The binding of microRNAs (miRNAs) to their target sites is a complex process, mediated by the Argonaute (Ago) family of proteins. The prediction of miRNA:target site binding is an important first step for any miRNA target prediction algorithm. To date, the potential for miRNA:target site binding is evaluated using either co-folding free energy measures or heuristic approaches, based on the identification of binding 'seeds', i.e., continuous stretches of binding corresponding to specific parts of the miRNA. The limitations of both these families of methods have produced generations of miRNA target prediction algorithms that are primarily focused on 'canonical' seed targets, even though unbiased experimental methods have shown that only approximately half of in vivo miRNA targets are 'canonical'. Herein, we present miRBind, a deep learning method and web server that can be used to accurately predict the potential of miRNA:target site binding. We trained our method using seed-agnostic experimental data and show that our method outperforms both seed-based approaches and co-fold free energy approaches. The full code for the development of miRBind and a freely accessible web server are freely available.
This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.
MicroRNAs (miRNAs) are noncoding regulatory sequences that govern posttranscriptional inhibition of genes through binding mainly at regulatory regions. The regulatory mechanism of miRNAs are influenced by complex crosstalk among single nucleotide polymorphisms (SNPs) within miRNA seed region and epigenetic modifications. Circulating miRNAs exhibit potential characteristics as stable biomarker. Functionally, miRNAs are involved in basic regulatory mechanisms of cells including inflammation. Thus, miRNA dysregulation, resulting in aberrant expression of a gene, is suggested to play an important role in disease susceptibility. This review focuses on the role of miRNA as diagnostic marker in pathogenesis of lung inflammatory diseases and in cardiac remodelling events during inflammation. From recent reports, In this context, the information about the models in which miRNAs expression were investigated including types of biological samples, as well as on the methods for miRNA validation and prediction/definition of their gene targets are emphasized in the review. Besides disease pathogenesis, promising role of miRNAs in early disease diagnosis and prognostication is also discussed. However, some miRNAs are also indicated with protective role. Thus, identifications and usage of such potential miRNAs as well as disruption of disease susceptible miRNAs using antagonists, antagomirs, are imperative and may provide a novel therapeutic approach towards combating the disease progression.
- MeSH
- Humans MeSH
- MicroRNAs genetics MeSH
- Myocardium immunology metabolism MeSH
- Lung immunology metabolism MeSH
- Inflammation immunology metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs. Due to the high specificity of miRNA sponges and strong inhibition of target miRNAs, these molecules have become increasingly applied in miRNA loss-of-function studies. However, improperly designed sponge constructs may sequester off-target miRNAs; thus, it has become increasingly important to develop a tool for miRNA sponge construct design and testing. In this study, we introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences. Furthermore, we also provide an example, supplemented with experimental data, of how to use this tool. Using miRNAsong, we designed and tested a sponge for miR-145 inhibition, and cloned the sequence into an inducible lentiviral vector. We found that established cell lines expressing miR-145 sponge strongly inhibited miR-145, thus demonstrating the usability of miRNAsong tool for sponge generation. URL: http://www.med.muni.cz/histology/miRNAsong/.
- MeSH
- HEK293 Cells MeSH
- Internet * MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- MicroRNAs genetics metabolism MeSH
- Computer Simulation MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Reduced DNA repair capacity and DNA damage accumulation may lead to cancer development. Regulation of and coordination between genes involved in DNA repair pathways is fundamental for maintaining genome stability, and post-transcriptional gene regulation by microRNAs (miRNAs) may therefore be of particular relevance. In this context, the presence of single nucleotide polymorphisms (SNPs) within the 3'untranslated regions of target DNA repair genes could alter the binding with specific miRNAs, modulating gene expression and ultimately affecting cancer susceptibility. In this study, we investigated the role of genetic variations in miRNA-binding sites of nucleotide excision repair (NER) genes in association with colorectal cancer (CRC) risk. From 28 NER genes, we screened among SNPs residing in their 3'untranslated regions and simultaneously located in miRNA-binding sites, with an in silico approach. Through the calculation of different binding free energy according to both alleles of identified SNPs, and with global binding free energies median providing a threshold, we selected nine NER gene variants. We tested those SNPs in 1098 colorectal cancer cases and 1469 healthy controls from the Czech Republic. Rs7356 in RPA2 and rs4596 in GTF2H1 were associated with colorectal cancer risk. After stratification for tumor location, the association of both SNPs was significant only for rectal cancer (rs7356: OR 1.52, 95% CI 1.02-2.26, P = 0.04 and rs4596: OR 0.69, 95% CI 0.50-0.94, P = 0.02; results not adjusted for multiple testing). Variation in miRNA target binding sites in the 3'untranslated region of NER genes may be important for modulating colorectal cancer risk, with a different relevance according to tumor location.
