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Background The majority of CAKUT-associated CNVs overlap at least one miRNA gene, thus affecting the cellular levels of the corresponding miRNA. We aimed to investigate the potency of restitution of CNV-affected miRNA levels to remediate the dysregulated expression of target genes involved in kidney physiology and development in vitro. Methods Heterozygous MIR484 knockout HEK293 and homozygous MIR185 knockout HEK293 cell lines were used as models depicting the deletion of the frequently affected miRNA genes by CAKUT-associated CNVs. After treatment with the corresponding miRNA mimics, the levels of the target genes have been compared to the non-targeting control treatment. For both investigated miRNAs, MDM2 and PKD1 were evaluated as common targets, while additional 3 genes were investigated as targets of each individual miRNA (NOTCH3, FIS1 and APAF1 as hsa-miR-484 targets and RHOA, ATF6 and CDC42 as hsa-miR-185-5p targets). Results Restitution of the corresponding miRNA levels in both knockout cell lines has induced a change in the mRNA levels of certain candidate target genes, thus confirming the potential to alleviate the CNV effect on miRNA expression. Intriguingly, HEK293 WT treatment with investigated miRNA mimics has triggered a more pronounced effect, thus suggesting the importance of miRNA interplay in different genomic contexts. Conclusions Dysregulation of multiple mRNA targets mediated by CNV-affected miRNAs could represent the underlying mechanism behind the unresolved CAKUT occurrence and phenotypic variability observed in CAKUT patients. Characterizing miRNAs located in CNVs and their potential to become molecular targets could eventually help in understanding and improving the management of CAKUT.

Background/Aim. The renin-angiotensin system (RAS) is linked to the development of atherosclerosis (As), including its initiation and progression. Besides the well-known angio-tensin-converting enzyme (ACE), two newer RAS family members are related to vascular remodeling - ACE2 as a homolog of ACE and collectrin [transmembrane protein 27 (TMEM27)] as a homolog of ACE2. Up to now, a limited number of studies have examined the expression of these RAS components in advanced carotid plaque (CP) tissue based on the sex of the patients and plaque phenotypes (PPs). There are two ultrasonographically defined PPs - the hypoechogenic plaque (HoP) and the hyperechogenic plaque (HerP) phenotype. The aim of the study was to investigate whether there was a correlation between the expression of RAS components in the CP and the sex and PPs of patients. Methods. We examined 74 patients with advanced CP who underwent carotid endarterectomy. The intraplaque expression of RAS components was determined with the real-time polymerase chain reaction, using the TaqMan® gene expression assays and Western blot. A two-way ANOVA followed by a post-hoc Tukey test was performed for the statistical analysis of results. Results. No interaction was recorded between the sex of the patients and PPs in influencing the relative expression of ACE and TMEM27 messenger RNA (mRNA) (p > 0.05). In 56.06% of plaque samples, no expression of ACE2 mRNA was detected. Among the plaques where ACE2 mRNA expression was detected, its expression level was higher in females with the HoP phenotype compared to females with the HerP phenotype (p < 0.001). In patients with the HoP phenotype, females had higher expression of ACE2 mRNA than males (p < 0.05). In the male study group, ACE protein levels were significantly lower in the HoP phenotype compared to the HerP phenotype (p < 0.001). Fe-males with the HoP phenotype had significantly higher ACE protein levels than males with the HoP phenotype (p < 0.0001). Conclusion. Ou, Renin-angiotenzin sistem (RAS) povezan je sa razvojem ateroskleroze (As), uključujući njen nastanak i progresiju. Pored dobro poznatog angiotenzinkonvertujućeg enzima (angiotensin-converting enzyme – ACE), dva nova člana RAS familije povezana su sa remodelovanjem zidova krvnih sudova – ACE2 kao homolog ACE i kolektrin [transmembrane protein 27 (TMEM27)] kao homolog ACE2. Do sada je mali broj studija ispitivao ekspresiju komponenti RAS sistema u tkivu uznapredovalog karotidnog plaka (KP) u odnosu na pol bolesnika i fenotip plaka (FP). Postoje dva tipa KP definisana primenom ultrazvuka – fenotip hipoehogenog plaka (HoP) i fenotip hiperehogenog plaka (HerP). Cilj rada bio je da se ispita da li postoji korelacija između ekspresije komponenti RAS u KP i pola i FP bolesnika. Metode. Ispitano je 74 bolesnika sa uznapredovalim KP koji su bili podvrgnuti operativnoj proceduri karotidne endarterektomije. Ekspresija komponenti RAS sistema u tkivu plaka utvrđena je lančanom reakcijom polimeraze u realnom vremenu (real-time polymerase chain reaction)primenom eseja TaqMan® tehnologije i metode Western blota. Dvosmerna analiza varijanse i Tukey post-hoc test korišćen su za statističku obradu rezultata. Rezultati. Nije utvrđena interakcija FP i pola bolesnika u uticaju na relativnu ekspresiju informacione RNK (iRNK) za ACE i TMEM27 (p > 0,05). U 56,06% uzoraka plaka nije detektovana ekspresija iRNK za ACE2. U plakovima u kojima je detektovana ekspresija iRNK za ACE2, njen nivo bio je viši kod žena sa HoP u poređenju sa ženama sa HerP (p < 0,001). U grupi bolesnika sa fenotipom HoP, žene su imale viši nivo iRNK za ACE2 nego muškarci (p < 0,05). U grupi muškaraca, nivoi ekspresije ACE proteina bili suznačajno niži u fenotipu HoP u poređenju sa HerP (p < 0,001). Žene sa fenotipom HoP imale su značajno viši nivo ACE proteina u poređenju sa muškarcima sa HoP (p < 0,0001). Zaključak. Naši rezultati pokazali su da postoje promene nivoa ekspresije ACE i ACE2, na nivou proteina i iRNK u uzn

