The Role Of MiR-346 In ER Stress Induced Autophagy And The Underlining Mechanism

[Objective] Endoplasmic reticulum(ER) is one of the most important organells in eukaryocyte, and the disturbance of ER homeostasis leading to the accumulation of misfolded or unfolded proteins in ER lumen would result in ER stress(ERS). ERS triggers unfolded protein response(UPR), trying to restore the homeostasis of ER. The UPR triggered by ERS participates in the initiation and progression of many human diseases, including neurodegenerative diseases, diabetes and cancers. Studies have indicated that both autophagy and miRNAs take part in the determination of cell fate under ER stress, but the associations between these two factors under ER stress is unclear yet. Our preliminary data showed that the expression of miR-346 is significantly increased under ERS, and miR-346 enhanced the cell viabilities under ERS. In the present study, we investigated the mechanisms by which miR-346 maintained the cell viabilities, and elucidated the role of miR-346 in the process autophagy and mitophagy under ERS, as well as its potential molecular mechanisms.[Methods] Firstly, we constructed ER stressed cell model with thapsigargin(Tg) treatment using human He La cells, and confirmed the induction of ERS via detecting the expression of ER chaperone protein GRP78 with western blot or the expression of XBP-1(U) and XBP-1(S) m RNA with RT-q PCR. We detected the expression of miR-346 with RT-q PCR and analyzed the impacts of miR-346 on cell apoptosis and viabilities with Annexin V-FITC/PI methods and MTT assay, respectively. Then, we determined the effects of miR-346 on autophagy via GFP-LC3 punctate formation assay or by detecting the expression of LC3-II/LC3-I with western blot, investigated the effects of miR-346 on mitophagy via the combination of GFP-LC3 punctate formation assay and Mito Tracker? Red CMXRos stainning, as well as determined the impacts of miR-346 on cellular ROS level with ROS sensitive DCFH-DA probe. Further, we predicted the potential candidate targets of miR-346 with bioinformatics analyses, and investigated its functional roles and validated the regulations by EGFP reporter assay, western blot and RT-q PCR assays, and we investigated the function of the target genes. Finally, we elucidated the potential mechanisms which were responsible for induction of autophagy by miR-346 via protein co-immunoprecipitation assay and protein ubiquitination assay.[Results] The expression level of miR-346 was significantly increased under ER stress, and miR-346 increased the cell viabilities by significantly decreasing cell apoptosis under ER stress. miR-346 enhanced the autophagy and mitophagy and reduce the ROS level within the cells under ER stress; Further, miR-346 improved the cell viabilities in an autophagy dependent manner under such circumstance. GSK3 B was identified as a direct functional target of miR-346 and its expression was upregulated by miR-346; GSK3 B significantly enhanced the autophagy flux and decreased the ROS level within the cells under ERS; rescue experiments further demonstrated that GSK3 B was a functional target of miR-346. miR-346 and GSK3 B enhanced the autophagy via increasing the ubiquitination level of BCL2 and dissociating BECN1 from BCL2.[Conclusion] The expression level of miR-346 is significantly increased under ER stress; miR-346 could dramatically improve the cell viabilities, decrease cell apoptosis, enhance the autophagy flux and reduce the ROS level within the cells under ER stress. Furthermore, miR-346 could directly targete and enhance the expression of GSK3 B, which resulted in reducing the BCL2 level by increasing its ubiquitination level. miR-346 and GSK3 B also increased the dissociation of BECN1 from BCL2 resulting in ER-stress induced autophagy.