DIANA miRPath Research Articles

MiRNAs as Epigenetic Biomarkers in the Study of the Bidirectional Relationship between Type 2 Diabetes Mellitus and Periodontitis: A Systematic Review.

The objective of this study is to analyze the miRNA expression of oral fluids such as gingival crevicular fluid (GCF) in patients with periodontitis and Type 2 diabetes mellitus, and how these epigenetic biomarkers can influence the bidirectional relationship of these two inflammatory diseases. This review was conducted following the PRISMA criteria. PubMed, Scopus, Cochrane Library, Embase, and Web of Science databases were searched for clinical studies conducted on humans investigating, through GCF miRNA expression, the relationship between periodontal diseases and type 2 diabetes mellitus. In addition, the etiopathogenic pathways of the studied miRNAs were analyzed using the DIANA MIR path tool. A total of 1436 references were identified in the initial literature search, and seven articles were finally included in this review. Most of the articles included in this review were case-control studies and examined the expression of miRNAs in patients with periodontitis with or without diabetes. Due to their characteristics, miRNAs appear to be the ideal biomarkers for improving the understanding and knowledge of the etiopathogenic pathways that link both diseases. Among all the studied miRNAs, miR-146a, miR-155, miR-200b, miR-223, and miR-203 showed strong involvement in inflammatory and metabolic pathways, making them potential good diagnostic and prognostic biomarkers.

Exosomal miRNAs as Potential Biomarkers for Preeclampsia: miR-1283 Has the Highest Expression, while miR-152-3p Has the Lowest Expression

BACKGROUND: Preeclampsia management is necessary, as it is one of the leading causes of death during pregnancy. Exosomal microRNAs (miRNAs) can serve as biomarkers for early detection, diagnosis, and prognosis of preeclampsia. NanoStrings is an effective method for identifying exosomal miRNA due to their high sensitivity and ability to work with small amounts of miRNA; however, the analysis using this method for determining preeclampsia biomarker is still limited. Therefore, this study was conducted to utilize the NanoStrings method in identifying preeclampsia biomarkers related to its underlying pathophysiology.METHODS: This study involved 12 pregnant women at 20–40 weeks of gestation, including 6 preeclampsia women and 6 normotension women. The miRNAs from plasma exosomes were processed using NanoStrings method with NanoString nCounter SPRINT Profiler. Enrichment analysis of The Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways were performed to examine the pathophysiological pathways of preeclampsia, using the DIANA–miRPath v3.0.RESULTS: Forty-eight miRNAs were downregulated and 7 were upregulated (miR-1283, miR-613, miR-520a-3p, miR-3185, miR-556-3p, miR-1973, and miR-598-3p) in women with preeclampsia. The highest expression was observed in miR-1283 (log fold-change: 3.69) and the most lowest expression was in miR-152-3p (log fold-change: 1.41). Enrichment analysis showed that the most upregulated miRNAs pathways was estrogen signaling pathway, and the most downregulated was Hippo signaling pathways.CONCLUSION: miR-1283 has the highest expression, while and miR-152-3p has the lowest expression in preeclampsia women. These miRNAs are shown to be linked to specific pathways, shedding light on the pathophysiology of preeclampsia, and may serve as promising biomarkers.KEYWORDS: exosomes, biomarker, miRNAs, pathophysiology, preeclampsia, pregnancy

A Pilot Study of Saliva MicroRNA Signatures in Children with Moderate-to-Severe Traumatic Brain Injury.

