Search

Autophagy is a cellular survival pathway that is necessary for the degradation of cellular constituents such as long-lived proteins and damaged organelles. Conditions resulting in cellular stress such as starvation or hypoxia might activate autophagy. Being at the crossroads of various cellular response pathways, dysregulation of autophagy might result in pathological states including cancer and neurodegenerative diseases. Autophagy has also been shown to participate in stemness. MicroRNAs were introduced as novel regulators of autophagy, and accumulating results underlined the fact that they constituted an important layer of biological control mechanism on the autophagic activity.MicroRNAs are protein noncoding small RNAs that control cellular levels of transcripts and proteins through posttrancriptional mechanisms. Novel miRNAs in human and mouse genomes are yet to be identified. Considering the emerging role of autophagy in health and disease, identification of novel autophagy-regulating miRNAs and determination of relations between miRNA expression and physiological and pathological conditions might contribute to a better understanding of mechanisms governing health and disease. High-throughput techniques were developed for miRNA profiling, yet for a thorough characterization and miRNA target determination, miRNA cloning remains as an important step. Here, we describe a modified miRNA cloning method for the characterization of novel autophagy-regulating miRNAs.

Macroautophagy (autophagy) is an evolutionarily conserved recycling and stress response mechanism. Active at basal levels in eukaryotes, autophagy is upregulated under stress providing cells with building blocks such as amino acids. A lysosome-integrated sensor system composed of RRAG GTPases and MTOR complex 1 (MTORC1) regulates lysosome biogenesis and autophagy in response to amino acid availability. Stress-mediated inhibition of MTORC1 results in the dephosphorylation and nuclear translocation of the TFE/MITF family of transcriptional factors, and triggers an autophagy- and lysosomal-related gene transcription program. The role of family members TFEB and TFE3 have been studied in detail, but the importance of MITF proteins in autophagy regulation is not clear so far. Here we introduce for the first time a specific role for MITF in autophagy control that involves upregulation of MIR211. We show that, under stress conditions including starvation and MTOR inhibition, a MITF-MIR211 axis constitutes a novel feed-forward loop that controls autophagic activity in cells. Direct targeting of the MTORC2 component RICTOR by MIR211 led to the inhibition of the MTORC1 pathway, further stimulating MITF translocation to the nucleus and completing an autophagy amplification loop. In line with a ubiquitous function, MITF and MIR211 were co-expressed in all tested cell lines and human tissues, and the effects on autophagy were observed in a cell-type independent manner. Thus, our study provides direct evidence that MITF has rate-limiting and specific functions in autophagy regulation. Collectively, the MITF-MIR211 axis constitutes a novel and universal autophagy amplification system that sustains autophagic activity under stress conditions. Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AKT1S1/PRAS40: AKT1 substrate 1; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; DEPTOR: DEP domain containing MTOR interacting protein; GABARAP: GABA type A receptor-associated protein; HIF1A: hypoxia inducible factor 1 subunit alpha; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPKAP1/SIN1: mitogen-activated protein kinase associated protein 1; MITF: melanogenesis associated transcription factor; MLST8: MTOR associated protein, LST8 homolog; MRE: miRNA response element; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; PRR5/Protor 1: proline rich 5; PRR5L/Protor 2: proline rich 5 like; RACK1: receptor for activated C kinase 1; RPTOR: regulatory associated protein of MTOR complex 1; RICTOR: RPTOR independent companion of MTOR complex 2; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT-qPCR: quantitative reverse transcription-polymerase chain reaction; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TSC1/2: TSC complex subunit 1/2; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VIM: vimentin; VPS11: VPS11, CORVET/HOPS core subunit; VPS18: VPS18, CORVET/HOPS core subunit; WIPI1: WD repeat domain, phosphoinositide interacting 1.

