIRT1. It suggests that autophagy may be involved in the regulation of aging by miR-34a. However, the exact role of miR-34 in aging in vivo remain unclear. The aim of the present study is to identify the role of miR-34 in aging in vivo, and to examine the mechanisms of autophagy in regulating aging by miR-34, both in vivo and in vitro.Part I. The role of miR-34 in aging of C.elegans. Real-time RT-PCR was employed to detect the expression of miR-34 in rats and C.elegans. To address the role of miR-34 in the longevity response, we also observed the lifespan in miR-34 loss-off-function mutant alleles (gk437 and n4276) under ad libitum (AL) and intermittent fasting (IF). The results showed that the expression of miR-34a in heart, brain, liver, thoracic aorta, and kidney from 24-month-old rats, compared with the 3-month-old rats, increased by 2.36,1.71,1.85,3.45 and 2.14-fold (p< 0.05～0.01), respectively. The expression of miR-34 wild type N2 worms also increased during aging, and there is a significant positive correlation between the expression of miR-34 and aging (r=0.856,p<0.0001). miR-34 mutant alleles markedly delayed the age-related physiological decline, and increased resistance to heat and oxidative stress. In ad libitum, the lifespan of miR-34 mutant alleles were increased by 42%～62% compared to wild type N2 worms (p＜0.0001) Intermittent fasting significantly extended lifespan of wild type N2 worms (p<0.0001), but didn't influenced the lifespan of miR-34 mutant alleles.PartⅡ. The role of autophagy in regulation of the aging process by miR-34. To further evaluate whether autophagy is required for extended lifespan in miR-34 mutant alleles, we also tested the effects on the lifespan of RNAi of 5 autophagy genes in C. elegans. In vitro, we examined the role of miR-34a on the autophagy process. We found that RNAi knockdown of the C.elegans orthologs of yeast atg4.1, atg4.2, lggl, bec-1 or atg9 didn't influenced the lifespan in wild type N2 worms, but they significantly suppressed the lifespan extending effect of miR-34 mutant (p＜0.0001). In vitro, Hela and HEK293 cells were respectively transfected with miR-34a mimics or inhibitors, and the results showed that miR-34a affected autophagy, and the effects weren't entirely dependent on SIRT1.PartⅢ. miR-34a regulating aging by inhibition of autophagy related protein Atg4B and Atg9A. To examine the role of the miR-34a on autophagy related gene (Atg4B or Atg9A) expression, we used mimics and inhibitors of miR-34a. We also used dual luciferase reporter gene assay system to indentify the relationship between the miR-34a and 3'-UTR of Atg4B or Atg9A. Overexpression of miR-34a in Hela cells decreased Atg4B and Atg9A protein levels. By contrast, inhibition of miR-34a in HEK293 cells increased Atg4B and Atg9A protein expression. But, miR-34a had no effect on the expression of Atg4B and Atg9A mRNA. Therefore, miR34a regulated the expression of Atg4B and Atg9A at the post-transcriptional level. Dual luciferase reporter assay confirmed that the miR-34a binding sequences in the 3'-UTR of Atg4B and Atg9A contributed to the modulation of Atg4B and Atg9A expression by miR-34a.In summary, we found that miR-34 accelerates aging in C.elegans, and autophagy might play a important role in the process. In vitro, our found that miR-34a suppress autophagy by indirectly inhitited autophagy related protein Atg4B and Atg9A.