Background/Objectives: Traumatic brain injury (TBI) is a leading cause of death and disability in children. Currently, no biological test can predict outcomes in pediatric TBI, complicating medical management. This study sought to identify brain-related micro-ribosomal nucleic acids (miRNAs) in saliva associated with moderate-to-severe TBI in children, offering a potential non-invasive, prognostic tool. Methods: A case-control design was used, enrolling participants ≤ 18 years old from three pediatric trauma centers. Participants were divided into moderate-to-severe TBI and non-TBI trauma control groups. Saliva samples were collected within 24 h of injury, with additional samples at 24-48 h and >48 h post-injury from the TBI group. miRNA profiles were visualized with partial least squares discriminant analysis (PLSDA) and hierarchical clustering. Mann-Whitney testing was used to compare miRNAs between groups, and mixed models were used to assess longitudinal expression patterns. DIANA miRPath v3.0 was used to interrogate the physiological functions of miRNAs. Results: Twenty-three participants were enrolled (14 TBI, nine controls). TBI and control groups displayed complete separation of miRNA profiles on PLSDA. Three miRNAs were elevated (adj. p < 0.05) in TBI (miR-1255b-5p, miR-3142, and miR-4320), and two were lower (miR-326 and miR-4646-5p). Three miRNAs (miR-3907, miR-4254, and miR-1273g-5p) showed temporal changes post-injury. Brain-related targets of these miRNAs included the glutamatergic synapse and GRIN2B. Conclusions: This study shows that saliva miRNA profiles in children with moderate-to-severe TBI may differ from those with non-TBI trauma and exhibit temporal changes post-injury. These miRNAs could serve as non-invasive biomarkers for prognosticating pediatric TBI outcomes. Further studies are needed to confirm these findings.

Differential microRNA expression in adolescent anxiety proneness.

Biological mechanisms underlying anxiety proneness (AP), the tendency to react fearfully to stressors due to the belief that experiencing anxiety has harmful consequences, remain unclear. Epigenetic mechanisms, such as microRNAs (small, non-coding RNAs 19-20 nucleotides long), may be contributory. This study investigated AP-associated differences in microRNA expression among South African adolescents with variable exposure to childhood trauma (CT). AP was assessed using a composite score reflecting trait anxiety and anxiety sensitivity, while CT exposure was assessed with the Childhood Trauma Questionnaire. High-quality total RNA (n = 88) extracted from whole blood underwent microRNA-sequencing. Differential microRNA expression analysis was conducted with DESeq2 in R, messenger RNA target prediction analysis was performed using TargetScan and DIANA-microT, and the DIANA mirPATH tool was used for KEGG pathway analysis. The majority of participants were female (75.86%) with an average age of 15 (±1.19) years. MicroRNA expression analysis identified upregulation of hsa-miR-28-5p and downregulation of hsa-miR-502-3p and hsa-miR-500a-3p in high-AP individuals, irrespective of CT. Four KEGG pathways, each with ≥10% of their constituent genes predicted to be targets of the differentially expressed microRNAs, were identified and were enriched for genes involved in calcineurin and glutamate signalling. These findings suggest that epigenetically mediated effects on neuronal function contribute to the molecular aetiology of AP.

MECHANISMS OF EPIGENETIC REGULATION IN THE DEVELOPMENT OF PREECLAMPSIA

Introduction. The etiology and pathophysiology of pre-eclampsia (PE) are unclear. Effective methods of its prognosis, prevention and treatment have not still been devised. Of great interest have been the prospects for the use microRNA molecules that epigenetically control the expression of target genes at the post-transcriptional level and of key importance in proliferation, differentiation, invasion, migration, apoptosis of trophoblast cells, regulation of angiogenesis, immune response and other processes which occur during pregnancy Aim. The objective of the study is to investigate the epigenetic development mechanisms of PE based on the evaluation of the expression of pathogenetically significant microRNAs in women’s blood plasma. Materials and methods. The study include 62 female patients divided into the main study group (42 pregnant women with PE) and the control group (20 healthy women with uncomplicated pregnancy, childbirth and post-natal period). All patients have undergone a general clinical, laboratory, instrumental examination. The expression 15 micRroNAs level in blood plasma has been evaluated using a quantitative real-time polymerase chain reaction.The software DIANA miRPath v. 3.0 has been used to evaluate the effect of differentially expressed microRNAs on the signalling pathways functioning . The statistical data processing has been carried out using the licensed software package Statistica 6.0. Results. The women with PE have been detected to have multidirectional changes in the expression of 13 out of 15 plasma microRNAs compared with the control group; however, there has been a statistically significant decrease in the expression levels of 8 microRNAs: hsa-miR-146a-5p, hsa-miR-181a-5p , hsa-miR-210-3p, hsa-miR-517a-3p , hsa-miR-517c-3p, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-1304-5p . The subgroup of pregnant women having PE proceeding with the symptoms of fetal growth retardation (FGR), has shown a significant decrease in the expression of hsa-miR-20a-5p and hsa-miR-143-3p molecules compared with the subgroup without FGR. Dysregulation of more than 40 signalling pathways has been shown. Conclusion. The development of PE proceeds alongside with significant epigenetic changes accompanied by changes in the microRNA expression profile which are associated with cardiovascular, cerebrovascular diseases, placental disorders. The detected differentially expressed microRNAs may be potential diagnostic markers of PE

Small RNA Deep Sequencing Uncovers microRNAs Associated with Hearing Loss in Vestibular Schwannoma.