Background Congenital anomalies of the kidney and urinary tract (CAKUT) represent a frequent cause of pediatric kidney failure. CNVs, as a major class of genomic variations, can also affect miRNA regions. Common CNV corresponding miRNAs (cCNV-miRNAs) are functional variants regulating crucial processes which could affect urinary system development. Thus, we hypothesize that cCNV-miRNAs are associated with CAKUT occurrence and its expressivity. Methods The extraction and filtering of common CNVs, identified in control samples deposited in publicly available databases gnomAD v2.1 and dbVar, were coupled with mapping of miRNA sequences using UCSC Genome Browser. After verification of the mapped miRNAs using referent miRBase V22.1, prioritization of cCNV-miRNA candidates has been performed using bioinformatic annotation and literature research. Genotyping of miRNA gene copy numbers for MIR9-3, MIR511, and MIR1299, was conducted on 221 CAKUT patients and 192 controls using TaqMan™ technology. Results We observed significantly different MIR9-3 and MIR1299 gene copy number distribution between CAKUT patients and controls (Chi-square, P = 0.006 and P = 0.0002, respectively), while difference of MIR511 copy number distribution showed nominal significance (Chi-square, P = 0.027). The counts of less and more than two of MIR1299 copy numbers were more frequent within CAKUT patients compared to controls (P = 0.01 and P = 0.008, respectively) and also in cohort of patients with anomalies of the urinary tract compared to controls (P = 0.016 and P = 0.003, respectively). Conclusions Copy number variations of miRNA genes represent a novel avenue in clarification of the inheritance complexity in CAKUT and provide potential evidence about the association of common genetic variation with CAKUT phenotypes.

Background The MMP-9 is a known player in atherosclerosis, yet associations of the MMP-9 -1562 C/T variant (rs3918242) with various atherosclerotic phenotypes and tissue mRNA expression are still contradictory. This study aimed to investigate the MMP-9 -1562 C/T variant, its mRNA and protein expression in carotid plaque (CP) tissue, as a risk factor for CP presence and as a marker of different plaque phenotypes (hyperechoic and hypoechoic) in patients undergoing carotid endarterectomy. The MnSOD as an MMP-9 negative regulator was also studied in relation to CP phenotypes. Methods and results Genotyping of 770 participants (285 controls/485 patients) was done by tetra-primer ARMS PCR. The MMP-9 mRNA expression in 88 human CP tissues was detected by TaqMan® technology. The protein levels of MMP-9 and MnSOD were assessed by Western blot analysis. The MMP-9 -1562 C/T variant was not recognized as a risk factor for plaque presence or in predisposing MMP-9 mRNA and protein levels in plaque tissue. Patients with hypoechoic plaques had significantly lower MMP-9 mRNA and protein levels than those with hyperechoic plaque (p = 0.008, p = 0.003, respectively). MnSOD protein level was significantly higher in hypoechoic plaque compared to hyperechoic (p = 0.039). MMP-9 protein expression in CP tissue was significantly affected by sex and plaque type interaction (p = 0.009). Conclusions Considering the differences of MMP-9 mRNA and protein expression in CP tissue regarding different plaque phenotypes and the observed sex-specific effect, the role of MMP-9 in human atherosclerotic plaques should be further elucidated.