Genetic variations in miRNAs binding site might participate in cancer risk. This study aimed to systematically review the association between miRNA-binding site polymorphisms and colorectal cancer (CRC). Electronic literature search was carried out on PubMed, Web of Science (WOS), Scopus, and Embase. All types of observational studies till 30 November 2018 were included. Overall 85 studies (21 SNPs) from two systematic searches were included analysis. The results showed that in the Middle East population, the minor allele of rs731236 was associated with decreased risk of CRC (heterozygote model: 0.76 [0.61-0.95]). The minor allele of rs3025039 was related to increased risk of CRC in East Asian population (allelic model: 1.25 [1.01-1.54]). Results for rs3212986 were significant in overall and subgroup analysis (P < .05). For rs1801157 in subgroup analysis the association was significant in Asian populations (including allelic model: 2.28 [1.11-4.69]). For rs712, subgroup analysis revealed a significant (allelic model: 1.41 [1.23-1.61]) and borderline (allelic model: 0.92 [0.84-1.00]) association in Chinese and Czech populations, respectively. The minor allele of rs17281995 increased risk of CRC in different genetic models (P < .05). Finally, rs5275, rs4648298, and rs61764370 did not show significant associations. In conclusion, minor allele of rs3025039, rs3212986, and rs712 polymorphisms increases the risk of CRC in the East Asian population, and heterozygote model of rs731236 polymorphism shows protective effect in the Middle East population. In Europeans, the minor allele of rs17281995 may increase the risk of CRC, while rs712 may have a protective effect. Further analysis based on population stratifications should be considered in future studies.
- MeSH
- 3' Untranslated Regions genetics MeSH
- Alleles MeSH
- Asian People genetics MeSH
- Genetic Predisposition to Disease * MeSH
- Polymorphism, Single Nucleotide MeSH
- Colorectal Neoplasms epidemiology genetics MeSH
- Humans MeSH
- MicroRNAs metabolism MeSH
- Observational Studies as Topic MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Risk Factors MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Meta-Analysis MeSH
- Research Support, Non-U.S. Gov't MeSH
- Systematic Review MeSH
- Geographicals
- Czech Republic MeSH
- Asia, Eastern MeSH
- Middle East MeSH
BACKGROUND: Great progress has been made in the understanding of inflammatory processes in psoriasis. However, clarifying the role of genetic variability in processes regulating inflammation, including post-transcriptional regulation by microRNA (miRNA), remains a challenge. OBJECTIVES: We therefore investigated single nucleotide polymorphisms (SNPs) with a predicted change in the miRNA/mRNA interaction of genes involved in the psoriasis inflammatory processes. METHODS: Studied SNPs rs2910164 C/G-miR-146a, rs4597342 T/C-ITGAM, rs1368439 G/T-IL12B, rs1468488 C/T-IL17RA were selected using a bioinformatics analysis of psoriasis inflammation-associated genes. These SNPs were then genotyped using a large cohort of women with psoriasis (n = 241) and healthy controls (n = 516). RESULTS: No significant association with psoriasis was observed for rs2910164, rs1368439, and rs1468488 genotypes. However, the major allele T of rs4597342 -ITGAM was associated with approximately 28% higher risk for psoriasis in comparison to the patients with the C allele (OR = 1.28, 95% CI 1.01-1.61, p = 0.037). In case of genotypes, the effect of the T allele indicates the dominant model of disease penetrance as the CT and TT genotypes increase the chance of psoriasis up to 42% in comparison to CC homozygotes of rs4597342 (OR = 1.42, 95% CI = 1.05-1.94, p = 0.025). CONCLUSION: SNP rs4597342 in 3'UTR of ITGAM influencing miR-21 binding may be considered a risk factor for psoriasis development. Upregulated miR-21 in psoriasis is likely to inhibit CD11b production in the case of the rs4597342 T allele which may lead to Mac-1 dysfunction, resulting in an aberrant function of innate immune cells and leading to the production of cytokines involved in psoriasis pathogenesis.
