PIWI-RNAs Small Noncoding RNAs with Smart Functions: Potential Theranostic Applications in Cancer.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of small noncoding RNAs (ncRNAs) that bind components of the PIWI protein family. piRNAs are specifically expressed in different human tissues and regulate important signaling pathways. Aberrant expressions of piRNAs and PIWI proteins have been associated with tumorigenesis and cancer progression. Recent studies reported that piRNAs are contained in extracellular vesicles (EVs), nanosized lipid particles, with key roles in cell-cell communication. EVs contain several bioactive molecules, such as proteins, lipids, and nucleic acids, including emerging ncRNAs. EVs are one of the components of liquid biopsy (LB) a non-invasive method for detecting specific molecular biomarkers in liquid samples. LB could become a crucial tool for cancer diagnosis with piRNAs as biomarkers in a precision oncology approach. This review summarizes the current findings on the roles of piRNAs in different cancer types, focusing on potential theranostic applications of piRNAs contained in EVs (EV-piRNAs). Their roles as non-invasive diagnostic and prognostic biomarkers and as new therapeutic options have been also discussed.

Could very low-calorie ketogenic diets turn off low grade inflammation in obesity? Emerging evidence.

Obesity is an emerging non-communicable disease associated with chronic low-grade inflammation and oxidative stress, compounded by the development of many obesity-related diseases, such as cardiovascular disease, type 2 diabetes mellitus, and a range of cancers. Originally developed for the treatment of epilepsy in drug non-responder children, the ketogenic diet (KD) is being increasingly used in the treatment of many diseases, including obesity and obesity-related conditions. The KD is a dietary pattern characterized by high fat intake, moderate to low protein consumption, and very low carbohydrate intake (<50 g) that has proved to be an effective and weight-loss tool. In addition, it also appears to be a dietary intervention capable of improving the inflammatory state and oxidative stress in individuals with obesity by means of several mechanisms. The main activity of the KD has been linked to improving mitochondrial function and decreasing oxidative stress. ß-hydroxybutyrate, the most studied ketone body, has been shown to reduce the production of reactive oxygen species, improving mitochondrial respiration. In addition, KDs exert anti-inflammatory activity through several mechanisms, e.g., by inhibiting activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, and the inflammatory nucleotide-binding, leucine-rich-containing family, pyrin domain-containing-3, and inhibiting histone deacetylases. Given the rising interest in the topic, this review looks at the underlying anti-inflammatory and antioxidant mechanisms of KDs and their possible recruitment in the treatment of obesity and obesity-related disorders.

Emerging noncoding RNAs contained in extracellular vesicles: rising stars as biomarkers in lung cancer liquid biopsy.

Lung cancer has a high morbidity and mortality rate, and affected patients have a poor prognosis and low survival. The therapeutic approaches for lung cancer treatment, including surgery, radiotherapy, and chemotherapy, are not completely effective, due to late diagnosis. Although the identification of genetic drivers has contributed to the improvement of lung cancer clinical management, the discovery of new diagnostic and prognostic tools remains a critical issue. Liquid biopsy (LB) represents a minimally invasive approach and practical alternative source to investigate tumor-derived alterations and to facilitate the selection of targeted therapies. LB allows for the testing of different analytes such as circulating tumor cells, extracellular vesicles (EVs), tumor-educated platelets, and cell-free nucleic acids including DNAs, RNAs, and noncoding RNAs (ncRNAs). Several regulatory factors control the key cellular oncogenic pathways involved in cancers. ncRNAs have a wide range of regulatory effects in lung cancers. This review focuses on emerging regulatory ncRNAs, freely circulating in body fluids or shuttled by EVs, such as circular-RNAs, small nucleolar-RNAs, small nuclear-RNAs, and piwi-RNAs, as new biomarkers for early detection, prognosis, and monitoring of therapeutic strategy of lung cancer treatment.

Mesoporous Silica Particles Functionalized with Newly Extracted Fish Oil (Omeg@Silica) Reducing IL-8 Counteract Cell Migration in NSCLC Cell Lines.