Background: Right ventricular (RV) dysfunction is recognized as a cardinal prognostic marker in heart failure (HF) patients. Myocardial infarction (MI) is often followed by unrecognized RV dysfunction, which can be associated with worse outcome. It is recently shown that the ratio between TAPSE and PASP (RV/PA) may depict cardiopulmonary hemodynamics better than the two parameters alone. Aim: To evaluate the interactions between left ventricular (LV) and RV function in early phase of MI and to assess the prognostic significance of RV/PA coupling in patients with first MI during 5 years follow up. Methods: The prospective study included 144 patients with the first MI treated with the primary percutaneous coronary intervention (p PCI) who underwent Doppler echocardiography within 2±1 days of MI. LV function analysis included: LV ejection fraction (EF), ratio between early diastolic velocity and tissue annular velocity (E/e) and global longitudinal strain (GLS). RV function and RV-PA interaction was expressed as ratio between TAPSE and PASP. During the five-year follow-up, major cardiovascular events and especially hospitalization for HF were analyzed. Results: Progressive RV/PA uncoupling was associated with higher degree of LV impairment and dysfunction (EF p<0.001, E/e p=0.002, GLS p<0.001) and severity of mitral regurgitation (p=0.013). Lower baseline RV/PA coupling significantly reflects the frequency of hospitalizations for HF in the population of patients with first MI during five-year follow-up (0.62 v.s.0.51, p=0.021). After multivariate adjustment RV/PA remained an independent predictor of all major cardiac events (MACE) after five years (OR 14.0 [1.5–130.8], p=0.019). Conclusion: A lower baseline RV-PA coupling, reflecting a higher degree of LV-induced pulmonary hypertension and secondary RV-dysfunction, is associated with decline of LV function in early phase of MI, and is independently associated with worse prognosis after five years. The value of RV-PA ra

Last decade provided multiple evidence that link disturbances in metabolic processes and energy metabolism with diseases of central nervous system and neurodegeneration. Initial phases of insulin resistance (IR) are present in natural course of multiple sclerosis (MS) and leptin was recognized as a player in MS pathophysiology and moreover cognitive decline. We aimed to investigate association of genetic variants in leptin (LEP) rs7799039, its receptor LEPR rs1137101 and proliferator-activated receptor gamma co-activator 1-alpha (PGCA1A) rs8192678 with IR parameters (HOMA-IR index, area under the curve for insulin and glucose, Cederholm insulin sensitivity index (ISIced), the insulinogenic index in the first 30 min of oral glucose tolerance test (OGTT) in patients with MS. Seventy eight relapsing-remitting patients in clinical remission, free of corticosteroids for at least three months, were included in the study. None of the 3 variants’ genotypes were associated with HOMA-IR index, area under the curve for insulin and glucose and the insulinogenic index in the first 30 min of OGTT. PGC1A variant was significantly associated with ISIced (Kruskal-Wallis ANOVA, p = 0.04). Leptin gene variant was significantly associated with impaired GT (p=0.029 adjusted for gender and other two variants). None of the variant showed association with IR. In conclusion, we found that genetic variants in leptin signalling pathway affect glucose tolerance and insulin sensitivity in patients with MS. As both, leptin and PGC1A have role in preventing neuronal death and reducing oxidative stress neuronal damage current results favour further investigation toward preserving cognitive status and neuroprotection in MS.

