Expression Profiling of Circulating MicroRNAs in Canine Myxomatous Mitral Valve Disease

MicroRNAs (miRNAs) are small non-coding RNAs that have shown promise as noninvasive biomarkers in cardiac disease. This study was undertaken to investigate the miRNA expression profile in dogs with myxomatous mitral valve disease (MMVD). 277 miRNAs were quantified using RT-qPCR from six normal dogs (American College of Veterinary Internal Medicine Stage A), six dogs with MMVD mild to moderate cardiac enlargement (ACVIM Stage B1/B2) and six dogs with MMVD and congestive heart failure (ACVIM Stage C/D). Eleven miRNAs were differentially expressed (False Discovery Rate < 0.05). Dogs in Stage B1/B2 or C/D had four upregulated miRNAs, including three cfa-let-7/cfa-miR-98 family members, while seven others were downregulated, compared to Stage A. Expression of six of the 11 miRNAs also were significantly different between dogs in Stage C/D and those in Stage B1/B2. The expression changes were greater as disease severity increased. These miRNAs may be candidates for novel biomarkers and may provide insights into genetic regulatory pathways in canine MMVD.     

Differential MicroRNA Expression Profile in Myxomatous Mitral Valve Prolapse and Fibroelastic Deficiency Valves

Myxomatous mitral valve prolapse (MMVP) and fibroelastic deficiency (FED) are two common variants of degenerative mitral valve disease (DMVD), which is a leading cause of mitral regurgitation worldwide. While pathohistological studies have revealed differences in extracellular matrix content in MMVP and FED, the molecular mechanisms underlying these two disease entities remain to be elucidated. By using surgically removed valvular specimens from MMVP and FED patients that were categorized on the basis of echocardiographic, clinical and operative findings, a cluster of microRNAs that expressed differentially were identified. The expressions of has-miR-500, -3174, -17, -1193, -646, -1273e, -4298, -203, -505, and -939 showed significant differences between MMVP and FED after applying Bonferroni correction (p < 0.002174). The possible involvement of microRNAs in the pathogenesis of DMVD were further suggested by the presences of in silico predicted target sites on a number of genes reported to be involved in extracellular matrix homeostasis and marker genes for cellular composition of mitral valves, including decorin (DCN), aggrecan (ACAN), fibromodulin (FMOD), α actin 2 (ACTA2), extracellular matrix protein 2 (ECM2), desmin (DES), endothelial cell specific molecule 1 (ESM1), and platelet/ endothelial cell adhesion molecule 1 (PECAM1), as well as inverse correlations of selected microRNA and mRNA expression in MMVP and FED groups. Our results provide evidence that distinct molecular mechanisms underlie MMVP and FED. Moreover, the microRNAs identified may be targets for the future development of diagnostic biomarkers and therapeutics.     

Quantitative Prediction of Steatosis in Patients with Non-Alcoholic Fatty Liver by Means of Hepatic MicroRNAs Present in Serum and Correlating with Hepatic Fat

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease worldwide, but a reliable non-invasive method to quantify liver steatosis in primary healthcare is not available. Circulating microRNAs have been proposed as biomarkers of severe/advanced NAFLD (steatohepatitis and fibrosis). However, the use of circulating miRNAs to quantitatively assess the % of liver fat in suspected NAFLD patients has not been investigated. We performed global miRNA sequencing in two sets of samples: human livers from organ donors (n = 20), and human sera from biopsy-proven NAFLD patients (n = 23), both with a wide range of steatosis quantified in their liver biopsies. Partial least squares (PLS) regression combined with recursive feature elimination (RFE) was used to select miRNAs associated with steatosis. Moreover, regression models with only 2 or 3 miRNAs, with high biological relevance, were built. Comprehensive microRNA sequencing of liver and serum samples resulted in two sets of abundantly expressed miRNAs (418 in liver and 351 in serum). Pearson correlation analyses indicated that 18% of miRNAs in liver and 14.5% in serum were significantly associated with the amount of liver fat. PLS-RFE models demonstrated that 50 was the number of miRNAs providing the lowest error in both liver and serum models predicting steatosis. Comparison of the two miRNA subsets showed 19 coincident miRNAs that were ranked according to biological significance (guide/passenger strand, relative abundance in liver and serum, number of predicted lipid metabolism target genes, correlation significance, etc.). Among them, miR-10a-5p, miR-98-5p, miR-19a-3p, miR-30e-5p, miR-32-5p and miR-145-5p showed the highest biological relevance. PLS regression models with serum levels of 2–3 of these miRNAs predicted the % of liver fat with errors <5%.     

