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

This monthly, peer-reviewed journal includes studies devoted to molecular medicine, underscoring aspects including pharmacology, pathology, genetics, neurosciences, infectious diseases, molecular cardiology and molecular surgery. In vitro and in vivo studies of experimental model systems pertaining to the mechanisms of a variety of diseases offer researchers the necessary tools and knowledge with which to aid the diagnosis and treatment of human diseases.

Molecular Medicine Reports

Arsenic exposure: A public health problem leading to several cancers.

Ferroptosis is a new type of oxidative regulated cell death (RCD) driven by iron-dependent lipid peroxidation. As major sites of iron utilization and master regulators of oxidative metabolism, mitochondria are the main source of reactive oxygen species (ROS) and, thus, play a role in this type of RCD. Ferroptosis is, indeed, associated with severe damage in mitochondrial morphology, bioenergetics, and metabolism. Furthermore, dysregulation of mitochondrial metabolism is considered a biochemical feature of neurodegenerative diseases linked to ferroptosis. Whether mitochondrial dysfunction can, per se, initiate ferroptosis and whether mitochondrial function in ferroptosis is context-dependent are still under debate. Cancer cells accumulate high levels of iron and ROS to promote their metabolic activity and growth. Of note, cancer cell metabolic rewiring is often associated with acquired sensitivity to ferroptosis. This strongly suggests that ferroptosis may act as an adaptive response to metabolic imbalance and, thus, may constitute a new promising way to eradicate malignant cells. Here, we review the current literature on the role of mitochondria in ferroptosis, and we discuss opportunities to potentially use mitochondria-mediated ferroptosis as a new strategy for cancer therapy.

Ferroptosis and Cancer: Mitochondria Meet the “Iron Maiden” Cell Death

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

Liver is the major organ for arsenic methylation metabolism and may be the potential target of arsenic-induced cancer. In this study, normal human liver cell was treated with arsenic trioxide, and detected using DNA methylation microarray. Some oncogenes, tumor suppressor genes, transcription factors (TF), and tumor-associated genes (TAG) that have aberrant DNA methylation have been identified. However, simple functional studies of genes adjacent to aberrant methylation sites cannot well reflect the regulatory relationship between DNA methylation and gene transcription during the pathogenesis of arsenic-induced liver cancer, whereas a further analysis of the cis-regulatory elements and their trans-acting factors adjacent to DNA methylation can more precisely reflect the relationship between them. MYC and MAX (MYC associated factor X) were found to participating cell cycle through a bioinformatics analysis. Additionally, it was found that the hypomethylation of cis-regulatory sites in the MYC promoter region and the hypermethylation of cis-regulatory sites in the MAX promoter region result in the up-regulation of MYC mRNA expression and the down-regulation of MAX mRNA, which increased the hepatocyte carcinogenesis tendency.

/pdf/analysis-of-the-transcriptional-regulation-of-cancer-related-2rorck9dp9.pdf

Analysis of the transcriptional regulation of cancer-related genes by aberrant DNA methylation of the cis-regulation sites in the promoter region during hepatocyte carcinogenesis caused by arsenic.

The aim of this study was to identify agents that regulate autophagy. A total of 544 differentially expressed genes were screened from the intersection set of GSE2435 and GSE31040, which was obtained from the Gene Expression Omnibus database and 19 differentially expressed kinases were selected according to a 'protein kinase database'. Gene ontology-biological process (GO-BP) enrichment analysis revealed that the 19 kinases were mainly associated with phosphorylation. The protein-protein interaction network exhibited 30 differentially expressed genes that interacted with BRAF, and GO-BP enrichment analysis showed the function of these genes were mainly involved in cell death and apoptosis. The kinase-kinase inhibitor regulatory network identified16 kinase inhibitors that specifically inhibited BRAF. Previous studies indicated that sorafenib is capable of regulating autophagy and regorafenib has also been reported; however, there have been no studies regarding the regulation of autophagy by afatinib, selumetinib, PD318088, axitinib, TAK-733, GDC-0980, GSK2126458, PLX-4720, AS703026, trametinib, GDC-0941 and PF-04217903. Thus, these kinase inhibitors are potential targets for further study on the regula- tion of autophagy in the future.

/pdf/screening-of-kinase-inhibitors-targeting-braf-for-regulating-1z5ecat4k0.pdf

Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways.

