<i>MIR376A</i> Is a Regulator of Starvation-Induced Autophagy

<div><p>Background</p><p>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. Hence, mechanisms of autophagy regulation are under exploration.</p><p>Methods</p><p>Over-expression of hsa-miR-376a1 (shortly <i>MIR376A</i>) 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 <i>MIR376A</i> and the related <i>MIR376B</i> was compared using TaqMan quantitative PCR.</p><p>Results</p><p>Here, we demonstrated that, a microRNA (miRNA) from the <i>DLK1/GTL2</i> gene cluster, <i>MIR376A</i>, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by <i>MIR376A</i> overexpression in MCF-7 and Huh7 cells. <i>MIR376A</i> shared the same seed sequence and had overlapping targets with <i>MIR376B</i>, 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 <i>MIR376A</i> in luciferase assays. Antagomir tests showed that, endogenous <i>MIR376A</i> was participating to the control of <i>ATG4C</i> and <i>BECN1</i> transcript and protein levels. Moreover, blockage of endogenous <i>MIR376A</i> accelerated starvation-induced autophagic activity. Interestingly, <i>MIR376A</i> and <i>MIR376B</i> 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.</p><p>Conclusions</p><p>Our findings underline the importance of miRNAs encoded by the <i>DLK1/GTL2</i> gene cluster in stress-response control mechanisms, and introduce <i>MIR376A</i> as a new regulator of autophagy.</p>