Introduction: Congenital anomalies of the kidney and urinary tracts(CAKUT) are a diverse spectrum of defects with complex etiology and not fully explained genetic background. miRNA-containing copy number variants (CNVs) are described as genetic risk factor for the disease development. We aimed to identify miRNAs with the maximum regulatory coverage of previously reported differentially expressed genes in CAKUT tissue compared to controls and bioinformatically characterize a set of these miRNAs which are located in common CNVs. Methods: Differentially expressed genes were identified from ureter tissue transcriptome open data GSE83946 from 15 CAKUT patients and 7 healthy controls, generated in house previously. miRPathDB v2.0 was used for identification of miRNAs with maximum coverage of DEGs(miRNAs which complimentarily regulate all DEGs). Mapping of maximum coverage miRNAs onto common CNVs (frequency >0.2) was performed using UCSC genome browser and gnomAD database. miRNA mapping common CNVs were further bioinformatically analyzed using miRPathDB v2.0. Results: In a maximum coverage set of 50 miRNAs interacting with DEGs in CAKUT, we have identified 3 miRNA geneslocated in the common CNVs(hsa-miR-663b, hsa-miR-3180-3p and hsa-miR-1302). Using Reactome database we identified all three miRNAsto be significantly enriched in the pathway Neuronal System: -log(p-value)>2.326 for hsa-miR-1302; -log(p-value)>1.556 for hsa-miR-3180-3p; and -log(pvalue)>1.703 for hsa-miR-663b. Conclusion: CAKUT is characterized with variable penetrability and expressivity and often followed with other comorbiditiessuch as neurodevelopmental disorders. miRNAsinvolved in DEG networks and prone to CNV effects could present modulating factors of the disease phenotype. Further studies should provide additional evidence about hsa-miR-1302, hsa-miR-3180-3p and hsa-miR-663b involvements in CAKUT etiology

Molecular background and biomarkers of highly heterogenous and hardly predictable disease progression among relapse-onset MS patients are of high research interest. In the current pilot study, we aimed to employ next-generation sequencing to investigate the expression of whole small non-coding microRNAs (miRNome) in two groups of MS patients with highly distinctive progression phenotype: one with fast progressing, severely disabling course vs. mild course of MS, longitudinally followed 10 years. Peripheral blood mononuclear cells (PBMC) miRNome data was obtained from mild phenotype MS (n=4 patients) and progressive phenotype MS (n=5 patients), using TakaraBio SMARTer smRNA-Seq Kit on iSeq100 (Illumina). Pre-processing of raw sequencing data, quality control and miRNA differential expression analysis was performed using sRNAtoolbox pipeline. Functional interpretation of differentially expressed miRNA target genes was done in DIANA-miRPathv3.0. Tarbase v8.0 served as a resource of miRNA:gene interactions. Achieved read depth was approximately 1 million raw reads/sample, allowing detection of up to 92 mature miRNAs after genome alignment and miRbase v22 annotation. Differential expression analysis identified the significant upregulation of hsa-miR-23c (log2FC=4.29, Padj= 0.03) in progressive phenotype. Top significantly enriched KEGG pathways in hsa-miR-23c targets suggested regulation of molecular pathways involved in autoimmunity (antigen presentation, Epstein-Barr virus infection) and cancer. In conclusion, this pilot study indicates phenotype-related differences in expression of miRNAs, molecules with high regulatory and biomarker properties. Although detected in PBMC, has-miR-23c is highly expressed in the brain and target MS relevant genes such as, HLA (A, B, C), transferrin receptor, Nrf2, recently proposed to play important role in neurodegeneration.

The significance of CD8+ T cell central nervous system migration and activation in the progression of glioblastoma is well documented. However, molecular signaling pathways regulation related to migration and activation in CD8+ cells in glioblastoma is scarce. Therefore we have analyzed the molecular pathway regulation in differentially expressed mRNAs of tumor infiltrating vs. peripheral blood CD8+ T cells from glioblastoma patients. Tumor-infiltrating vs. peripheral blood differentially expressed mRNAs were obtained by analyzing the FASTAQ files on the Galaxy platform using the LimmaVoom tool with filtering low count mRNAs (CPM > 2). We used publically avaliable FASTAQ files with CD8+ T cells mRNA sequencing data deposited at NCBI’s GEO database (accession number GSE171197). The differentially expressed mRNA were analyzed with Qiagen’s Ingenuity pathway analysis (p adj. cutoff 0.05). Protein-protein interaction network was constructed on the NetworkAnalyst platform using the IMeX database with minimal order parameters. The top rated disease canonical pathway was the multiple sclerosis (MS) signaling pathway, with 18 differentially expressed mRNA hits (out of possible 222), p adj. = 0.0009 and Z score = 2.828, implying significant upregulation of this pathway in tumor-infiltrating CD8+ T cells. The MS signaling pathway describes the molecular cascade which leads to the autoimmune phenotype in lymphocytes, including activation and central nervous tissue infiltration. To further specify the aspects of the canonical MS signaling pathway which might influence tumor infiltrating phenotype we have constructed a minimal order protein-protein interaction network. Results showed a number of lymphocyte migration and activation KEGG terms within the network, such as: TNF signaling pathway (p adj. = 0.0000115), IL-17 signaling pathway (p adj. = 0.00000427), sphingolipid signaling pathway (p adj. = 0.00171), NF-kappa B signaling pathway (p adj. = 0.0000694) and TCR signaling pa