Autophagy is a cellular survival pathway that is necessary for the degradation of cellular constituents such as long-lived proteins and damaged organelles. Conditions resulting in cellular stress such as starvation or hypoxia might activate autophagy. Being at the crossroads of various cellular response pathways, dysregulation of autophagy might result in pathological states including cancer and neurodegenerative diseases. Autophagy has also been shown to participate in stemness. MicroRNAs were introduced as novel regulators of autophagy, and accumulating results underlined the fact that they constituted an important layer of biological control mechanism on the autophagic activity.MicroRNAs are protein noncoding small RNAs that control cellular levels of transcripts and proteins through posttrancriptional mechanisms. Novel miRNAs in human and mouse genomes are yet to be identified. Considering the emerging role of autophagy in health and disease, identification of novel autophagy-regulating miRNAs and determination of relations between miRNA expression and physiological and pathological conditions might contribute to a better understanding of mechanisms governing health and disease. High-throughput techniques were developed for miRNA profiling, yet for a thorough characterization and miRNA target determination, miRNA cloning remains as an important step. Here, we describe a modified miRNA cloning method for the characterization of novel autophagy-regulating miRNAs.

Macroautophagy (autophagy herein) is a cellular stress response and a survival pathway that is responsible for the degradation of long-lived proteins, protein aggregates, as well as damaged organelles in order to maintain cellular homeostasis. Consequently, abnormalities of autophagy are associated with a number of diseases, including Alzheimers’s disease, Parkinson’s disease, and cancer. According to the current view, autophagy seems to serve as a tumor suppressor in the early phases of cancer formation, yet in later phases, autophagy may support and/or facilitate tumor growth, spread, and contribute to treatment resistance. Therefore, autophagy is considered as a stage-dependent dual player in cancer. microRNAs (miRNAs) are endogenous non-coding small RNAs that negatively regulate gene expression at a post-transcriptional level. miRNAs control several fundamental biological processes, and autophagy is no exception. Furthermore, accumulating data in the literature indicate that dysregulation of miRNA expression contribute to the mechanisms of cancer formation, invasion, metastasis, and affect responses to chemotherapy or radiotherapy. Therefore, considering the importance of autophagy for cancer biology, study of autophagy-regulating miRNA in cancer will allow a better understanding of malignancies and lead to the development of novel disease markers and therapeutic strategies. The potential to provide study of some of these cancer-related miRNAs were also implicated in autophagy regulation. In this review, we will focus on autophagy, miRNA, and cancer connection, and discuss its implications for cancer biology and cancer treatment.

The development of non-toxic theranostic nanoparticles capable of delivering a therapeutic cargo and providing a means for diagnosis is one of the most challenging tasks in nano-biotechnology. Gene therapy is a very important mode of therapy and polyethyleneimine (PEI) is one of the most successful vehicles for gene transfection, yet poses significant toxicity. Optical imaging utilizing quantum dots is one of the newer but fast growing diagnostic modalities, which requires non-toxic, highly luminescent materials, preferentially active in the near infrared region. Ag2S NIRQDs fit to this profile perfectly. Here, we demonstrate the aqueous synthesis of cationic Ag2S NIRQDs with a mixed coating of 2-mercaptopropionic acid (2MPA) and PEI (branched, 25 kDa), which are highly luminescent in the NIR-I window (lambda(em) = 810-840 nm) as new theranostic nanoparticles. Synergistic stabilization of the QD surface via the simultaneous use of a small molecule and a polymeric material provided the highest quantum yield, 150% (with respect to LDS 798 at pH 7.4), reported in the literature for Ag2S. These cationic particles show a dramatic improvement in cytocompatibility even without PEGylation, a strong optical signal easily detected by confocal laser microscopy and effective conjugation and transfection of the green fluorescence protein plasmid (pGFP) to HeLa and MCF-7 cell lines (40% efficiency). Overall, these Ag2S NIRQDs show great potential as new theranostics., NA

To cope with stress factors including nutrient deprivation, toxins, abnormal protein accumulation, cells developed mechanisms to repair damage caused by stress or, in cases where the damage is irrepairable, cell death responses. Autophagy and apoptosis are among the most important stress and death response mechanisms. Autophagy is defined by sequestration and transport of cytoplasm pieces and organelles by double- or multi-membrane vesicles and, degradation of the cargo following fusion with lysosomes. Consequently, autophagy plays an important role in elimination of long-lived proteins, damaged organelles, mutant proteins and even intracellular bacteria and viruses, helping to survive stressfull conditions. Paradoxically, depending stress and cell type, autophagy may kill cells by a non-apoptotic programmed cell death mechanism called “autophagic cell death”. Recent studies including our publications suggest that miRNAs add a novel layer of regulation to autophagy pathways. Molecular details of autophagy regulation by miRNAs started to emerge in the last 2 years. In this chapter we will summarize acccumulating data on this novel mechanism of autophagy regulation.