Sjögren's syndrome: novel insights from proteomics and miRNA expression analysis.

Introduction: Sjögren's syndrome (SS) is a systemic autoimmune disease, which affects the exocrine glands leading to glandular dysfunction and, particularly, symptoms of oral and ocular dryness. The aetiology of SS remains unclear, and the disease lacks distinctive clinical features. The current diagnostic work-up is complex, invasive and often time-consuming. Thus, there is an emerging need for identifying disease-specific and, ideally, non-invasive immunological and molecular biomarkers that can simplify the diagnostic process, allow stratification of patients, and assist in monitoring the disease course and outcome of therapeutic intervention in SS. Methods: This systematic review addresses the use of proteomics and miRNA-expression profile analyses in this regard. Results and discussion: Out of 272 papers that were identified and 108 reviewed, a total of 42 papers on proteomics and 23 papers on miRNA analyses in saliva, blood and salivary gland tissue were included in this review. Overall, the proteomic and miRNA studies revealed considerable variations with regard to candidate biomarker proteins and miRNAs, most likely due to variation in sample size, processing and analytical methods, but also reflecting the complexity of SS and patient heterogeneity. However, interesting novel knowledge has emerged and further validation is needed to confirm their potential role as biomarkers in SS.

Serum microRNAs associated with concussion in football players.

Mild Traumatic Brain Injury (mild TBI)/concussion is a common sports injury, especially common in football players. Repeated concussions are thought to lead to long-term brain damage including chronic traumatic encephalopathy (CTE). With the worldwide growing interest in studying sport-related concussion the search for biomarkers for early diagnosis and progression of neuronal injury has also became priority. MicroRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally. Due to their high stability in biological fluids, microRNAs can serve as biomarkers in a variety of diseases including pathologies of the nervous system. In this exploratory study, we have evaluated changes in the expression of selected serum miRNAs in collegiate football players obtained during a full practice and game season. We found a miRNA signature that can distinguish with good specificity and sensitivity players with concussions from non-concussed players. Furthermore, we found miRNAs associated with the acute phase (let-7c-5p, miR-16-5p, miR-181c-5p, miR-146a-5p, miR-154-5p, miR-431-5p, miR-151a-5p, miR-181d-5p, miR-487b-3p, miR-377-3p, miR-17-5p, miR-22-3p, and miR-126-5p) and those whose changes persist up to 4 months after concussion (miR-17-5p and miR-22-3p).

Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model.

Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.

Association of circulating miRNAS in patients with Alstrom and Bardet-Biedl syndromes with clinical course parameters.

Background: Patients with the rare syndromic forms of monogenic diabetes: Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) have multiple metabolic abnormalities, including early-onset obesity, insulin resistance, lipid disorders and type 2 diabetes mellitus. The aim of this study was to determine if the expression of circulating miRNAs in patients with ALMS and BBS differs from that in healthy and obese individuals and determine if miRNA levels correlate with metabolic tests, BMI-SDS and patient age. Methods: We quantified miRNA expression (Qiagen, Germany) in four groups of patients: with ALMS (n=13), with BBS (n=7), patients with obesity (n=19) and controls (n=23). Clinical parameters including lipids profile, serum creatinine, cystatin C, fasting glucose, insulin and C-peptide levels, HbA1c values and insulin resistance (HOMA-IR) were assessed in patients with ALMS and BBS. Results: We observed multiple up- or downregulated miRNAs in both ALMS and BBS patients compared to obese patients and controls, but only 1 miRNA (miR-301a-3p) differed significantly and in the same direction in ALMS and BBS relative to the other groups. Similarly, 1 miRNA (miR-92b-3p) was dysregulated in the opposite directions in ALMS and BBS patients, but diverged from 2 other groups. We found eight miRNAs (miR-30a-5p, miR-92b-3p, miR-99a-5p, miR-122-5p, miR-192-5p, miR-193a-5p, miR-199a-3p and miR-205-5p) that significantly correlated with at least of the analyzed clinical variables representing an association with the course of the diseases. Conclusions: Our results show for the first time that serum miRNAs can be used as available indicators of disease course in patients with ALMS and BBS syndromes.

Impact of differing methodologies for serum miRNA-371a-3p assessment in stage I testicular germ cell cancer recurrence.