Congenital anomalies of the kidney and urinary tract (CAKUT) represent structural and functional urinary system malformations and take place as one of the most common congenital malformations with an incidence of 1:500. Ureteral obstruction-induced hydronephrosis is associated with renal fibrosis and chronic kidney diseases in the pediatric CAKUT. We aimed to construct interaction network of previously bioinformatically associated miRNAs with CAKUT differentially expressed genes in order to prioritize those associated with fibrotic process and to experimentally validate the expression of selected miRNAs in CAKUT patients compared to control group. We constructed interaction network of hsa-miR-101-3p, hsa-miR-101-5p and hsa-miR-29c-3p that showed significant association with fibrosis. The top enriched molecular pathway was extracellular matrix-receptor interaction (adjusted p = .0000263). We experimentally confirmed expression of three miRNAs (hsa-miR-29c-3p, hsa-miR-101-3p and hsa-miR-101-5p) in obstructed ureters (ureteropelvic junction obstruction and primary obstructive megaureter) and vesicoureteral reflux. The hsa-miR-29c-3p was shown to have lower expression in both patient groups compared to controls. Relative levels of hsa-miR-101-5p and hsa-miR-101-3p showed significant positive correlations in both groups of patients. Statistically significant correlation was observed between hsa-miR-101 (-3p and -5p) and hsa-miR-29c-3p only in the obstructed group. The significant downregulation of anti-fibrotic hsa-miR-29c-3p in obstructive CAKUT could explain activation of genes involved in fibrotic processes. As miRNAs are promising candidates in therapeutic approaches our results need further measurement of fibrotic markers or assessment of extent of fibrosis and functional evaluation of hsa-miR-29c.

Galectin-3 is encoded by LGALS-3, located in a unique haplotype block in Caucasians. According to the Tagger server, rs4040064, rs11628437, and rs7159490 cover 82% (r2 > 0.8) of the genetic variance of this HapBlock. Our aims were to examine the association of their haplotypes with first myocardial infarction (MI), changes in left ventricular echocardiographic parameters over time, and impact on plasma galectin-3 and LGALS-3 mRNA in peripheral blood mononuclear cells, both 6 months post-MI. The study group consisted of 546 MI patients and 323 controls. Gene expression was assessed in 92 patients and plasma galectin-3 in 189 patients. Rs4040064, rs11628437, rs7159490, and LGALS-3 mRNA expression were detected using TaqMan® technology. Plasma galectin-3 concentrations were determined by the ELISA method. We found that the TGC haplotype could have a protective effect against MI (adjusted OR 0.19 [0.05–0.72], p = 0.015) and that the GAC haplotype had significantly higher galectin-3 concentrations (48.3 [37.3–59.4] ng/mL vs. 18.9 [14.5–23.4] ng/mL, p < 0.0001), both in males and compared to the referent haplotype GGC. Higher plasma Gal-3 was also associated with higher NYHA class and systolic dysfunction. Our results suggest that variants tagging LGALS-3 HapBlock could reflect plasma Gal-3 levels 6 months post-MI and may have a potential protective effect against MI in men. Further replication, validation, and functional studies are needed.

ObjectivesGalectin-3 affects a variety of biological processes. It is encoded by LGALS-3, located in unique haplotype block in Caucasians. Most of the studies regarding the gal-3 role in atherosclerosis are focused exclusively on protein/mRNA levels. Genetic analyses of LGALS-3 are scarce. We sought to thoroughly examine the genetic background of gal-3 and to analyze tag variants that cover more than 80% variability of the LGALS-3 containing hap-block in association with carotid plaque presence (CPP). According to Tagger server, rs4040064 G/T, rs11628437 G/A and rs7159490 C/T cover 82% (r2 > 0.8) of the genetic variance of this hap-block. Our aims were to investigate possible association of rs4040064, rs11628437 and rs7159490 haplotypes with CPP in patients with advanced carotid atherosclerosis (CA) and to analyze their possible effect on LGALS-3 mRNA expression in carotid plaques.Materials and methodsStudy group consisted of 468 patients and 296 controls. Rs4040064, rs11628437, rs7159490 and LGALS-3 mRNA expression were detected by TaqMan® technology.ResultsWe have found that haplotype TAC was associated with the cerebrovascular insult (CVI) occurrence (OR = 1.68, 95% CI = 1.09-2.58, p = 0.02), compared to the referent haplotype. OR was adjusted for hypertension, age and BMI. TAC also showed higher, but not statistically significant, LGALS-3 expression in carotid plaques.ConclusionsOur results suggest that rs4040064, rs11628437 and rs7159490 bear no association with CPP, neither they affect LGALS-3 mRNA in carotid plaques. However, we showed a significant association of haplotype TAC with the CVI occurrence in CA patients from Serbia. Replication and validation of our results are required.