To analyze the correlation between the miRNA expression profile in vestibular schwannoma (VS) tumor tissue and preoperative patient's hearing status, using the RNA-seq technique. Nineteen tumor samples were collected from patients operated for VS in a Tertiary Academic Center. Samples were classified into "good hearing" and "poor hearing" study group based on the results of audiometric studies. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, using NovaSeq 6000 Illumina system. Functional analysis was performed with the use of DIANA miRpath v. 4.0 online tool. The most overexpressed miRNAs in VS samples derived from poor hearing patients belonged to miR 449a/b, miR 15/16-1, and hypoxamiR families. Functional analysis showed that the differentially expressed miRNAs regulate cellular pathways associated with hypoxia, adherence junction functions, and signaling pathways such as Hippo, FOXO, MAPK, and Wnt signaling pathway. Our study identified a specific miRNA expression profile in VS tumor tissues that correlates with hearing impairment. These results suggest potential new molecular mechanisms related to hearing loss in the course of VS. 3 (cohort study) Laryngoscope, 134:3778-3785, 2024.

High-throughput RNA sequencing identifies the miRNA expression profile, target genes, and molecular pathways contributing to growth of sporadic vestibular schwannomas.

To assess the differences in the miRNA expression profile between small (stage I Koos classification) and large solid vestibular schwannoma (VS) tumors, using the RNA-seq technique. Twenty tumor samples (10 small and 10 large tumors) were collected from patients operated for VS in a Tertiary Academic Center. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, with NovaSeq 6000 Illumina system. Bioinformatics analysis was done using statistical software R. Gene enrichment and functional analysis was performed using miRTargetLink 2.0 and DIANA miRpath 3.0 online tools. We identified 9 differentially expressed miRNAs in large VS samples: miR-7, miR-142 (-3p and -5p), miR-155, miR-342, miR-1269, miR-4664, and miR-6503 were upregulated, whereas miR-204 was significantly down-regulated in comparison to small VS samples. Gene enrichment analysis showed that the most enriched target genes were SCD, TMEM43, LMNB2, JARID2, and CCND1. The most enriched functional pathways were associated with lipid metabolism, along with signaling pathways such as Hippo and FOXO signaling pathway. We identified a set of 9 miRNAs that are significantly deregulated in large VS in comparison to small, intracanalicular tumors. The functional enrichment analysis of these miRNAs suggests novel mechanisms, such as that lipid metabolism, as well as Hippo and FOxO signaling pathways that may play an important role in VS growth regulation.

Medication-Related Osteonecrosis of the Jaw: A Systematic Review and a Bioinformatic Analysis

The objective was to evaluate the current evidence regarding the etiology of medication-related osteonecrosis of the jaw (MRONJ). This study systematically reviewed the literature by searching PubMed, Web of Science, and ProQuest databases for genes, proteins, and microRNAs associated with MRONJ from the earliest records through April 2023. Conference abstracts, letters, review articles, non-human studies, and non-English publications were excluded. Twelve studies meeting the inclusion criteria involving exposure of human oral mucosa, blood, serum, saliva, or adjacent bone or periodontium to anti-resorptive or anti-angiogenic agents were analyzed. The Cochrane Collaboration risk assessment tool was used to assess the quality of the studies. A total of 824 differentially expressed genes/proteins (DEGs) and 22 microRNAs were extracted for further bioinformatic analysis using Cytoscape, STRING, BiNGO, cytoHubba, MCODE, and ReactomeFI software packages and web-based platforms: DIANA mirPath, OmicsNet, and miRNet tools. The analysis yielded an interactome consisting of 17 hub genes and hsa-mir-16-1, hsa-mir-21, hsa-mir-23a, hsa-mir-145, hsa-mir-186, hsa-mir-221, and hsa-mir-424. A dominance of cytokine pathways was observed in both the cluster of hub DEGs and the interactome of hub genes with dysregulated miRNAs. In conclusion, a panel of genes, miRNAs, and related pathways were found, which is a step toward understanding the complexity of the disease.