Lung cancer is one of the leading forms of cancer in developed countries. Interleukin-8 (IL-8), a pro-inflammatory cytokine, exerts relevant effects in cancer growth and progression, including angiogenesis and metastasis in lung cancer. Mesoporous silica particles, functionalized with newly extracted fish oil (Omeg@Silica), are more effective than the fish oil alone in anti-proliferative and pro-apoptotic effects in non-small cell lung cancer (NSCLC) cell lines. The mechanisms that explain this efficacy are not yet understood. The aim of the present study is therefore to decipher the anti-cancer effects of a formulation of Omeg@Silica in aqueous ethanol (FOS) in adenocarcinoma (A549) and muco-epidermoid (NCI-H292) lung cancer cells, evaluating cell migration, as well as IL-8, NF-κB, and miRNA-21 expression. Results show that in both cell lines, FOS was more efficient than oil alone, in decreasing cell migration and IL-8 gene expression. FOS reduced IL-8 protein release in both cell lines, but this effect was only stronger than the oil alone in A549. In A549, FOS was able to reduce miRNA-21 and transcription factor NF-κB nuclear expression. Taken together, these data support the potential use of the Omeg@Silica as an add-on therapy for NSCLC. Dedicated studies which prove clinical efficacy are needed.

Generation and Characterization of Novel iPSC Lines from a Portuguese Family Bearing Heterozygous and Homozygous GRN Mutations.

Mutations in granulin (GRN) have been associated with neurodegenerative diseases, such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). In Portugal, GRN mutations account for around half of all FTLD cases with known genetic origin. Here, we describe the generation and characterization of three human-induced pluripotent stem cell (hiPSC) lines from a Portuguese family harboring heterozygous and homozygous GRN mutation. hiPSCs were reprogrammed from human dermal fibroblasts by episomal nucleofection of the Yamanaka factors. The new generated lines were positive for pluripotency markers, could be further differentiated to cells expressing all trilineage markers, and presented a normal karyotype. They were also capable of differentiating into 3D brain organoids and presented a significant decrease in progranulin protein levels. Hence, these cell lines constitute suitable new tools to elucidate the pathophysiological mechanisms associated with the GRN mutations in the context of FTLD.

Extracellular Vesicles-ceRNAs as Ovarian Cancer Biomarkers: Looking into circRNA-miRNA-mRNA Code.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies in females worldwide. OC is frequently diagnosed at an advanced stage due to a lack of specific symptoms and effective screening tests, resulting in a poor prognosis for patients. Age, genetic alterations, and family history are the major risk factors for OC pathogenesis. Understanding the molecular mechanisms underlying OC progression, identifying new biomarkers for early detection, and discovering potential targets for new drugs are urgent needs. Liquid biopsy (LB), used for cancer detection and management, consists of a minimally invasive approach and practical alternative source to investigate tumor alterations by testing extracellular vesicles (EVs), circulating tumor cells, tumor-educated platelets, and cell-free nucleic acids. EVs are nanosize vesicles shuttling proteins, lipids, and nucleic acids, such as DNA, RNA, and non-coding RNAs (ncRNAs), that can induce phenotypic reprogramming of target cells. EVs are natural intercellular shuttles for ncRNAs, such as microRNAs (miRNAs) and circular-RNAs (circRNAs), known to have regulatory effects in OC. Here we focus on the involvement of circRNAs and miRNAs in OC cancer progression. The circRNA-microRNA-mRNA axis has been investigated with Circbank and miRwalk analysis, unraveling the intricate and detailed regulatory network created by EVs, ncRNAs, and mRNAs in OC.

miR-126-3p and miR-21-5p as Hallmarks of Bio-Positive Ageing; Correlation Analysis and Machine Learning Prediction in Young to Ultra-Centenarian Sicilian Population.

Human ageing can be characterized by a profile of circulating microRNAs (miRNAs), which are potentially predictors of biological age. They can be used as a biomarker of risk for age-related inflammatory outcomes, and senescent endothelial cells (ECs) have emerged as a possible source of circulating miRNAs. In this paper, a panel of four circulating miRNAs including miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p, involved in several pathways related to inflammation, and ECs senescence that seem to be characteristic of the healthy ageing phenotype. The circulating levels of these miRNAs were determined in 78 healthy subjects aged between 22 to 111 years. Contextually, extracellular miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p levels were measured in human ECs in vitro model, undergoing senescence. We found that the levels of the four miRNAs, using ex vivo and in vitro models, progressively increase with age, apart from ultra-centenarians that showed levels comparable to those measured in young individuals. Our results contribute to the development of knowledge regarding the identification of miRNAs as biomarkers of successful and unsuccessful ageing. Indeed, they might have diagnostic/prognostic relevance for age-related diseases.

High Mobility Group Box 1: Biological Functions and Relevance in Oxidative Stress Related Chronic Diseases.