Background/Objectives: Rare copy number variants (rCNVs) are the common genetic cause of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).1 miRNAs located in rCNVs represent well-founded functional variants for human CAKUT research. However, the impact of rCNVs on miRNA genes in CAKUT is unknown. Thus, burden assessment was performed to identify chromosomes with non-random representation of miRNA genes in rCNVs associated with CAKUT. Methods: A comprehensive literature mining of rCNV regions associated with CAKUT was performed. The total cumulative length of rCNVs per chromosome was the sum of corresponding CNV-DNA regions, taking into account overlapping. Mapping of miRNAs onto cumulative rCNV regions gave counts of affected miRNA loci. The correlation analysis was performed between the number of miRNA genes overlapping rCNVs, and the fractional lengths of cumulative rCNVs regions in relation to the chromosome size. Results: A statistically significant positive correlation was observed for duplications and deletions respectively (Spearman correlation p<0.0001, r=0.9, r=0.8). However, a deviation from the best fit line for chromosome 16, for both rare duplications and deletions, was observed due to the high overrepresentation of miRNA genes in identified rCNVs. Conclusion: The current finding of the high overall burden of rCNVs on miRNA genes in chromosome 16 suggests that miRNAs located on this chromosome could serve as candidates for the investigation of miRNA role in CAKUT development.

Introduction: miR-548 family members, located on all human chromosomes except chr19 and chrY, regulate podocyte differentiation in vitro, important for kidney development. Rare copy number variants (rCNVs) are the common genetic cause of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) and could harbour miRNAs. The aim of this study was to investigate to which extent rCNVs associated with CAKUT harbour miR-548 members. Materials and Methods: Extensive literature review was conducted to collect data of pathogenic and likely pathogenic rCNVs in CAKUT patients. UCSC genome browser tool was employed for mapping of miR-548 members onto collected rCNV regions and gnomAD SV controls database. Bioinformatic analysis was conducted using miRPathDB2 tool. Results: We generated CAKUT database of pathogenic CNVs in 79 chromosome regions from 191 patient and likely pathogenic CNVs in 74 regions from 87 patients. Pathogenic rCNVs of seventeen patients, located on 7 chromosomes, contained at least one miR-548 member. Likely pathogenic rCNVs of 4 patients, located on 3 chromosomes, contained one of miR-548 members. Bioinformatic analysis implied the role of mapped miRNAs in the regulation of processes associated with CAKUT. In controls, only hsa-mir-548i-3 (out of 73 precursors) was mapped on polymorphic CNVs (af>1%) and wasn’t identified in patients. Conclusions: miR-548 members located in rCNVs should be investigated in future studies as potential genetic drivers of CAKUT development, beyond protein coding genes.

Introduction: Genetic studies of congenital anomalies of the kidney and urinary tract (CAKUT) have demonstrated variable penetrability and expressivity of the associated genetic defects. Previously, it was shown that deletions of 17q12 and 22q11.2 regions were specific for kidney anomalies (KA) while 16p11.2 and 1q21.1 loci showed extensive pleiotropy in CAKUT phenotypes. CNVs affecting miRNA gene dosage have been described to have functional influence on gene expression. We aimed to conduct comprehensive in silico analysis using publicly available databases to analyze miRNA content of CAKUT-associated CNVs in quoted chromosomal loci with regard to pleiotropy. Methods: Extensive literature review was conducted to collect data about pathogenic rCNVs associated with CAKUT. UCSC genome browser tool was employed for mapping miRNAs onto collected rCNV regions. Results: Analysis of CNVs in CAKUT included four studies counting more than 2500 patients. In further analysis we included 191 patients harboring pathogenic CNVs. Surprisingly, CAKUT pleiotropic regions (16p11.2, 1q21.2) did not contain any miRNA. 22q11.2 showed the densest miRNAs content (n = 21). Conclusions: Absence of miRNAs may potentially pronounce the pleiotropy of the CAKUT genetic defects, thus leading to the variety of phenotypes. Contrary, abundancy of miRNAs in 22q11.2 might be associated with reproducible phenotype, such as KA, producing the functional effect when deleted. This assumption agrees with recent results of miRNA expression variability in 22q11.2 deletion syndrome.