Discovering the Biological Significance and Therapeutic Potential of miR-29b-3p in Triple-Negative Breast Cancer.

The lack of estrogen or progesterone receptors and absence of HER2 amplification/overexpression in triple-negative breast cancer (TNBC) restricts therapeutic options used in clinical management. MicroRNAs (miRNAs) are small, non-coding transcripts which affect important cellular mechanisms by regulating gene expression at the post-transcriptional level. Among this class, attention was focused on miR-29b-3p with a high profile in TNBC and correlated with the overall survival rates, as TCGA data revealed. This study aims to investigate the implication of the miR-29b-3p inhibitor in TNBC cell lines by identifying a potential therapeutic transcript, improving the clinical outcomes of this disease. The experiments were performed on two TNBC cell lines (MDA-MB-231 and BT549) as in vitro models. An established dose of 50 nM was used for all functional assays performed on the miR-29b-3p inhibitor. A decreased level of miR-29b-3p determined a significant reduction in cell proliferation and colony-forming capacity. At the same time, the changes occurring at the molecular and cellular levels were highlighted. We observed that, when inhibiting the expression level of miR-29b-3p, processes such as apoptosis and autophagy were activated. Further, microarray data revealed that the miRNA expression pattern was altered after miR-29b-3p inhibition, pointing out 8 overexpressed and 11 downregulated miRNAs specific for BT549 cells and 33 upregulated and 10 downregulated miRNAs that were specific for MDA-MB-231 cells. As a common signature for both cell lines, three transcripts were observed, two downregulated, miR-29b-3p and miR-29a, and one upregulated, miR-1229-5p. According to DIANA miRPath, the main predicted targets are related to ECM (extracellular matrix) receptor interaction and TP53 signaling. An additional validation step through qRT-PCR was performed, which showed an upregulation of MCL1 and TGFB1. By inhibiting the expression level of miR-29b-3p, it was shown that complex regulatory pathways targeted this transcript in TNBC cells.

Could Long Non-Coding RNA MEG3 and PTENP1 Interact with miR-21 in the Pathogenesis of Non-Alcoholic Fatty Liver Disease?

NAFLD is the most common cause of chronic liver disease worldwide. The miRNAs and lncRNAs are important endogenous ncRNAs families that can regulate molecular mechanisms. The aim of this study was to analyze the miRNA and lncRNA expression profiles in serum samples of NAFLD patients with different types of hepatosteatosis compared to healthy controls by the qPCR method. A total of180 NAFLD patients and 60 healthy controls were included. miRCURY LNA miRNA miRNome PCR human panel I + II kit and LncProfiler qPCR Array Kit were used to detect miRNA and lncRNA expression, respectively. DIANA miRPath and DIANA-lncBase web servers were used for interaction analysis. As a result, 75 miRNA and 24 lncRNA expression changes were determined. For miRNAs and lncRNAs, 30 and 5 were downregulated and 45 and 19 were upregulated, respectively. hsa-miR-21 was upregulated 2-fold whereas miR-197 was downregulated 0.25-fold. Among lncRNAs, NEAT1 was upregulated 2.9-fold while lncRNA MEG3 was downregulated 0.41-fold. A weak correlation was found between hsa-miR-122 and lncRNA MALAT1. As a conclusion, it is clear that lncRNA-miRNA interaction is involved in the molecular mechanisms of the emergence of NAFLD. The lncRNAs MEG3 and PTENP1 interacted with hsa-miR-21. It was thought that this interaction should be investigated as a biomarker for the development of NAFLD.

