In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

MicroRNAs as major regulators of the autophagy pathway

MicroRNAs (MiRNAs, MiRs) represent a class of conserved small non-coding RNAs that affect post-transcriptional gene regulation and play a vital role in angiogenesis, proliferation, apoptosis, migration and invasion. They are essential for a wide range of physiological and pathological processes, especially for vascular diseases. However, data concerning miRNAs in endothelial progenitor cells (EPCs) and deep vein thrombosis (DVT) remain incomplete. We explored miRNAs that modulate angiogenesis in EPCs and thrombolysis, and analysed their underlying mechanisms using a DVT model, dual-luciferase reporter assay, qRT-PCR, Western blot, immunofluorescence staining, flow cytometry analysis, CCK-8 assay, angiogenesis assay, wound healing and Transwell assay. We found that miR-205 enhanced the homing ability of EPCs to DVT sites and promoted thrombosis resolution and recanalization, which significantly reduced venous thrombus. Additionally, we demonstrated that miR-205 overexpression significantly enhanced angiogenesis in vivo and in vitro, migration, invasion, F-actin filaments and proliferation in EPCs, and inhibited cell apoptosis. Conversely, down-regulation of miR-205 played the opposite role in EPCs. Importantly, this study demonstrated that miR-205 directly targeted PTEN to modulate the Akt/autophagy pathway and MMP2 expression, subsequently playing a key role in EPC function and DVT recanalization and resolution. These results elucidated the pro-angiogenesis effects of miR-205 in EPCs and established it as a potential target for DVT treatment.

MiR-205 promotes endothelial progenitor cell angiogenesis and deep vein thrombosis recanalization and resolution by targeting PTEN to regulate Akt/autophagy pathway and MMP2 expression.

Long non-coding RNAs (lncRNAs) play an essential role in multitudinous physiological and pathological processes, including vascular disease. We previously showed that lncRNA GUSBP5-AS (enst00000511042) is upregulated in endothelial progenitor cells (EPCs) of deep veni thrombosis (DVT) patients. Here, we investigate the role and mechanism of GUSBP5-AS in EPCs and DVT. Using the DVT model, we found that GUSBP5-AS significantly reduced the thrombus size and weight and enhanced the homing ability of EPC to DVT sites to promote resolution and recanalization of thrombus. GUSBP5-AS promoted cell cycle progression, proliferation, migration and invasion in EPCs, enhanced EPC angiogenesis in vitro and in vivo, and inhibited apoptosis. Strikingly, this study showed that GUSBP5-AS was unbalanced and modulated Forkhead Box Protein O1 (FOXO1) in EPCs in patients with DVT by interacting with miR-223-3p. Mechanistically, GUSBP5-AS functions as a sponge of miR-223-3p, which targets FOXO1. Both GUSBP5-AS knockdown and miR-223-3p overexpression remarkably inhibited angiogenesis, migration and invasion in EPCs. Additionally, our data suggested that GUSBP-AS activated the Akt pathway and enhanced fibroblast growth factor 2 (FGF2), matrix metalloproteinase-2/9 (MMP2/9) and F-actin expression. Taken together, this study indicates that GUSBP5-AS modulates angiogenesis, proliferation and homing ability of EPCs via regulating FGF2 and MMP2/9 expression through the miR-223-3p/FOXO1/Akt pathway, which may provide a new direction for the development of DVT therapeutics.

LncRNA GUSBP5-AS promotes EPC migration and angiogenesis and deep vein thrombosis resolution by regulating FGF2 and MMP2/9 through the miR-223-3p/FOXO1/Akt pathway.

Lysosome is a crucial organelle in charge of degrading proteins and damaged organelles to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master transcription factor regulating lysosomal biogenesis and autophagy. Under external stimuli such as starvation, dephosphorylated TFEB transports into the nucleus to specifically recognize and bind to the coordinated lysosomal expression and regulation (CLEAR) elements at the promotors of autophagy and lysosomal biogenesis-related genes. The function of TFEB in the nucleus is fine regulated but the molecular mechanism is not fully elucidated. In this study, we discovered that miR-30b-5p, a small RNA which is known to regulate a series of genes through posttranscriptional regulation in the cytoplasm, was translocated into the nucleus, bound to the CLEAR elements, suppressed the transcription of TFEB-dependent downstream genes, and further inhibited the lysosomal biogenesis and the autophagic flux; meanwhile, knocking out the endogenous miR-30b-5p by CRISPR/Cas9 technique significantly increased the TFEB-mediated transactivation, resulting in the increased expression of autophagy and lysosomal biogenesis-related genes. Overexpressing miR-30b-5p in mice livers showed a decrease in lysosomal biogenesis and autophagy. These in vitro and in vivo data indicate that miR-30b-5p may inhibit the TFEB-dependent transactivation by binding to the CLEAR elements in the nucleus to regulate the lysosomal biogenesis and autophagy. This novel mechanism of nuclear miRNA regulating gene transcription is conducive to further elucidating the roles of miRNAs in the lysosomal physiological functions and helps to understand the pathogenesis of abnormal autophagy-related diseases.

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Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy

Autophagy plays an important role in maintaining cell function. Abnormal autophagy leads to cell dysfunction and is associated with many diseases such as tumors, immunodeficiency diseases, lysosomal storage disorders, and neurodegenerative diseases. Autophagy is precisely regulated, and PTEN plays an important role in regulating autophagy. As noncoding small RNAs, miRNAs play an important role in the fine regulation of cellular processes. However, the mechanism of the miRNA regulation of PTEN-related autophagy has not been fully elucidated. In this study, our results showed that miR-4465 significantly inhibited the expression of PTEN, upregulated phosphorylated AKT, and thereby inhibited autophagy by activating mTOR in HEK293, HeLa, and SH-SY5Y cells. Further studies indicated that miR-4465 reduced PTEN mRNA levels through posttranscriptional regulation via directly targeting the 3'-UTR. Our novel findings provide useful hints for the comprehensive elucidation of the molecular mechanism of miRNA-regulated PTEN-related autophagy and may also provide some new insights for the exploration of miRNAs in the treatment of PTEN-related diseases.

Huijie Guo

Papers

Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy

MiR-4465 directly targets PTEN to inhibit AKT/mTOR pathway-mediated autophagy.

miR-34a-5p regulates PINK1-mediated mitophagy via multiple modes.