Objectives This study aimed to experimentally validate dysregulated expression of miRNA candidates selected through updated meta-analysis of most commonly deregulated miRNAs in oral cancer and to explore their diagnostic and prognostic potential. Materials and methods Five miRNAs (miR-31-3p, miR-135b-5p, miR-18a-5p, miR-30a-5p and miR-139-5p) from updated meta-signature were selected for validation by qRT-PCR method in 35 oral cancer clinical specimens and adjacent non-cancerous tissue. Results Updated meta-analysis has identified 13 most commonly deregulated miRNAs in oral cancer. Seven miRNAs were consistently up-regulated (miR-21-5p, miR-31-3p, miR-135b-5p, miR-31-5p, miR-424-5p, miR-18a-5p and miR-21-3p), while five were down-regulated (miR-139-5p, miR-30a-3p, miR-375-3p, miR-376c-3p and miR-30a-5p). Increased expression of miR-31-3p and miR-135b-5p, and decreased expression of miR-139-5p and miR-30a-5p were confirmed in oral cancer compared to adjacent non-cancerous tissue. A three miRNAs combination (miR-31-3p, miR-139-5p and miR-30a-5p) gave the most promising diagnostic potential for discriminating oral cancer from non-cancerous tissue (AUC: 0.780 [95% CI: 0.673–0.886], p < 0.0005, sensitivity 94.3%, specificity 51.4%). High expression of miR-135b-5p, miR-18a-5p and miR-30a-5p was associated with poor survival (p = 0.003, p = 0.048, p = 0.016 respectively). Conclusion miR-31-3p, miR-139-5p and miR-30a-5p panel was confirmed as a potential diagnostic biomarker when distinguishing oral cancer from non-cancerous tissue. miR-135b-5p, miR-18a-5p and miR-30a-5p might serve as potential biomarkers of poor survival of oral cancer patients.

Rare copy number variants (CNVs) are among the most common genomic disorders underlying CAKUT. miRNAs located in rare CNVs represent well-founded functional variants for human CAKUT research. The study aimed to identify and functionally interpret miRNAs most frequently affected by rare CNVs in CAKUT and to estimate the overall burden of rare CNVs on miRNA genes in CAKUT. The additional aim of this study was to experimentally confirm the effect of a rare CNV in CAKUT on candidate miRNA’s expression and the subsequent change in mRNA levels of selected target genes. A database of CAKUT-associated rare CNV regions, created by literature mining, was used for mapping of the miRNA precursors. miRNAs and miRNA families, most frequently affected by rare CAKUT-associated CNVs, have been subjected to bioinformatic analysis. CNV burden analysis was performed to identify chromosomes with over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT. A functional study was performed on HEK293 MIR484+/- KO and HEK293 WT cell lines, followed by the analysis of relative miRNA and mRNA target gene levels. 80% of CAKUT patients with underlying rare CNV had at least one miRNA gene overlapping the identified CNV. Network analysis of the most frequently affected miRNAs has revealed the dominant regulation of the two miRNAs, hsa-miR-484 and hsa-miR-185-5p. Additionally, miR-548 family members have shown substantial enrichment in rare CNVs in CAKUT. An over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT was observed in multiple chromosomes, such as chr16, chr20, and chr21. A significant 0.37 fold downregulation of hsa-miR-484, followed by a notable upregulation of MDM2 and APAF1 and downregulation of NOTCH3 was detected in HEK293 MIR484+/- KO compared to HEK293 WT cell lines, supporting the study hypothesis. miRNA genes are frequently affected by rare CNVs in CAKUT patients. Understanding the potential of CNV-affected miRNAs to participate in CAKUT as ge