IDENTIFICATION AND FUNCTIONAL ANALYSIS OF microRNA AS PROSPECTIVE PROGNOSTIC MARKERS OF COLORECTAL CANCER

Summary. Colorectal cancer (CRC) is one of the main causes of death from malignant neoplasms. Despite the progress achieved in the diagnosis and treatment of CRC, long-term results still remain unsatisfactory. That is why the search for specific biomarkers for predicting the course of the tumor process and personalizing the therapy of this form of cancer is relevant. Aim: to identify microRNAs and determine their functional role in the occurrence and progression of CRC, with the aim of further using them as prognostic markers of this oncopathology. Object and methods: KEGG analysis of signaling pathways was performed using DIANA miRPath v.3.0. Analysis of the functional role of the target genes of the microRNAs was carried out using the DAVID online tool. The study of overall survival rates of patients with CRC depending on the expression level of the miRNAs was conducted using the ENCORI Pan-Cancer Analysis Platform databases. Results: based on the data presented in the databases miR2Disease, HMDD v.3.2, PhenomiR v.2.0, we identified that 3 microRNAs are involved in the occurrence and progression of CRC and can be considered as prognostic markers — hsa-mir-100-5p, hsa-mir-125b- 5p, hsa-mir- 200b-3p. It was established that the studied microRNAs are involved in the regulation of 24 signaling pathways, among which the strongest signaling associations were characteristic of Proteoglycans in cancer (hsa05205), Fatty acid biosynthesis (hsa00061), Fatty acid metabolism (hsa01212) and ErbB signaling pathway (hsa04012). It was found that high expression rates of hsa-mir-200b- 3p in CRC tissue are associated with better rates of overall patient survival. Conclusions: the results obtained by us indicate the need for further research of the role of hsamir- 100-5p, hsa-mir-125b-5p and hsa-mir-200b-3p in the mechanisms of the occurrence and progression of CRC and indicate the perspective of using hsa-mir-200b-3p as a prognostic marker associated with the aggressiveness of the tumor process.

Bioinformatics and Expression Analyses of miR-639, miR-641, miR-1915-3p and miR-3613-3p in Colorectal Cancer Pathogenesis.

Objectives: MicroRNAs (miRNAs) have important function in cancer development and progression. This study aims to determine the expression levels of miR-639, miR-641, miR-1915-3p, and miR-3613-3p in tissues of colorectal cancer (CRC) patients and the role of these miRNAs in the CRC pathogenesis. Methods: Tumor and non-tumor tissues were collected from a total of 59 CRC patients. qRT-PCR was used to identify the expressions of miR-639, miR-641, miR-1915-3p and miR-3613-3p. Through bioinformatics analysis, the target genes of miRNAs were identified by using DIANA mirPath v.3. Signaling pathways were generated using KEGG pathway database. Biological pathway, cellular component analysis, and analysis of Protein-Protein Interactions (PPI) Networks were performed using FunRich and STRING database. Results: Our findings revealed that miR-639, miR-641 and miR-3613-3p were significantly downregulated, and miR-1915-3p was significantly upregulated in tumor tissues compared to non-tumor tissues (p˂0.05). Furthermore, MAPK signaling pathway was the most enriched KEGG pathway regulated by miR-639, miR-641, miR-1915-3p and miR-3613-p. According to the FunRich, it was demonstrated that the targeted genes by miRNAs related to the cellular component and biological pathways such as beta-catenin-TCF7L2, axin-APC-beta-catenin-GSK3B complexes, Arf6 signaling, Class I PI3K signaling, etc. And, by the PPI analysis, it was established that the target genes were clustered on CTNNB1 and KRAS. Conclusions: These outcomes imply that miR-639, miR-641 and miR-3613-3p have tumor suppressor roles, while miR-1915-3p has an oncogenic role in the pathogenesis of CRC. According to the results of the current study, dysregulated miR-639, miR-641, miR-1915-3p, and miR-3613-3p might contribute to the development of CRC.