In the early 1970s, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and named high-mobility group (HMG) proteins. High-mobility group box 1 (HMGB1) is the most studied HMG protein that detects and coordinates cellular stress response. The biological function of HMGB1 depends on its subcellular localization and expression. It plays a critical role in the nucleus and cytoplasm as DNA chaperone, chromosome gatekeeper, autophagy maintainer, and protector from apoptotic cell death. HMGB1 also functions as an extracellular alarmin acting as a damage-associated molecular pattern molecule (DAMP). Recent findings describe HMGB1 as a sophisticated signal of danger, with a pleiotropic function, which is useful as a clinical biomarker for several disorders. HMGB1 has emerged as a mediator in acute and chronic inflammation. Furthermore, HMGB1 targeting can induce beneficial effects on oxidative stress related diseases. This review focus on HMGB1 redox status, localization, mechanisms of release, binding with receptors, and its activities in different oxidative stress-related chronic diseases. Since a growing number of reports show the key role of HMGB1 in socially relevant pathological conditions, to our knowledge, for the first time, here we analyze the scientific literature, evaluating the number of publications focusing on HMGB1 in humans and animal models, per year, from 2006 to 2021 and the number of records published, yearly, per disease and category (studies on humans and animal models).

Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) "young" and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

Pompe disease: pathogenesis, molecular genetics and diagnosis.

Pompe disease (PD) is a rare autosomal recessive disorder caused by mutations in the GAA gene, localized on chromosome 17 and encoding for acid alpha-1,4-glucosidase (GAA). Currently, more than 560 mutations spread throughout GAA gene have been reported. GAA catalyzes the hydrolysis of α-1,4 and α-1,6-glucosidic bonds of glycogen and its deficiency leads to lysosomal storage of glycogen in several tissues, particularly in muscle. PD is a chronic and progressive pathology usually characterized by limb-girdle muscle weakness and respiratory failure. PD is classified as infantile and childhood/adult forms. PD patients exhibit a multisystemic manifestation that depends on age of onset.Early diagnosis is essential to prevent or reduce the irreversible organ damage associated with PD progression. Here, we make an overview of PD focusing on pathogenesis, clinical phenotypes, molecular genetics, diagnosis, therapies, autophagy and the role of miRNAs as potential biomarkers for PD.

The Phenotypic Characterization of the Cammalleri Sisters, an Example of Exceptional Longevity.

This article shows demographic, clinical, anamnestic, cognitive, and functional data as well as biochemical, genetic, and epigenetic parameters of two exceptional siblings: Diega (supercentenarian) and Filippa (semisupercentenarian) Cammalleri. The purpose of this study is to provide new insights into the extreme phenotypes represented by semisupercentenarians and supercentenarians. Different studies have been published on supercentenarians, but to the best of our knowledge, this is the only concerning two sisters and the most detailed from a phenotypic point of view. Our findings agree with the suggestion that supercentenarians have an increasing relative resistance to age-related diseases, approximating the limits of the functional human reserve to address successfully the acute causes of death. More interestingly, our data agree with, and extend, the suggestion that inflammation and oxidative stress predict centenarian mortality.

Circulating miRNAs in Successful and Unsuccessful Aging. A Mini-review.

Aging is a multifactorial process that affects the organisms at genetic, molecular and cellular levels. This process modifies several tissues with a negative impact on cells physiology, tissues and organs functionality, altering their regeneration capacity. The chronic low-grade inflammation typical of aging, defined as inflammaging, is a common biological factor responsible for the decline and beginning of the disease in age. A murine parabiosis model that combines the vascular system of old and young animals, suggests that soluble factors released by young individuals may improve the regenerative potential of old tissue. Therefore, circulating factors have a key role in the induction of aging phenotype. Moreover, lifestyle can influence the physiological status of multiple organs, via epigenetic mechanisms. Recently, microRNAs are considered potential sensors of aging.

Mutations in the GLA Gene and LysoGb3: Is It Really Anderson-Fabry Disease?