ObjectivesGalectin-3 affects a variety of biological processes. It is encoded by LGALS-3, located in unique haplotype block in Caucasians. Most of the studies regarding the gal-3 role in atherosclerosis are focused exclusively on protein/mRNA levels. Genetic analyses of LGALS-3 are scarce. We sought to thoroughly examine the genetic background of gal-3 and to analyze tag variants that cover more than 80% variability of the LGALS-3 containing hap-block in association with carotid plaque presence (CPP). According to Tagger server, rs4040064 G/T, rs11628437 G/A and rs7159490 C/T cover 82% (r2 > 0.8) of the genetic variance of this hap-block. Our aims were to investigate possible association of rs4040064, rs11628437 and rs7159490 haplotypes with CPP in patients with advanced carotid atherosclerosis (CA) and to analyze their possible effect on LGALS-3 mRNA expression in carotid plaques.Materials and methodsStudy group consisted of 468 patients and 296 controls. Rs4040064, rs11628437, rs7159490 and LGALS-3 mRNA expression were detected by TaqMan® technology.ResultsWe have found that haplotype TAC was associated with the cerebrovascular insult (CVI) occurrence (OR = 1.68, 95% CI = 1.09-2.58, p = 0.02), compared to the referent haplotype. OR was adjusted for hypertension, age and BMI. TAC also showed higher, but not statistically significant, LGALS-3 expression in carotid plaques.ConclusionsOur results suggest that rs4040064, rs11628437 and rs7159490 bear no association with CPP, neither they affect LGALS-3 mRNA in carotid plaques. However, we showed a significant association of haplotype TAC with the CVI occurrence in CA patients from Serbia. Replication and validation of our results are required.

Objective Cholesteatoma is a challenging chronic pathology of the middle ear for which pharmacologic therapies have not been developed yet. Cholesteatoma occurrence depends on the interplay between genetic and environmental factors while master regulators orchestrating disease progression are still unknown. Therefore, in this review, we will discuss the diagnostic and therapeutic potential of non-coding RNAs (ncRNA) as a new class of regulatory molecules. Methods We have comprehensively reviewed all articles investigating ncRNAs, specifically micro RNAs (miRNAs) and long ncRNAs (lncRNA/circRNA) in cholesteatoma tissue. Results Candidate miRNA approaches indicated that miR-21 and let-7a are the major miRNAs involved in cholesteatoma growth, migration, proliferation, bone destruction, and apoptosis. Regulatory potential for the same biological processes was also observed for miR-203a. The NF-kB/miR-802/PTEN regulatory network was in relation to observed miR-21 activity in cholesteatoma as well. High throughput approaches revealed additional ncRNAs implicated in cholesteatoma pathology. Competitive endogenous RNA (ceRNA) analysis highlighted lncRNA/circRNA that could be “endogenous sponge” for miR-21 and let-7a based on the hypothesis that RNA transcripts can communicate with and regulate each other by using shared miRNA response elements. Conclusion In this review, we summarize the discoveries and role of ncRNA in major pathways in cholesteatoma and highlight the potential of miRNA-based therapeutics in the treatment of cholesteatoma. Level of Evidence: NA.

Background Myocardial infarction (MI) leads to ischemia and afterward to left ventricular (LV) remodeling. Matrix metalloproteinase−1 (MMP1) and −3 (MMP3) belong to the family of endopeptidases and together they can dissolve most of the components of the extracellular matrix. MMP1 and MMP3 variants have been investigated solely in association with ischemic heart disease and LV dysfunction, but not in haplotype. The aims of this study were to investigate the association of haplotypes inferred from MMP1 rs1799750 (−1607 1G/2G; NC_000011.9:g.102670497del) and MMP3 rs35068180 (−1612 5A/6A; NC_000011.9:g.102715952dup) with MI and their effect on the change in echocardiographic parameters of LV structure and function in patients within 6 months after MI. Methods The study included 325 patients with the first MI and 283 healthy controls. Gene variants were detected by PCR-RFLP method. Parameters of LV structure and function were assessed by conventional 2D echocardiography, 3–5 days and 6 months after the first MI, on a subgroup of 160 patients. Haplotype analysis was performed with Thesias software. Results Haplotypes 2G-5A and 1G-6A were significantly and independently associated with MI compared with the reference haplotype 2G-6A (adjusted, p = 0.009 and p = 0.026, respectively). After Bonferroni correction for multiple testing, MMP1 and MMP3 haplotypes lost their association with the change in LV long diameter and stroke volume within 6 months after MI. Conclusion MMP1 and MMP3 haplotypes are strongly associated with MI. Further studies are needed to validate this result and to examine their association with echocardiographic parameters of LV structure and function after MI.