Introduction: Current evidence shows that serum miR-371a-3p can identify disease recurrence in testicular germ cell tumour (TGCT) patients and correlates with tumour load. Despite convincing evidence showing the advantages of including miR-371a-3p testing to complement and overcome the classical serum tumour markers limitations, the successful introduction of a serum miRNA based test into clinical practice has been impeded by a lack of consensus regarding optimal methodologies and lack of a universal protocol and thresholds. Herein, we investigate two quantitative real-time PCR (qRT-PCR) based pipelines in detecting disease recurrence in stage I TGCT patients under active surveillance, and compare the sensitivity and specificity for each method. Methods: Sequential serum samples collected from 33 stage I TGCT patients undergoing active surveillance were analysed for miR-371a-3p via qRT-PCR with and without an amplification step included. Results: Using a pre-amplified protocol, all known recurrences were detected via elevated miR-371a-3p expression, while without pre-amplification, we failed to detect recurrence in 3/10 known recurrence patients. For pre-amplified analysis, sensitivity and specificity was 90% and 94.4% respectively. Without amplification, sensitivity dropped to 60%, but exhibited 100% specificity. Discussion: We conclude that incorporating pre-amplification increases sensitivity of miR-371a-3p detection, but produces more false positive results. The ideal protocol for quantification of miR-371a-3p still needs to be determined. TGCT patients undergoing active surveillance may benefit from serum miR-371a-3p quantification with earlier detection of recurrences compared to current standard methods. However, larger cross-institutional studies where samples are processed and data is analysed in a standardised manner are required prior to its routine clinical implementation.

Advances in vitiligo: Update on therapeutic targets.

Vitiligo, whose treatment remains a serious concern and challenge, is an autoimmune skin disease characterized by patches of depigmentation. The increasing application of molecular-targeted therapy in skin diseases, such as psoriasis and systemic lupus erythematosus, has dramatically improved their condition. Besides, there is a favorable effect of repigmentation in the treatment of the above diseases combined with vitiligo, implying that molecular-targeted therapy may also have utility in vitiligo treatment. Recently, the role of cytokine and signaling pathways in vitiligo pathogenesis are increasingly recognized. Thus, investigations are underway targeting the molecules described above. In this paper, we present a synopsis of current practices in vitiligo treatment and introduce the improvement in identifying new molecular targets and applying molecular-targeted therapies, including those under development in vitiligo treatment, providing valuable insight into establishing further precision medicine for vitiligo patients.

Long Non-Coding RNAs in the Cell Fate Determination of Neoplastic Thymic Epithelial Cells.

Thymic Epithelial Tumors (TETs) arise from epithelial cells of the thymus and are very rare neoplasms comprising Thymoma, Thymic carcinoma, and Thymic Neuroendocrine tumors that still require in-depth molecular characterization. Long non-coding RNAs (lncRNAs) are emerging as relevant gene expression modulators involved in the deregulation of several networks in almost all types of human cancer, including TETs. LncRNAs act at different control levels in the regulation of gene expression, from transcription to translation, and modulate several pathways relevant to cell fate determination under normal and pathological conditions. The activity of lncRNAs is strongly dependent on their expression, localization, and post-transcriptional modifications. Starting from our recently published studies, this review focuses on the involvement of lncRNAs in the acquisition of malignant traits by neoplastic thymic epithelial cells, and describes the possible use of these molecules as targets for the design of novel therapeutic approaches specific for TET. Furthermore, the involvement of lncRNAs in myasthenia gravis (MG)-related thymoma, which is still under investigation, is discussed.

The Role of Extracellular Vesicles as a Shared Disease Mechanism Contributing to Multimorbidity in Patients With COPD.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. Individuals with COPD typically experience a progressive, debilitating decline in lung function as well as systemic manifestations of the disease. Multimorbidity, is common in COPD patients and increases the risk of hospitalisation and mortality. Central to the genesis of multimorbidity in COPD patients is a self-perpetuating, abnormal immune and inflammatory response driven by factors including ageing, pollutant inhalation (including smoking) and infection. As many patients with COPD have multiple concurrent chronic conditions, which require an integrative management approach, there is a need to greater understand the shared disease mechanisms contributing to multimorbidity. The intercellular transfer of extracellular vesicles (EVs) has recently been proposed as an important method of local and distal cell-to-cell communication mediating both homeostatic and pathological conditions. EVs have been identified in many biological fluids and provide a stable capsule for the transfer of cargo including proteins, lipids and nucleic acids. Of these cargo, microRNAs (miRNAs), which are short 17-24 nucleotide non-coding RNA molecules, have been amongst the most extensively studied. There is evidence to support that miRNA are selectively packaged into EVs and can regulate recipient cell gene expression including major pathways involved in inflammation, apoptosis and fibrosis. Furthermore changes in EV cargo including miRNA have been reported in many chronic diseases and in response to risk factors including respiratory infections, noxious stimuli and ageing. In this review, we discuss the potential of EVs and EV-associated miRNA to modulate shared pathological processes in chronic diseases. Further delineating these may lead to the identification of novel biomarkers and therapeutic targets for patients with COPD and multimorbidities.

