Abstract
BackgroundAutophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration.MethodsOver-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3′ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR.ResultsHere, we demonstrated that, a microRNA (miRNA) from the DLK1/GTL2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3′ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role.ConclusionsOur findings underline the importance of miRNAs encoded by the DLK1/GTL2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy.
Highlights
IntroductionNamely macroautophagy (autophagy ) and the ubiquitin-proteasome system, are operational in the maintenance of cellular homeostasis
Two major degradation pathways, namely macroautophagy and the ubiquitin-proteasome system, are operational in the maintenance of cellular homeostasis
Similar to the results obtained in MCF-7 cells, MIR376A overexpression could block starvation-induced GFP-LC3 dot formation (Figure 3A and B) and LC3 lipidation (Figure 3C) in Huh7 cells as well. All these results showed that, MIR376A, another gene encoded by the miRNA gene cluster in the DLK1/GTL2 genomic region, was a new miRNA regulator of starvation-activated autophagy
Summary
Namely macroautophagy (autophagy ) and the ubiquitin-proteasome system, are operational in the maintenance of cellular homeostasis. BECN1 was discovered as a master regulator of the VPS34 activity and autophagosome formation.[2] Autophagic vesicle membrane elongation, growth and closure occur through the action of two ubiqituination-like protein conjugation systems.[5] The first system is rather regulatory, resulting in the covalent conjugation of a ubiquitinlike protein ATG12 to ATG5, and in the eventual formation of a larger complex including the ATG16 protein. Autophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases.
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