Hsa-let-7g-5p, a circulating microRNA, as a biomarker for Alzheimer's disease

Effective prevention and treatment of Alzheimer's disease depends on the identification of accurate, rapid and non-invasive biomarkers. Peripheral blood and biological fluids such as plasma, serum, and cerebrospinal fluid are valuable samples for the detection of biomarkers because they cause little discomfort to the patient and facilitate the diagnosis of the disease in the early stages. Small non-coding RNAs known as microRNA (miRNA) play a vital role in regulating various signaling pathways and are involved in various diseases. More than 70% of experimentally confirmed miRNAs are expressed in the brain which can be used as potential diagnostic biomarkers of AD. In silico pathway analysis by DIANA-Mir Path confirmed that miR-let-7g is involved in chronic myeloid leukemia, cell cycle, melanoma and pancreatic cancer pathways. This study aimed to evaluate the hsa-let7g-5p miRNA as a possible blood biomarker for the early detection of AD.Samples of blood were taken from 30 AD patients and 30 healthy people. RNA total was isolated from serum samples and converted to cDNA and the expression level of Let7 miRNAs was measured by the real-time PCR method.The results of the study showed that the expression of hsa-let7g-5p was significantly decreased in patients with AD compared to the normal cases. The AROC for hsa-let-7g-5p transcript levels was 0.4032 compared to the 0.7869 from the normal control group (P = 0.035; 95% CI, 0.27–0.90). These findings suggest that hsa-let7g-5p miRNAs may be used as a potential biomarker for AD diagnosis.

Epigenetic Profiling in the Saliva of Obese Pregnant Women.

Maternal obesity is associated with inflammation and oxidative stress, strongly impacting the intrauterine environment with detrimental consequences for both mother and offspring. The saliva is a non-invasive biofluid reflecting both local and systemic health status. This observational study aimed to profile the epigenetic signature in the saliva of Obese (OB) and Normal-Weight (NW) pregnant women. Sixteen NW and sixteen OB Caucasian women with singleton spontaneous pregnancies were enrolled. microRNAs were quantified by the OpenArray Platform. The promoter region methylation of Suppressor of Cytokine Signaling 3 (SOCS3) and Transforming Growth Factor Beta 1 (TGF-Beta1) was assessed by pyrosequencing. There were 754 microRNAs evaluated: 20 microRNAs resulted in being differentially expressed between OB and NW. microRNA pathway enrichment analysis showed a significant association with the TGF-Beta signaling pathway (miTALOS) and with fatty acids biosynthesis/metabolism, lysine degradation, and ECM–receptor interaction pathways (DIANA–miRPath). Both SOCS3 and TGF-Beta1 were significantly down-methylated in OB vs. NW. These results help to clarify impaired mechanisms involved in obesity and pave the way for the understanding of specific damaged pathways. The characterization of the epigenetic profile in saliva of pregnant women can represent a promising tool for the identification of obesity-related altered mechanisms and of possible biomarkers for early diagnosis and treatment of pregnancy-adverse conditions.

Circular RNA detection identifies circPSEN1 alterations in brain specific to autosomal dominant Alzheimer's disease

BackgroundAutosomal-dominant Alzheimer's disease (ADAD) is caused by pathogenic mutations in APP, PSEN1, and PSEN2, which usually lead to an early age at onset (< 65). Circular RNAs are a family of non-coding RNAs highly expressed in the nervous system and especially in synapses. We aimed to investigate differences in brain gene expression of linear and circular transcripts from the three ADAD genes in controls, sporadic AD, and ADAD.MethodsWe obtained and sequenced RNA from brain cortex using standard protocols. Linear counts were obtained using the TOPMed pipeline; circular counts, using python package DCC. After stringent quality control (QC), we obtained the counts for PSEN1, PSEN2 and APP genes. Only circPSEN1 passed QC. We used DESeq2 to compare the counts across groups, correcting for biological and technical variables. Finally, we performed in-silico functional analyses using the Circular RNA interactome website and DIANA mirPath software.ResultsOur results show significant differences in gene counts of circPSEN1 in ADAD individuals, when compared to sporadic AD and controls (ADAD = 21, AD = 253, Controls = 23—ADADvsCO: log2FC = 0.794, p = 1.63 × 10–04, ADADvsAD: log2FC = 0.602, p = 8.22 × 10–04). The high gene counts are contributed by two circPSEN1 species (hsa_circ_0008521 and hsa_circ_0003848). No significant differences were observed in linear PSEN1 gene expression between cases and controls, indicating that this finding is specific to the circular forms. In addition, the high circPSEN1 levels do not seem to be specific to PSEN1 mutation carriers; the counts are also elevated in APP and PSEN2 mutation carriers. In-silico functional analyses suggest that circPSEN1 is involved in several pathways such as axon guidance (p = 3.39 × 10–07), hippo signaling pathway (p = 7.38 × 10–07), lysine degradation (p = 2.48 × 10–05) or Wnt signaling pathway (p = 5.58 × 10–04) among other KEGG pathways. Additionally, circPSEN1 counts were able to discriminate ADAD from sporadic AD and controls with an AUC above 0.70.ConclusionsOur findings show the differential expression of circPSEN1 is increased in ADAD. Given the biological function previously ascribed to circular RNAs and the results of our in-silico analyses, we hypothesize that this finding might be related to neuroinflammatory events that lead or that are caused by the accumulation of amyloid-beta.

