Knockdown of Long Non-Coding RNA GAS5 Increases miR-23a by Targeting ATG3 Involved in Autophagy and Cell Viability

Autophagy is a process of evolutionarily conservative degradation, which could maintain cellular homeostasis and cope with various types of stress. LncRNAs are considered as competing endogenous RNAs (ceRNAs) contributing to autophagy. GAS5 has been suggested as a new potential factor to mediate autophagy pathway and the underlying mechanism remains to be further confirmed. This study was taken to identify the effect of GAS5/miR-23a/ATG3 axis on autophagy and cell viability.

The western blotting assay was used to detecte the protein levels of LC3, mTOR, Beclin-1, ATG3, ATG5-ATG12 complex and p62. The mRNA level of Pre-miR-23a, Pri-miR-23a, miR-23a, GAS5, LC3, mTOR and ATG3 were quantified by real-time RT-PCR. Dual-luciferase reporter assays were performed to confirm the direct binding of miR-23a and ATG3 or GAS5. Cell viability was evaluated by CCK-8 and flow cytometry.

We showed that miR-23a could directly suppress ATG3 expression in 293T cells, which suggested that ATG3 was identified as a target of miR-23a. MiR-23a mimics could restrain LC3 II, Beclin1 levles and ATG5-ATG12 complex formation. Meanwhile, miR-23a also increased the expression of mTOR and p62. Notably, there was a putative miR-23a-binding site in GAS5. MiR-23a overexpression might suppress the GAS5 expression, but the repressive effect was abolished by mutation of binding sites. Importantly, overexpression of GAS5 could inhibit the mature miR-23a and has no effect on miR-23a precursors. Knockdown of GAS5 suppressed the expression of LC3 II, ATG3 and ATG5-ATG12 complex formation, whereas p62 and mTOR levels were promoted. The further results showed that miR-23a overexpression and GAS5 inhibition both significantly suppressed cell viability and promoted the apoptosis rate following LPS stimulation, and knockdown of miR-23a exhibited the opposite effects.

Our study revealed that down-regulation GAS5 attenuated cell viability and inhibited autophagy through ATG3-dependent autophagy by regulating miR-23a expression. The results suggested that GAS5/miR-23a/ATG3 axis might be a novel regulatory network contributing to a better understanding of regulation on autophagy program and cell viability.