Anderson-Fabry disease (FD) is a rare, progressive, multisystem storage disorder caused by the partial or total deficit of the lysosomal enzyme α-galactosidase A (α-Gal A). It is an X-linked, lysosomal enzymopathy due to mutations in the galactosidase alpha gene (GLA), encoding the α-Gal A. To date, more than 900 mutations in this gene have been described. In our laboratories, the study of genetic and enzymatic alterations related to FD was performed in about 17,000 subjects with a symptomatology referable to this disorder. The accumulation of globotriaosylsphingosine (LysoGb3) was determined in blood of positives. Exonic mutations in the GLA gene were detected in 471 patients (207 Probands and 264 relatives): 71.6% of mutations were associated with the classic phenotype, 19.8% were associated with the late-onset phenotype, and 8.6% of genetic variants were of unknown significance (GVUS). The accumulation of LysoGb3 was found in all male patients with a mutation responsible for classic or late-onset FD. LysoGb3 levels were consistent with the type of mutations and the symptomatology of patients. α-Gal A activity in these patients is absent or dramatically reduced. In recent years, confusion about the pathogenicity of some mutations led to an association between non-causative mutations and FD. Our study shows that the identification of FD patients is possible by associating clinical history, GLA gene analysis, α-Gal A assay, and blood accumulation of LysoGB3. In our experience, LysoGB3 can be considered a reliable marker, which is very useful to confirm the diagnosis of Fabry disease.

Systematic DNA Study for Fabry Disease in the End Stage Renal Disease Patients from a Southern Italy Area.

BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by pervasive renal involvement. However, this disease is underdiagnosed in patient with chronic kidney disease (CKD), including those with end stage renal disease (ESRD), so their investigation represents an unexploited opportunity for early diagnosis of the disease and for its identification in relatives of affected patients. METHODS: We investigated Fabry disease in a clinical and biological database including ESRD patients of unknown cause in a geographical area with 2 million residents. The study was based on state of art GLA gene sequencing and was extended to relatives of affected ESRD patients. RESULTS: Among ESRD patients qualified for enrollment into this study, a previously undiagnosed young man harboring the mutation p.I91T was identified. The study of the proband's family led to the identification of 8 additional cases. In another ESRD male patient, we identified the functional polymorphism p.D313Y. Furthermore, in 55 ESRD patients (24.2%) we found intronic polymorphisms of uncertain functional relevance in the non-coding regions of the GLA gene. CONCLUSION: A comprehensive survey of ESRD patients in a geographical area of 2 million residents identified one undiagnosed case of Fabry disease and led to the identification of 8 additional cases among his relatives. Screening protocols starting from the dialysis population and upstream extended to families of affected individuals may be an effective strategy to maximize the early identification of subjects with Fabry disease.

A pilot study of circulating microRNAs as potential biomarkers of Fabry disease.

Patients suffering from Fabry disease (FD), a lysosomal storage disorder, show a broad range of symptoms and the diagnosis followed by the therapeutic decision remains a great challenge. The biomarkers available today have not proven to be useful for predicting the evolution of the disease and for assessing response to therapy in many patients. Here, we used high-throughput microRNA profiling methodology to identify a specific circulating microRNA profile in FD patients. We discovered a pattern of 10 microRNAs able to identify FD patients when compared to healthy controls. Notably, two of these: the miR199a-5p and the miR-126-3p are able to discriminate FDs from the control subjects with left ventricular hypertrophy, a frequent but non-specific FD symptom. These same microRNAs are also sensitive to enzyme replacement therapy showing variation in the subjects under treatment. Furthermore, two other microRNAs of the profile, the miR-423-5p and the miR-451a, seem useful to highlight cardiac involvement in FD patients. A literature and database search revealed that miR-199a-5p, miR-126-3p, miR-423-5p and miR-451a are known to be linked to pathological states that occur during the FD development. In particular, miR-199a-5p, and miR-126-3p are involved in endothelial dysfunction and miR-423-5p and miR-451a in myocardial remodeling. In conclusion, in this study we identified a common plasma microRNA profile in FD patients, useful not only for the correct classification of Fabry patients regardless of sex and age, but also to evaluate the response to therapy. Furthermore, our observations suggest that some microRNAs of this profile demonstrate prognostic qualities.

Fabry disease and multiple sclerosis misdiagnosis: the role of family history and neurological signs.

Fabry disease (FD) is an X-linked inherited lysosomal storage disorder caused by α galactosidase A (α-gal A) deficiency. Central nervous system involvement and chronic white matter lesions are observed in both FD and multiple sclerosis (MS), which can confound the differential diagnosis. We analyzed the GLA gene, which encodes α-gal A, in 86 patients with clinical and neuroradiological findings consistent with MS to determine whether they had FD. We identified four women initially diagnosed with MS who had GLA mutations associated with FD. Our results indicate that family history besides neurological findings should be evaluated in patients with an uncertain diagnosis of MS. Also the involvement of organs outside the central nervous system can support the FD diagnosis.

Molecular and clinical studies in five index cases with novel mutations in the GLA gene.

