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

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

After the initial investigations into applications of mesenchymal stem cells (MSCs) for cell therapy, there was increased interest in their secreted soluble factors. Following studies of MSCs and their secreted factors, extracellular vesicles (EVs) released from MSCs have emerged as a new mode of intercellular crosstalk. MSC-derived EVs have been identified as essential signaling mediators under both physiological and pathological conditions, and they appear to be responsible for many of the therapeutic effects of MSCs. In several in vitro and in vivo models, EVs have been observed to have supportive functions in modulating the immune system, mainly mediated by EV-associated proteins and nucleic acids. Moreover, stimulation of MSCs with biophysical or biochemical cues, including EVs from other cells, has been shown to influence the contents and biological activities of subsequent MSC-derived EVs. This review provides on overview of the contents of MSC-derived EVs in terms of their supportive effects, and it provides different perspectives on the manipulation of MSCs to improve the secretion of EVs and subsequent EV-mediated activities. In this review, we discuss the possibilities for manipulating MSCs for EV-based cell therapy and for using EVs to affect the expression of elements of interest in MSCs. In this way, we provide a clear perspective on the state of the art of EVs in cell therapy focusing on MSCs, and we raise pertinent questions and suggestions for knowledge gaps to be filled.

/pdf/enhancement-of-therapeutic-potential-of-mesenchymal-stem-3ryeqg9tgs.pdf

Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles

Regulation of Nuclear Factor-KappaB (NF-κB) signaling pathway by non-coding RNAs in cancer: Inhibiting or promoting carcinogenesis?

Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.

Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis.

MicroRNAs (miRNAs) play important roles in the progression of human cancer. Although previous reports have shown that miR-145-5p is down-regulated in esophageal squamous cell carcinoma (ESCC), the roles and mechanisms of down-regulation of miR-145-5p in ESCC are still largely unknown. Using microRNA microarray and Gene Expression Omnibus (GEO) datasets, we confirmed that miR-145-5p was down-regulated in ESCC tissues. In vitro assays revealed that ectopic miR-145-5p expression repressed cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT). miR-145-5p also reduced the expressions of cell cycle genes including cyclin A2 (CCNA2), cyclin D1 (CCND1) and cyclin E1 (CCNE1), the EMT-associated transcription factor Slug, and matrix metalloproteinases (MMPs) including MMP2, MMP7 and MMP13. Furthermore, miR-145-5p mimics reduced candidate target gene specificity protein 1 (Sp1) and nuclear factor κ B (NF-κB) (p65) both in mRNA and protein levels. Knockdown of Sp1 phenocopied the effects of miR-145-5p overexpression on cell cycle regulators, EMT and the expression of NF-κB (p65). Importantly, inhibition of the NF-κB signaling pathway or knockdown of NF-κB (p65) phenocopied the effects of miR-145-5p on the migration, invasion and EMT of ESCC cells. In conclusion, our results suggested that miR-145-5p plays tumor-suppressive roles by inhibiting esophageal cancer cell migration, invasion and EMT through regulating the Sp1/NF-κB signaling pathway.

miR-145-5p Suppresses Tumor Cell Migration, Invasion and Epithelial to Mesenchymal Transition by Regulating the Sp1/NF-κB Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Ferroptosis is a new form of programmed cell death with characteristic accumulation of reactive oxygen species (ROS) resulting from iron accumulation and lipid peroxidation. Ferroptosis is involved in many diseases, including cancer, and induction of ferroptosis has shown attractive antitumour activities. In this review, we summarize recent findings on the regulatory mechanisms of key regulators of ferroptosis, including the catalytic subunit solute carrier family 7 member 11 (SLC7A11), the glutathione peroxidase 4 (GPX4), p53 and non-coding RNAs, the correlations between ferroptosis and iron homeostasis or autophagy, ferroptosis-inducing agents and nanomaterials and the diagnostic and prognostic value of ferroptosis-associated genes in TCGA data.