Identification of miRNAs and their potential effects on multiple sclerosis related pathways using ın silico analysis

BackgroundMicroRNAs (miRNAs) regulate gene expression post-transcriptionally via mRNA degradation. As a result, they have an impact on a variety of pathways in organisms that are important for both health and disease. miRNAs can be used as potential diagnostic, prognostic and therapeutic biomarkers for immune and nervous system-related diseases such as MS. MethodDifferentially expressed miRNAs from peripheral blood samples of patient and control groups were selected from NCBI GEO Datasets using GEO2R. Common miRNAs and their related pathways were analyzed using miRNet, miRWalk, DIANA mirpath, KEGG pathway. Target genes and their protein-protein interactions were also evaluated using STRING and GeneMANIA. ResultsWe found 12 common miRNAs, four of which were determined to be more important in MS-related pathways such as the immune and neural signaling networks. These include pathways neurotrophin, JAK-STAT, B cell receptor, ErbB, MAPK, Fc gamma R-mediated phagocytosis, Chemokine and T cell receptor signaling pathways. Moreover, target gene analyses were performed and MAPK1, PIK3CD, PIK3R1, PIK3R2, PIK3R3, PIK3R5, AKT2, SOS2, RAF1 genes were found. Further analysis showed that the identified genes and related pathways have interactions at multiple points, and that the overlapping points are commonly found in the PI3K-Akt signaling pathway. Conclusion: In this paper, MS-related miRNAs and their potential effects on related pathways were evaluated. This study can be used for understanding MS pathogenesis and provides new tools for the discovery of new therapies.

Competing endogenous RNA network mediated by circ_3205 in SARS-CoV-2 infected cells

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA–miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman’s correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student’s t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman’s correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.

Transient Hyperglycemia and Hypoxia Induce Memory Effects in AngiomiR Expression Profiles of Feto-Placental Endothelial Cells.

Gestational diabetes (GDM) and preeclampsia (PE) are associated with fetal hyperglycemia, fetal hypoxia, or both. These adverse conditions may compromise fetal and placental endothelial cells. In fact, GDM and PE affect feto-placental endothelial function and also program endothelial function and cardiovascular disease risk of the offspring in the long-term. MicroRNAs are short, non-coding RNAs that regulate protein translation and fine tune biological processes. A group of microRNAs termed angiomiRs is particularly involved in the regulation of endothelial function. We hypothesized that transient hyperglycemia and hypoxia may alter angiomiR expression in feto-placental endothelial cells (fpEC). Thus, we isolated primary fpEC after normal, uncomplicated pregnancy, and induced hyperglycemia (25 mM) and hypoxia (6.5%) for 72 h, followed by reversal to normal conditions for another 72 h. Current vs. transient effects on angiomiR profiles were analyzed by RT-qPCR and subjected to miRNA pathway analyses using DIANA miRPath, MIENTURNET and miRPathDB. Both current and transient hypoxia affected angiomiR profile stronger than current and transient hyperglycemia. Both stimuli altered more angiomiRs transiently, i.e., followed by 72 h culture at control conditions. Pathway analysis revealed that hypoxia significantly altered the pathway ‘Proteoglycans in cancer’. Transient hypoxia specifically affected miRNAs related to ‘adherens junction’. Our data reveal that hyperglycemia and hypoxia induce memory effects on angiomiR expression in fpEC. Such memory effects may contribute to long-term adaption and maladaption to hyperglycemia and hypoxia.