Fabry disease is a metabolic and lysosomal storage disorder caused by the functional defect of the α-galactosidase A enzyme; this defect is due to mutations in the GLA gene, that is composed of seven exons and is located on the long arm of the X-chromosome (Xq21-22). The enzymatic deficit is responsible for the accumulation of glycosphingolipids in lysosomes of different cellular types, mainly in those ones of vascular endothelium. It consequently causes a cellular and microvascular dysfunction. In this paper, we described five novel mutations in the GLA gene, related to absent enzymatic activity and typical manifestations of Fabry disease. We identified three mutations (c.846_847delTC, p.E341X and p.C382X) that lead to the introduction of a stop codon in positions 297, 341 and 382. Moreover we found a missense mutation (p.R227P) in the exon 5 of the GLA gene and a single point mutation (c.639+5 G>T) occurring five base pairs beyond the end of the exon 4. These mutations have never been found in our group of healthy control subjects >2300. The studied patients presented some clinical manifestations, such as cornea verticillata, hypo-anhidrosis, left ventricular hypertrophy, cerebrovascular disorders and renal failure, that, considering the null enzymatic activity, suggest that the new mutations reported here are related to the classic form of Fabry disease. The identification of novel mutations in patients with symptomatology referable to FD increases the molecular knowledge of the GLA gene and it gives clinicians an important support for the proper diagnosis of the disease.

miR-155 regulative network in FLT3 mutated acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) represents a heterogeneous disorder with recurrent chromosomal alterations and molecular abnormalities. Among AML with normal karyotype (NK-AML) FLT3 activating mutation, internal tandem duplication (FLT3-ITD), is present in about 30% of patients, conferring unfavorable outcome. Our previous data demonstrated specific up-regulation of miR-155 in FLT3-ITD+ AML. miR-155 is known to be directly implicated in normal hematopoiesis and in some pathologies such as myeloid hyperplasia and acute lymphoblastic leukemia. METHODS AND RESULTS: To investigate about the potential influence of miR-155 de-regulation in FLT3-mutated AML we generated a transcription factors regulatory network and combined this with data from multiple sources that predict miR-155 interactions. From these analyses, we derived a sub-network, called "miR-155 module" that describes functional relationship among miR-155 and transcription factors in FLT3-mutated AML. We found that "miR-155 module" is characterized by the presence of six transcription factors as central hubs: four miR-155 regulators (JUN, RUNX1, FOSb, JUNB) and two targets of miR-155 (SPI1, CEBPB) all known to be "master" genes of myelopoiesis. We found, in FLT3-mutated AML, a significant down-regulation of miR-155 target genes CEBPB and SPI1 and up-regulation of miR-155 regulator genes JUN and RUNX1. We also showed that PKC412-related FLT3 inhibition, in MV4-11 cell line, causes down-regulation of miR-155 and increased level of mRNA and protein of miR-155 target SPI1. We showed in experiments of miR-155 mimic in K562 cell line, a high increase of miR-155 and an inverse correlation with the mRNA levels of its targets SPI1 and CEBPB. Moreover silencing of miR-155 in primary AMLs causes mRNA up-regulation of its target SPI1 and CEBPB. CONCLUSION: Our results suggest that activating mutation of FLT3 in AML can lead, through the induction of JUN, to an increased expression of miR-155, which then causes down-regulation of SPI1 and CEBPB and consequently may causes block of myeloid differentiation.

High variability of Fabry disease manifestations in an extended Italian family.

Fabry disease (FD) is an inherited metabolic disorder caused by partial or full inactivation of the lysosomal hydrolase α-galactosidase A (α-GAL). The impairment of α-GAL results in the accumulation of undegraded glycosphingolipids in lysosomes and subsequent cell and microvascular dysfunctions. This study reports the clinical, biochemical, and molecular characterization of 15 members of the same family. Eight members showed the exonic mutation M51I in the GLA gene, a disease-causing mutation associated with the atypical phenotype. The clinical history of this family highlights a wide phenotypic variability, in terms of involved organs and severity. The phenotypic variability of two male patients is not related to differences in α-GAL enzymatic activity: though both have no enzymatic activity, the youngest shows severe symptoms, while the eldest is asymptomatic. It is noticeable that for two female patients with the M51I mutation the initial clinical diagnosis was different from FD. One of them was diagnosed with Familial Mediterranean Fever, the other with Multiple Sclerosis. Overall, this study confirms that the extreme variability of the clinical manifestations of FD is not entirely attributable to different mutations in the GLA gene and emphasizes the need to consider other factors or mechanisms involved in the pathogenesis of Fabry Disease.