- MeSH
- 3' Untranslated Regions genetics MeSH
- Alleles MeSH
- CD11b Antigen genetics MeSH
- Asian People MeSH
- Genetic Predisposition to Disease MeSH
- Genetic Association Studies MeSH
- Genotype MeSH
- Polymorphism, Single Nucleotide genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- MicroRNAs genetics MeSH
- Psoriasis genetics pathology MeSH
- Risk Factors MeSH
- Binding Sites genetics MeSH
- Inflammation genetics pathology MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Polymorphisms in microRNA (miRNA) binding sites may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and susceptibility to common diseases. Mucins have been identified as markers of adverse prognosis. We hypothesized that genetic variations in miRNA binding sites located in mucin genes may modulate signaling response and the maintenance of genomic stability ultimately affecting cancer susceptibility, efficacy of chemotherapy and survival. In this study, we analyzed the association of single nucleotide polymorphisms in predicted miRNA target sites (miRSNPs) of mucin genes with colorectal cancer (CRC) risk and clinical outcome. Thirteen miRSNPs in 9 genes were assessed in 1111 cases and 1469 controls. No strongly significant associations were observed in the case-control study. Patients carrying the CC genotype of rs886403 in MUC21 displayed a shorter survival and higher recurrence risk when compared with TT carriers [overall survival (OS): hazard ratios (HR) 1.69; 95% confidence intervals (CI) 1.13-2.46; P = 0.01 and event-free survival (EFS): HR 1.99; 95% CI 1.38-2.84; P = 0.0002, respectively]. The observed associations were more striking after stratification for tumor site (in patients with colon cancer, OS: HR 2.63; 95% CI 1.69-4.10; P < 0.0001 and EFS: HR 2.65; 95% CI 1.72-4.07; P < 0.0001). In contrast, rectal cancer cases carrying the CC genotype of rs4729655 in MUC17 displayed a longer survival (OS: HR 0.27; 95% CI 0.14-0.54; P = 0.0002) than those with the most common genotype. To our knowledge, this is the first study investigating miRSNPs potentially affecting miRNA binding to mucin genes and revealing their impact on CRC susceptibility or patient's survival.
- MeSH
- 3' Untranslated Regions genetics MeSH
- Genetic Predisposition to Disease MeSH
- Genotype MeSH
- Polymorphism, Single Nucleotide * MeSH
- Colorectal Neoplasms genetics mortality pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Neoplasm Recurrence, Local genetics mortality pathology MeSH
- MicroRNAs genetics MeSH
- Survival Rate MeSH
- Mucins genetics metabolism MeSH
- Biomarkers, Tumor genetics MeSH
- Follow-Up Studies MeSH
- Prognosis MeSH
- Aged MeSH
- Neoplasm Staging MeSH
- Case-Control Studies MeSH
- Binding Sites MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
PURPOSE: Colorectal cancer is routinely treated with a 5-fluorouracil (5-FU)-based chemotherapy. 5-FU incorporates into DNA, and the base excision repair (BER) pathway specifically recognizes such damage. We investigated the association of single-nucleotide polymorphisms (SNP) in the 3'-untranslated regions (UTR) of BER genes, and potentially affecting the microRNA (miRNA) binding, on the risk of colorectal cancer, its progression, and prognosis. SNPs in miRNA-binding sites may modulate the posttranscriptional regulation of gene expression operated by miRNAs and explain interindividual variability in BER capacity and response to 5-FU. EXPERIMENTAL DESIGN: We tested 12 SNPs in the 3'-UTRs of five BER genes for colorectal cancer susceptibility in a case-control study (1,098 cases and 1,459 healthy controls). Subsequently, we analyzed the role of these SNPs on clinical outcomes of patients (866 in the Training set and 232 in the Replication set). RESULTS: SNPs in the SMUG1 and NEIL2 genes were associated with overall survival. In particular, SMUG1 rs2233921 TT carriers showed increased survival compared with those with GT/GG genotypes [HR, 0.54; 95% confidence interval (CI), 0.36-0.81; P = 0.003] in the Training set and after pooling results from the Replication set. The association was more significant following stratification for 5-FU-based chemotherapy (P = 5.6 × 10(-5)). A reduced expression of the reporter gene for the T allele of rs2233921 was observed when compared with the common G allele by in vitro assay. None of the genotyped BER polymorphisms were associated with colorectal cancer risk. CONCLUSIONS: We provide the first evidence that variations in miRNA-binding sites in BER genes 3'-UTR may modulate colorectal cancer prognosis and therapy response.
- MeSH
- 3' Untranslated Regions * MeSH
- Adult MeSH
- Genetic Variation * MeSH
- Polymorphism, Single Nucleotide MeSH
- Colorectal Neoplasms drug therapy genetics mortality MeSH
- Middle Aged MeSH
- Humans MeSH
- MicroRNAs genetics metabolism MeSH
- DNA Repair genetics MeSH
- Prognosis MeSH
- Antineoplastic Combined Chemotherapy Protocols therapeutic use MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Case-Control Studies MeSH
- Binding Sites MeSH
- Treatment Outcome MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs-PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer-1⋅Loqs-PB heterodimer. The Dicer-1 dsRBD and three Loqs-PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer-1⋅Loqs-PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
- MeSH
- Drosophila genetics MeSH
- MicroRNAs * genetics metabolism MeSH
- Drosophila Proteins * genetics metabolism MeSH
- RNA-Binding Proteins metabolism MeSH
- Ribonuclease III genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