Identification of the Association Between Toll-Like Receptors and T-Cell Activation in Takayasu's Arteritis.

To explore the relationships between Toll-like receptors (TLRs) and the activation and differentiation of T-cells in Takayasu's arteritis (TAK), using real-time fluorescence quantitative polymerase chain reaction, mRNA abundance of 29 target genes in peripheral blood mononuclear cells (PBMCs) were detected from 27 TAK patients and 10 healthy controls. Compared with the healthy control group, the untreated TAK group and the treated TAK group had an increased mRNA level of TLR2 and TLR4. A sample-to-sample matrix revealed that 80% of healthy controls could be separated from the TAK patients. Correlation analysis showed that the inactive-treated TAK group exhibited a unique pattern of inverse correlations between the TLRs gene clusters (including TLR1/2/4/6/8, BCL6, TIGIT, NR4A1, etc) and the gene cluster associated with T-cell activation and differentiation (including TCR, CD28, T-bet, GATA3, FOXP3, CCL5, etc). The dynamic gene co-expression network indicated the TAK groups had more active communication between TLRs and T-cell activation than healthy controls. BCL6, CCL5, FOXP3, GATA3, CD28, T-bet, TIGIT, IκBα, and NR4A1 were likely to have a close functional relation with TLRs at the inactive stage. The co-expression of TLR4 and TLR6 could serve as a biomarker of disease activity in treated TAK (the area under curve/sensitivity/specificity, 0.919/100%/90.9%). The largest gene co-expression cluster of the inactive-treated TAK group was associated with TLR signaling pathways, while the largest gene co-expression cluster of the active-treated TAK group was associated with the activation and differentiation of T-cells. The miRNA sequencing of the plasma exosomes combining miRDB, DIANA-TarBase, and miRTarBase databases suggested that the miR-548 family miR-584, miR-3613, and miR-335 might play an important role in the cross-talk between TLRs and T-cells at the inactive stage. This study found a novel relation between TLRs and T-cell in the pathogenesis of autoimmune diseases, proposed a new concept of TLR-co-expression signature which might distinguish different disease activity of TAK, and highlighted the miRNA of exosomes in TLR signaling pathway in TAK.

Whole Blood Holding Time Prior to Plasma Processing Alters microRNA Expression Profile.

MicroRNAs (miRNAs) can exhibit aberrant expression under different physiological and pathological conditions. Therefore, differentially expressed circulating miRNAs have been a focus of biomarker discovery research. However, the use of circulating miRNAs comes with challenges which may hinder the reliability for their clinical application. These include varied sample collection protocols, storage times/conditions, sample processing and analysis methods. This study focused on examining the effect of whole blood holding time on the stability of plasma miRNA expression profiles. Whole blood samples were collected from healthy pregnant women and were held at 4°C for 30 min, 2 h, 6 h or 24 h prior to processing for plasma isolation. Plasma RNA was extracted and the expression of 179 miRNAs were analyzed. Unsupervised principal component analysis demonstrated that whole blood holding time was a major source of variation in miRNA expression profiles with 53 of 179 miRNAs showing significant changes in expression. Levels of specific miRNAs previously reported to be associated with pregnancy-associated complications such as hsa-miR-150-5p, hsa-miR-191-5p, and hsa-miR-29a-3p, as well as commonly used endogenous miRNA controls, hsa-miR-16-5p, hsa-miR-25-3p, and hsa-miR-223-3p were significantly altered with increase in blood holding time. Current protocols for plasma-based miRNA profiling for diagnostics describe major differences in whole blood holding periods ranging from immediately after collection to 26 h after. Our results demonstrate holding time can have dramatic effects on analytical reliability and reproducibility. This highlights the importance of standardization of blood holding time prior to processing for plasma in order to minimize introduction of non-biological variance in miRNA profiles.

Ovarian Aging: Role of Pituitary-Ovarian Axis Hormones and ncRNAs in Regulating Ovarian Mitochondrial Activity.