Circulating miRNA Expression Profiles and Machine Learning Models in Association with Response to Irinotecan-Based Treatment in Metastatic Colorectal Cancer

Colorectal cancer represents a leading cause of cancer-related morbidity and mortality. Despite improvements, chemotherapy remains the backbone of colorectal cancer treatment. The aim of this study is to investigate the variation of circulating microRNA expression profiles and the response to irinotecan-based treatment in metastatic colorectal cancer and to identify relevant target genes and molecular functions. Serum samples from 95 metastatic colorectal cancer patients were analyzed. The microRNA expression was tested with a NucleoSpin miRNA kit (Machnery-Nagel, Germany), and a machine learning approach was subsequently applied for microRNA profiling. The top 10 upregulated microRNAs in the non-responders group were hsa-miR-181b-5p, hsa-miR-10b-5p, hsa-let-7f-5p, hsa-miR-181a-5p, hsa-miR-181d-5p, hsa-miR-301a-3p, hsa-miR-92a-3p, hsa-miR-155-5p, hsa-miR-30c-5p, and hsa-let-7i-5p. Similarly, the top 10 downregulated microRNAs were hsa-let-7d-5p, hsa-let-7c-5p, hsa-miR-215-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-10a-5p, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-122-5p, and hsa-miR-17-5p. The upregulation of microRNAs in the miR-181 family and the downregulation of those in the let-7 family appear to be mostly involved with non-responsiveness to irinotecan-based treatment.     

Hepato-Protective Effects of Delta-Tocotrienol and Alpha-Tocopherol in Patients with Non-Alcoholic Fatty Liver Disease: Regulation of Circulating MicroRNA Expression

MicroRNAs (miRNAs) play a key role in the regulation of genes for normal metabolism in the liver. Dysregulation of miRNAs is involved in the development and progression of non-alcoholic fatty liver disease (NAFLD). We aimed to explore changes in circulating miRNA expression in response to delta-tocotrienol (δT3) and alpha-tocopherol (αTF) supplementation and correlate them with relevant biochemical markers in patients with NAFLD. In total, 100 patients with NAFLD were randomized to either receive δT3 (n = 50) 300 mg or αTF (n = 50) 268 mg twice/day for 48 weeks. Plasma expression of miRNA-122, -21, -103a-2, -421, -375 and -34a were determined at baseline, 24 and 48 weeks of intervention using RT-qPCR. Both δT3 and αTF significantly downregulated expression of miRNA-122, -21, -103a-2, -421, -375 and -34a. Moreover, δT3 was more effective than αTF in reducing expression of miRNA-375 and -34a. A significant correlation was observed between miRNA expression and biochemical markers of hepatic steatosis, insulin resistance (IR), oxidative stress (OS), inflammation and apoptosis. δT3 and αTF exert hepato-protective effects by downregulating miRNAs involved in hepatic steatosis, IR, OS, inflammation and apoptosis in patients with NAFLD. Furthermore, δT3 has more pronounced effects than αTF in reducing miR-375 and miR-34a, which are linked to regulation of inflammation and apoptosis.     