Acute pancreatitis (AP) is a common acute digestive tract disease, with increased morbidity and mortality, and an unclear pathogenesis. Trypsinogen activation in pancreatic acinar cells may be the primary mechanism underlying the development of AP. Previous studies reported that autophagy participates in the formation of acinar cell vacuoles in AP and in the process of trypsinogen activation as an important cause of AP. Furthermore, microRNAs (miRNAs) maintain the autophagy process by regulating the expression of autophagy‑associated genes. In the present study, an in vitro pancreatic acinar cell autophagy model was established using the AR42J starvation‑induced pancreatic acinar cell line. Twenty differentially expressed microRNAs were identified using miRNA microarray. Bioinformatics analysis was used to predict the target genes of miRNAs and analyze the functions of differentially expressed miRNAs. The results demonstrated that only the downregulated miRNA rno‑miR‑148b‑3p predicted 593 target genes with a statistical significance (P<0.05), from which 10 genes were autophagy‑associated. The results of gene ontology and pathway analyses demonstrated that the target genes of miRNAs were enriched in the Response to insulin stimulus, Regulation of cell death and the Insulin signaling pathways (P<0.05, FDR<0.05). In addition, protein‑protein interaction network analysis demonstrated a widespread interaction among the 593 target genes. The results of the present study may provide novel targets for research on the mechanisms of autophagy-promoted AP and AP treatment.

/pdf/differentially-expressed-microrna-identification-and-target-18nyjo9ywr.pdf

Differentially expressed microRNA identification and target gene function analysis in starvation-induced autophagy of AR42J pancreatic acinar cells

Trypsinogen activation is the initial factor involved in the development of all types of acute pancreatitis (AP) and has been suggested to be regulated by protein kinases. In the present study, AR42J rat pancreatic acinar cells were treated with taurolithocholic acid 3-sulfate (TLC-S), and trypsinogen activation was detected with bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) staining and flow cytometry. Differentially expressed protein kinase genes were screened by Gene Chip analysis, and the functions of these kinases were analyzed. A significantly increased activation of trypsinogen in AR42J cells following treatment with TLC-S was observed. A total of 22 differentially expressed protein kinase genes were found in the TLC-S group, among which 19 genes were upregulated and 3 were downregulated. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, kinase genes of the same KEGG pathways were connected to create a network through signaling pathways, and 10 nodes of kinases were identified, which were mitogen-activated protein kinase (Mapk)8, Mapk14, Map2k4, interleukin-1 receptor-associated kinase 3 (Irak3), ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (Rps6ka2), protein kinase C, alpha (Prkca), v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (Lyn), protein tyrosine kinase 2 beta (Ptk2b), p21 protein (Cdc42/Rac)-activated kinase 4 (Pak4) and FYN oncogene related to SRC, FGR, YES (Fyn). The interactions between signaling pathways were further analyzed and a network was created. MAPK and calcium signaling pathways were found to be located at the center of the network. Thus, protein kinases constitute potential drug targets for AP treatment.

https://www.spandidos-publications.com/10.3892/mmr.2013.1355/download

Differentially expressed kinase genes associated with trypsinogen activation in rat pancreatic acinar cells treated with taurolithocholic acid 3-sulfate

Acute pancreatitis (AP) exhibits high morbidity and mortality rates. The onset of AP is characterized by early trypsinogen activation.The present study aimed to investigate the expression of microRNA (miR)‑92a‑3p and early growth response protein 1 (Egr1), and the effect of miR‑92a‑3p on trypsinogen activation in the pancreatic exocrine cell line AR42J. mRNA and miRNA microarrays were used to identify differentially expressed mRNAs and miRNAs in AR42J cells. A miRNA‑mRNA network was constructed using bioinformatics software, and Egr1 and its regulated miRNA subnetworks were identified by reviewing previous literature. The results suggested that miR‑92a‑3p could bind to Egr1 3'untranslated region sequence. Subsequently, miR‑92a‑3p mimic and inhibitor were used to transfect AR42J cells. Following transfection, reverse transcription‑quantitative PCR and western blotting were performed to detect Egr1 expression. Furthermore, AR42J cells were cotransfected with miR‑92a‑3p inhibitor and small interfering (si)‑Egr1. The trypsinogen activation rate of AR42J cells was measured by flow cytometry. Microarrays and bioinformatics results indicated that Egr1 may be a target gene of miR‑92a‑3p. In addition, the present study suggested that miR‑92a‑3p downregulated Egr1 in vitro and that miR‑92a‑3p and Egr1 expression was associated with trypsinogen activation. Furthermore, miR‑92a‑3p inhibitor reversed the effect of si‑Egr1 on trypsinogen activation. In conclusion, miR‑92a‑3p may negatively regulate the activation of trypsinogen in AR42J cells via Egr1.

/pdf/mir-92a-3p-regulates-trypsinogen-activation-via-egr1-in-3awosa48wy.pdf

Bo Gao

Papers

Analysis of the transcriptional regulation of cancer-related genes by aberrant DNA methylation of the cis-regulation sites in the promoter region during hepatocyte carcinogenesis caused by arsenic.

Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways.

Differentially expressed microRNA identification and target gene function analysis in starvation-induced autophagy of AR42J pancreatic acinar cells

Differentially expressed kinase genes associated with trypsinogen activation in rat pancreatic acinar cells treated with taurolithocholic acid 3-sulfate

miR‑92a‑3p regulates trypsinogen activation via Egr1 in AR42J cells.