The number of mitochondria in the oocyte along with their functions (e.g., energy production, scavenger activity) decline with age progression. Such multifaceted functions support several processes during oocyte maturation, ranging from energy supply to synthesis of the steroid hormones. Hence, it is hardly surprising that their impairment has been reported in both physiological and premature ovarian aging, wherein they are crucial players in the apoptotic processes that arise in aged ovaries. In any form, ovarian aging implies the progressive damage of the mitochondrial structure and activities as regards to ovarian germ and somatic cells. The imbalance in the circulating hormones and peptides (e.g., gonadotropins, estrogens, AMH, activins, and inhibins), active along the pituitary-ovarian axis, represents the biochemical sign of ovarian aging. Despite the progress accomplished in determining the key role of the mitochondria in preserving ovarian follicular number and health, their modulation by the hormonal signalling pathways involved in ovarian aging has been poorly and randomly explored. Yet characterizing this mechanism is pivotal to molecularly define the implication of mitochondrial dysfunction in physiological and premature ovarian aging, respectively. However, it is fairly difficult considering that the pathways associated with ovarian aging might affect mitochondria directly or by altering the activity, stability and localization of proteins controlling mitochondrial dynamics and functions, either unbalancing other cellular mediators, released by the mitochondria, such as non-coding RNAs (ncRNAs). We will focus on the mitochondrial ncRNAs (i.e., mitomiRs and mtlncRNAs), that retranslocate from the mitochondria to the nucleus, as active players in aging and describe their role in the nuclear-mitochondrial crosstalk and its modulation by the pituitary-ovarian hormone dependent pathways. In this review, we will illustrate mitochondria as targets of the signaling pathways dependent on hormones and peptides active along the pituitary/ovarian axis and as transducers, with a particular focus on the molecules retrieved in the mitochondria, mainly ncRNAs. Given their regulatory function in cellular activities we propose them as potential diagnostic markers and/or therapeutic targets.

Evidence for Training-Induced Changes in miRNA Levels in the Skeletal Muscle of Patients With Type 2 Diabetes Mellitus.

Physical training can improve glycemic control in patients with type 2 diabetes mellitus (T2DM). However, the underlying mechanisms are not entirely clear. An interesting piece of the puzzle could be the regulation of micro-RNAs (miRNAs). They are important modulators of protein expression. Some miRNAs were found to be both linked to poor glycemic control/insulin resistance (with evidence from in vivo and/or in vitro studies) and dysregulated in the skeletal muscle of T2DM patients. This pilot study examines whether a 3-month endurance training program [three times a week, 70-80% peak heart rate (HRpeak)] can down-regulate their levels in T2DM men (n = 7). One skeletal muscle biopsy sample was obtained from each patient at T1 (6 weeks pre-intervention), one at T2 (1 week pre-intervention) and one at T3 (3-4 days post-intervention). miRNA-27a-3p, -29a-3p, -29b-3p, -29c-3p, -106b-5p, -135a-5p, -143-3p, -144-3p, -194-5p, and - 206 levels were determined by RT-qPCR. Friedman ANOVA and post-hoc tests showed that miRNA-29b-3p, -29c-3p and -135a-5p levels were significantly reduced post-training (T3 vs. T2 and/or T1). Glycated hemoglobin (HbA1c) and HOMA insulin resistance index did not change significantly. However, HbA1c was reduced in 6 of 7 patients post-training. Furthermore, Spearman's rank correlation analyses with all values from all time points showed significant negative associations between miRNA-29c-3p, -106b-5p, -144-3p and -194-5p levels and cardiorespiratory fitness (VO2peak). The study results imply that regular exercise and improving one's physical fitness is helpful for the regulation of skeletal muscle miRNAs in T2DM patients. Whether or not changes in the miRNA profile can affect the clinical situation of T2DM patients warrants further research.

Distinguishable Prognostic miRNA Signatures of Head and Neck Squamous Cell Cancer With or Without HPV Infection.

Since their discovery in the 1990's, microRNAs (miRNA) have opened up new vistas in the field of cancer biology and are found to have fundamental roles in tumorigenesis and progression. As head and neck squamous cell carcinoma (HNSCC) with positive human papillomavirus (HPV+) is significantly distinct from its HPV negative (HPV-) counterpart in terms of both molecular mechanisms and clinical prognosis, the current study aimed to separately develop miRNA signatures for HPV+ and HPV- HNSCC as well as to explore the potential functions. Both signatures were reliable for the prediction of prognosis in their respective groups. Then Enrichment analysis was performed to predict the potential biological functions of the signatures. Importantly, combining previous studies and our results, we speculated that HPV+ HNSCC patients with low signature score had better immunity against the tumors and enhanced the sensitivity of therapies leading to improved prognosis, while HPV- HNSCC patients with high signature score acquired resistance to therapeutic approaches as well as dysregulation of cell metabolism leading to poor prognosis. Hence, we believe that the identified signatures respectively for HPV+ and HPV- HNSCC, are of great significance in accessing patient outcomes as well as uncovering new biomarkers and therapeutic targets, which are worth further investigation through molecular biology experiments.