Plasma Proteomic Profile of Patients with Tick-Borne Encephalitis and Co-Infections

Despite the increasing number of patients suffering from tick-borne encephalitis (TBE), Lyme disease, and their co-infection, the mechanisms of the development of these diseases and their effects on the human body are still unknown. Therefore, the aim of this study was to evaluate the changes in the proteomic profile of human plasma induced by the development of TBE and to compare it with changes in TBE patients co-infected with other tick-borne pathogens. The results obtained by proteomic analysis using a nanoLC-Q Exactive HF mass spectrometer showed that the most highly elevated groups of proteins in the plasma of TBE patients with co-infection were involved in the pro-inflammatory response and protein degradation, while the antioxidant proteins and factors responsible for protein biosynthesis were mainly downregulated. These results were accompanied by enhanced GSH- and 4-HNE-protein adducts formation, observed in TBE and co-infected patients at a higher level than in the case of patients with only TBE. In conclusion, the differences in the proteomic profiles between patients with TBE and co-infected patients indicate that these diseases are significantly diverse and, consequently, require different treatment, which is particularly important for further research, including the development of novel diagnostics tools.     

Translational Read-Through Drugs (TRIDs) Are Able to Restore Protein Expression and Ciliogenesis in Fibroblasts of Patients with Retinitis Pigmentosa Caused by a Premature Termination Codon in FAM161A

Ataluren and Gentamicin are translational readthrough drugs (TRIDs) that induce premature termination codon (PTC) readthrough, resulting in the production of full-length proteins that usually harbor a single missense substitution. FAM161A is a ciliary protein which is expressed in photoreceptors, and pathogenic variants in this gene cause retinitis pigmentosa (RP). Applying TRIDs on fibroblasts from RP patients due to PTC in the FAM161A (p.Arg523*) gene may uncover whether TRIDs can restore expression, localization and function of this protein. Fibroblasts from six patients and five age-matched controls were starved prior to treatment with ataluren or gentamicin, and later FAM161A expression, ciliogenesis and cilia length were analyzed. In contrast to control cells, fibroblasts of patients did not express the FAM161A protein, showed a lower percentage of ciliated cells and grew shorter cilia after starvation. Ataluren and Gentamicin treatment were able to restore FAM161A expression, localization and co-localization with α-tubulin. Ciliogenesis and cilia length were restored following Ataluren treatment almost up to a level which was observed in control cells. Gentamicin was less efficient in ciliogenesis compared to Ataluren. Our results provide a proof-of-concept that PTCs in FAM161A can be effectively suppressed by Ataluren or Gentamicin, resulting in a full-length functional protein.     

CD28null and Regulatory T Cells Are Substantially Disrupted in Patients with End-Stage Renal Disease Due to Diabetes Mellitus

Background: End-stage renal disease (ESRD) is associated with alterations in T-cell immunity, including increased CD28null and reduced regulatory T cells (Tregs). However, whether immune disturbances are due to ESRD or primary disease is not yet clear. As diabetes mellitus is the leading cause of ESRD, we evaluated its impact on the immune profile of ESRD patients. Methods: CD28null, Tregs, and natural killer cells were initially analyzed by flow cytometry in 30 predialysis ESRD patients due to diabetes (DM), 30 non-DM (NDM), and 25 healthy controls. Measurements were repeated after 6 months on hemodialysis (HD) or peritoneal dialysis (CAPD). Results: The percentage of CD4 + CD28null cells, CD8 + CD28null cells, and Tregs showed significant differences in DM, NDM, and controls; mean rank 33.71 vs. 25.68 vs. 18.88, p = 0.006, 37.79 vs. 28.82 vs. 17.08, p = 0.008, and 20.79 vs. 26.12 vs. 41.33, p = 0.001, respectively. DM vs. NDM had increased CD4 + CD28null and CD8 + CD28null cells, 11.5% (1.5%–24%) vs. 4.1% (0–42.3%), p = 0.02 and 61.3% (24%–76%) vs. 43% (5.7%–85%), p = 0.04, respectively. After 6 months on HD but not CAPD, DM showed a significant further increase in CD4 + CD28null cells, from 30 (14–100) to 52.7 (15–203), p = 0.02; and CD8 + CD28null cells, from 137 (56–275) to 266 (103–456), p = 0.01. Conclusions: Diabetes mellitus affects T-cell subtypes even at predialysis stage, though changes become more prominent after commencement on HD.