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Ferroptosis in Carcinoma: Regulatory Mechanisms and New Method for Cancer Therapy

MicroRNAs (miRNAs) play important roles in the progression of human cancer including esophageal squamous cell carcinoma (ESCC). Although previous reports showed that miR-125b-5p was down-regulated in ESCC, the roles and mechanisms of loss of function of miR-125b-5p in ESCC were still unknown. Using microRNA microarray and GEO datasets, we found and confirmed that miR-125b-5p was down-regulated in ESCC tissues. In-vitro assays showed that ectopic miR-125b-5p expression repressed cell proliferation, migration and invasion, and induced cell senescence. We also found that miR-125b-5p reduced the expressions of cell cycle regulatory genes including CCNA2, CCND1 and CCNE1, and regulated the markers of epithelial to mesenchymal transition (EMT) including E-cadherin, N-cadherin and EMT associated transcription factor Slug, and also decreased the MMPs including MMP2, MMP7 and MMP13. Furthermore, the candidate target gene HMGA2 was negatively regulated by miR-125b-5p both in mRNA and protein levels. Importantly, knockdown of HMGA2 partially phenocopied the effects of miR-125b-5p overexpression on cell cycle regulators and EMT markers. In conclusion, our results suggested that overexpression of miR-125b-5p inhibited cell proliferation, migration and invasion partially by down-regulating HMGA2 in ESCC.

/pdf/mir-125b-5p-functions-as-a-tumor-suppressor-gene-partially-1wap1tlr5a.pdf

miR-125b-5p functions as a tumor suppressor gene partially by regulating HMGA2 in esophageal squamous cell carcinoma.

The roles and mechanisms of mini-chromosome maintenance complex component 7 (MCM7) amplification and overexpression in esophageal carcinogenesis were investigated. By analyzing the TCGA datasets, we found that MCM7 was amplified in approximately 12% of esophageal squamous cell carcinomas (ESCCs), and in more than 4% of head and neck squamous cell carcinomas and stomach carcinomas. Overexpression of MCM7 was further verified in three independent GEO datasets of esophageal cancer. Knockdown of MCM7 using two siRNAs significantly inhibited cell proliferation, colony formation and migration of KYSE510 and EC9706 cells in vitro. Noteworthy, we further found that silencing of MCM7 suppressed the phosphorylation of AKT1 and mTOR both in KYSE510 and EC9706 cells, and reduced the cell cycle regulatory proteins cyclin D1, cyclin E2 and CDK2. Taken together, our findings suggested that MCM7 promoted tumor cell proliferation, colony formation and migration of ESCC cells via activating AKT1/mTOR signaling pathway.

/pdf/mcm7-amplification-and-overexpression-promote-cell-4s0g84rufh.pdf

MCM7 amplification and overexpression promote cell proliferation, colony formation and migration in esophageal squamous cell carcinoma by activating the AKT1/mTOR signaling pathway

miR-99a is down-regulated in esophageal squamous cell carcinoma (ESCC), however the role and underlying mechanism are still unknown. We aim to explore the role and mechanism of miR-99a down-regulation in ESCC. The expression of miR-99a in ESCC tissues and cell lines was detected by Human miRNA Microarrays and Real-time PCR. The effects of miR-99a on cell proliferation, migration and invasion were determined by Cell Counting Kit-8 (CCK-8) assay, transwell migration and invasion assay. Target gene of miR-99a were analyzed by target prediction software and validated by Real-time PCR and Western blotting assay. Our microarray results and four Gene Expression Omnibus (GEO) datasets showed lower expression level of miR-99a in ESCC tissues. Overexpression of miR-99a using mimics significantly suppressed cell proliferation, and decreased expressions of CCND1, CCNA2 and CCNE1. We also found that enhanced miR-99a significantly inhibited migration, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells, and down-regulated EMT associated transcription factor Slug, and MMPs including MMP2, MMP7 and MMP13. TargetScan predicted insulin-like growth factor 1 receptor (IGF1R) as the cadidate target gene of miR-99a, and western blotting confirmed the negative correlation between miR-99a and IGF1R. Importantly, we further found that knockdown of IGF1R also significantly inhibited the proliferation, migration, invasion and slug-induced EMT of ESCC cells, and reduced the cell cycle regulatory proteins and MMPs. In conclusion, our findings suggested that loss of miR-99a in ESCC promoted the tumor cell proliferation, migration, invasion and slug-induced EMT through activating IGF1R signaling pathway.

Wen-Jun Wang

Papers

miR-145-5p Suppresses Tumor Cell Migration, Invasion and Epithelial to Mesenchymal Transition by Regulating the Sp1/NF-κB Signaling Pathway in Esophageal Squamous Cell Carcinoma.

Ferroptosis in Carcinoma: Regulatory Mechanisms and New Method for Cancer Therapy

miR-125b-5p functions as a tumor suppressor gene partially by regulating HMGA2 in esophageal squamous cell carcinoma.

MCM7 amplification and overexpression promote cell proliferation, colony formation and migration in esophageal squamous cell carcinoma by activating the AKT1/mTOR signaling pathway

MiR-99a suppresses proliferation, migration and invasion of esophageal squamous cell carcinoma cells through inhibiting the IGF1R signaling pathway.