Extracellular Vesicles as Delivery Shippers for Noncoding RNA-Based Modulation of Angiogenesis: Insights from Ischemic Stroke and Cancer

Ischemic stroke and systemic cancer are two of the leading causes of mortality. Hypoxia is a central pathophysiological component in ischemic stroke and cancer, representing a joint medical function. This function includes angiogenesis regulation. Vascular remodeling coupled with axonal outgrowth following cerebral ischemia is critical in improving poststroke neurological functional recovery. Antiangiogenic strategies can inhibit cancer vascularization and play a vital role in impeding cancer growth, invasion, and metastasis. Although there are significant differences in the cause of angiogenesis across both pathophysiological conditions, emerging evidence states that common signaling structures, such as extracellular vesicles (EVs) and noncoding RNAs (ncRNAs), are involved in this context. EVs, heterogeneous membrane vesicles encapsulating proteomic genetic information from parental cells, act as multifunctional regulators of intercellular communication. Among the multifaceted roles in modulating biological responses, exhaustive evidence shows that ncRNAs are selectively sorted into EVs, modulating common specific aspects of cancer development and stroke prognosis, namely, angiogenesis. This review will discuss recent advancements in the EV-facilitated/inhibited progression of specific elements of angiogenesis with a particular concern about ncRNAs within these vesicles. The review is concluded by underlining the clinical opportunities of EV-derived ncRNAs as diagnostic, prognostic, and therapeutic agents.     

Role of Nitric Oxide in Gene Expression Regulation during Cancer: Epigenetic Modifications and Non-Coding RNAs

Nitric oxide (NO) has been identified and described as a dual mediator in cancer according to dose-, time- and compartment-dependent NO generation. The present review addresses the different epigenetic mechanisms, such as histone modifications and non-coding RNAs (ncRNAs), miRNA and lncRNA, which regulate directly or indirectly nitric oxide synthase (NOS) expression and NO production, impacting all hallmarks of the oncogenic process. Among lncRNA, HEIH and UCA1 develop their oncogenic functions by inhibiting their target miRNAs and consequently reversing the inhibition of NOS and promoting tumor proliferation. The connection between miRNAs and NO is also involved in two important features in cancer, such as the tumor microenvironment that includes key cellular components such as tumor-associated macrophages (TAMs), cancer associated fibroblasts (CAFs) and cancer stem cells (CSCs).     

Long Non-Coding RNA Epigenetics

Long noncoding RNAs exceeding a length of 200 nucleotides play an important role in ensuring cell functions and proper organism development by interacting with cellular compounds such as miRNA, mRNA, DNA and proteins. However, there is an additional level of lncRNA regulation, called lncRNA epigenetics, in gene expression control. In this review, we describe the most common modified nucleosides found in lncRNA, 6-methyladenosine, 5-methylcytidine, pseudouridine and inosine. The biosynthetic pathways of these nucleosides modified by the writer, eraser and reader enzymes are important to understanding these processes. The characteristics of the individual methylases, pseudouridine synthases and adenine–inosine editing enzymes and the methods of lncRNA epigenetics for the detection of modified nucleosides, as well as the advantages and disadvantages of these methods, are discussed in detail. The final sections are devoted to the role of modifications in the most abundant lncRNAs and their functions in pathogenic processes.     

Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9

Background: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients’, matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. Results: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. Conclusions: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.