Long Noncoding RNAs: Recent Insights into Their Role in Male Infertility and Their Potential as Biomarkers and Therapeutic Targets

Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in length. LncRNAs fulfill important functions in a variety of biological processes, including genome imprinting, cell differentiation, apoptosis, stem cell pluripotency, X chromosome inactivation and nuclear transport. As high throughput sequencing technology develops, a substantial number of lncRNAs have been found to be related to a variety of biological processes, such as development of the testes, maintaining the self-renewal and differentiation of spermatogonial stem cells, and regulating spermatocyte meiosis. These indicate that lncRNAs can be used as biomarkers and potential therapeutic targets for male infertility. However, only a few comprehensive reviews have described the role of lncRNAs in male reproduction. In this paper, we summarize recent findings relating to the role of lncRNAs in spermatogenesis, their potential as biomarkers for male infertility and the relationship between reproductive arrest and transgenerational effects. Finally, we suggest specific targets for the treatment of male infertility from the perspective of lncRNAs.     

Foam Cells as Therapeutic Targets in Atherosclerosis with a Focus on the Regulatory Roles of Non-Coding RNAs

Atherosclerosis is a major cause of human cardiovascular disease, which is the leading cause of mortality around the world. Various physiological and pathological processes are involved, including chronic inflammation, dysregulation of lipid metabolism, development of an environment characterized by oxidative stress and improper immune responses. Accordingly, the expansion of novel targets for the treatment of atherosclerosis is necessary. In this study, we focus on the role of foam cells in the development of atherosclerosis. The specific therapeutic goals associated with each stage in the formation of foam cells and the development of atherosclerosis will be considered. Processing and metabolism of cholesterol in the macrophage is one of the main steps in foam cell formation. Cholesterol processing involves lipid uptake, cholesterol esterification and cholesterol efflux, which ultimately leads to cholesterol equilibrium in the macrophage. Recently, many preclinical studies have appeared concerning the role of non-encoding RNAs in the formation of atherosclerotic lesions. Non-encoding RNAs, especially microRNAs, are considered regulators of lipid metabolism by affecting the expression of genes involved in the uptake (e.g., CD36 and LOX1) esterification (ACAT1) and efflux (ABCA1, ABCG1) of cholesterol. They are also able to regulate inflammatory pathways, produce cytokines and mediate foam cell apoptosis. We have reviewed important preclinical evidence of their therapeutic targeting in atherosclerosis, with a special focus on foam cell formation.     

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.     

Long Non-Coding RNAs,Extracellular Vesicles and Inflammation in Alzheimer’s Disease

Alzheimer’s Disease (AD) has currently no effective treatment; however, preventive measures have the potential to reduce AD risk. Thus, accurate and early prediction of risk is an important strategy to alleviate the AD burden. Neuroinflammation is a major factor prompting the onset of the disease. Inflammation exerts its toxic effect via multiple mechanisms. Amongst others, it is affecting gene expression via modulation of non-coding RNAs (ncRNAs), such as miRNAs. Recent evidence supports that inflammation can also affect long non-coding RNA (lncRNA) expression. While the association between miRNAs and inflammation in AD has been studied, the role of lncRNAs in neurodegenerative diseases has been less explored. In this review, we focus on lncRNAs and inflammation in the context of AD. Furthermore, since plasma-isolated extracellular vesicles (EVs) are increasingly recognized as an effective monitoring strategy for brain pathologies, we have focused on the studies reporting dysregulated lncRNAs in EVs isolated from AD patients and controls. The revised literature shows a positive association between pro-inflammatory lncRNAs and AD. However, the reports evaluating lncRNA alterations in EVs isolated from the plasma of patients and controls, although still limited, confirm the value of specific lncRNAs associated with AD as reliable biomarkers. This is an emerging field that will open new avenues to improve risk prediction and patient stratification, and may lead to the discovery of potential novel therapeutic targets for AD.     

Circulating U13 Small Nucleolar RNA as a Potential Biomarker in Huntington’s Disease: A Pilot Study

Plasma small RNAs have been recently explored as biomarkers in Huntington’s disease (HD). We performed an exploratory study on nine HD patients, eight healthy subjects (HS), and five psychiatric patients (PP; to control for iatrogenic confounder effects) through an Affymetrix-Gene-Chip-miRNA-Array. We validated the results in an independent population of 23 HD, 15 pre-HD, 24 PP, 28 Alzheimer’s disease (AD) patients (to control the disease-specificity) and 22 HS through real-time PCR. The microarray results showed higher levels of U13 small nucleolar RNA (SNORD13) in HD patients than controls (fold change 1.54, p = 0.003 HD vs. HS, and 1.44, p = 0.0026 HD vs. PP). In the validation population, a significant increase emerged with respect to both pre-HD and the control groups (p < 0.0001). SNORD13 correlated with the status of the mutant huntingtin carrier (r = 0.73; p < 0.001) and the disease duration (r = 0.59; p = 0.003). The receiver operating characteristic (ROC) curve analysis showed the high accuracy of SNORD13 in discriminating HD patients from other groups (AUC = 0.963). An interactome and pathway analysis on SNORD13 revealed enrichments for factors relevant to HD pathogenesis. We report the unprecedented finding of a potential disease-specific role of SNORD13 in HD. It seems to peripherally report a ‘tipping point’ in the pathogenic cascade at the neuronal level.