Sustained Morphine Delivery Suppresses Bone Formation and Alters Metabolic and Circulating miRNA Profiles in Mice

Opioid use is associated with a three to four-fold increase in fracture risk, which is higher in males compared to females. Although one component of that risk may be falls, there is also evidence of altered bone remodeling. However, the mechanisms of these effects are not entirely clear. Our aim was to develop a mouse model of opioid-induced bone loss and study the impact on bone turnover and potential miRNA-mediated regulatory mechanisms. We evaluated the effects of sustained morphine treatment on the skeleton and metabolism of male and female C57BL/6J mice by treating with vehicle (0.9% saline) (n=9–11) or morphine (18 mg/kg) (n=11) using subcutaneous osmotic minipumps for 25 days. Morphine did not influence body weight or food intake, but did reduce fat mass in both sexes. All mice treated with morphine had higher resting energy expenditure and respiratory quotient, indicating a shift toward carbohydrate metabolism. After 25 days of treatment, microComputed Tomography (µCT) analyses indicated that morphine-treated male mice lost 15% of trabecular bone volume fraction (Tb.BV/TV) and 14% of Tb. bone mineral density (BMD) (p<0.05) in the distal femur compared to vehicle, but there were no changes in cortical bone. Females did not lose bone, suggesting differences may be hormone-related. Despite these sex differences, males and females had similar levels of morphine exposure, measured by LC-MS/MS. Histomorphometric analyses demonstrated that in males, morphine reduced bone formation rate (p<0.05) compared to vehicle, but did not impact osteoclast parameters. Consistent with this, morphine reduced bone formation marker gene expression in the tibia of males (including Bglap and Dmp1). Circulating miRNA profiles were interrogated in serum collected at day 12 from vehicle- and morphine-treated male and female mice. While very few changes were present in females, there were 14 differentially expressed miRNAs in males treated with morphine that reached a threshold of ≥ 2-fold change and p<0.01. After target pathway analysis (DIANA miR Path V.3 using experimentally validated miRNA/mRNA targets (Tarbase V.7)), we found that the four upregulated miRNAs (miR-484, -223-3p, -328-3p, and -3107-3p) were associated with 13 enriched KEGG pathways (p<0.05), including fatty acid metabolism pathways (p<0.001). Fatty acid metabolism has recently been linked to osteoblast function, and suppression of such pathways is consistent with the finding of increased respiratory quotient. In summary, morphine leads to trabecular bone loss due to reduced bone formation in males. miRNA findings indicate this may be due to altered metabolic control of mineralization. Further investigation into hormonal and metabolic dependency of morphine-induced bone formation changes could lead to clinical mitigation strategies for preventing the adverse effects of opioids on bone health.

A comparative study of single or dual treatment of theranostic 188Re-Liposome on microRNA expressive profiles of orthotopic human head and neck tumor model

Background: 188Re-liposome has been used for evaluating the theranostic efficacy on human head and neck squamous cell carcinoma (HNSCC) at preclinical stages. Here we furthercompared the microRNA expressive profile in orthtopic HNSCC tumor model exposed to 188Re-liposome. Methods: A single dose or dual doses of 188Re-liposome was intravenously injected into tumor-bearing mice followed by the Cerenkov luminescent imaging (CLI) for monitoring the accumulation of 188Re-liposome in tumors. The microRNA expressive profile was generated using the Taqman® OpenArray® Human MicroRNA Panel followed by the DIANA mirPath analysis, KEGG signaling pathways prediction, and Kaplan-Meier survival analysis for predicting the prognostic role of 188Re-liposome affected microRNAs. Results: Dual doses of 188Re-liposome exhibited a better tumor suppression than a single dose of 188Re-liposome, including reduced tumor size, Ki-67 proliferative marker, and epithelial-mesenchymal transition (EMT) related factors. The microRNA expressive profiles showed that 22 microRNAs and 19 microRNAs were up-regulated and down-regulated by dual doses of 188Re-liposome, respectively. Concomitantly, these two groups of microRNAs were inversely regulated by a single dose of 188Re-liposome accordingly. These microRNAs influenced most downstream genes involved in cancer related signaling pathways. Further, miR-520e and miR-522-3p were down-regulated whereas miR-186-5p and miR-543 were up-regulated by dual doses of 188Re-liposome, and they separately affected most of genes involved in their corresponding pathways with high significance. Additionally, high expressions of miR-520e and miR-522-3p were associated with lower survival rate of HNSCC patients. Conclusion: MicroRNA expression could be used to evaluate the therapeutic efficacy and regarded prognostic factors using different doses of 188Re-liposome.