miRNA Binding Research Articles

Upregulated miRNAs on the TP53 and RB1 Binding Seedless Regions in High-Risk HPV-Associated Penile Cancer.

Cancer development by the human papillomavirus (HPV) infection can occur through the canonical HPV/p53/RB1 pathway mediated by the E2/E6/E7 viral oncoproteins. During the transformation process, HPV inserts its genetic material into host Integration Sites (IS), affecting coding genes and miRNAs. In penile cancer (PeCa) there is limited data on the miRNAs that regulate mRNA targets associated with HPV, such as the TP53 and RB1 genes. Considering the high frequency of HPV infection in PeCa patients in Northeast Brazil, global miRNA expression profiling was performed in high-risk HPV-associated PeCa that presented with TP53 and RB1 mRNA downregulated expression. The miRNA expression profile of 22 PeCa tissue samples and five non-tumor penile tissues showed 507 differentially expressed miRNAs: 494 downregulated and 13 upregulated (let-7a-5p, miR-130a-3p, miR-142-3p, miR-15b-5p miR-16-5p, miR-200c-3p, miR-205-5p, miR-21-5p, miR-223-3p, miR-22-3p, miR-25-3p, miR-31-5p and miR-93-5p), of which 11 were identified to be in HPV16-IS and targeting TP53 and RB1 genes. One hundred and thirty-one and 490 miRNA binding sites were observed for TP53 and RB1, respectively, most of which were in seedless regions. These findings suggest that up-regulation of miRNA expression can directly repress TP53 and RB1 expression by their binding sites in the non-canonical seedless regions.

CircRNAs in Xiang pig ovaries among diestrus and estrus stages

BackgroundThe fecundity of sows is a trait of major economic in pig industry. The molecular regulation of estrus cycles can affect the fecundity of female animals. Compared with the other pig breeds, Xiang pig exhibits the special estrus behaviors. CircRNAs are thought to involve in regulation of multiple biological processes. However, the potential roles of circRNAs in ovary regulation on Xiang pig estrus are largely unknown.Results8,937 circRNAs were identified from eight libraries constructed from the ovarian samples of Xiang pig at estrus and diestrus stages by RNA sequencing method. Of which, 1,995 were high confidence circRNAs detected at least two junction reads in each ovary sample and seven circRNAs were validated by RT-PCR method. Furthermore, we identified 290 upregulated and 15 downregulated circRNAs in estrus ovaries. These differentially expressed circRNAs (DECs) derived from 273 host genes. And 207 miRNAs were identified to be targets sponged by 156 DECs with 432 binding sites, containing more than one miRNA binding site in each circRNA. Function enrichment analysis revealed that the host genes and the targets of miRNAs sponged by DECs were enriched in several reproduction-related signaling pathways, such as ovarian steroidogenesis, oocyte maturation, circadian rhythm, estrogen signaling pathway, GnRH signaling pathway, circadian entrainment, and oocyte meiosis. The circRNA-miRNA-mRNA networks revealed that 153 miRNAs interacting with 122 DECs and 86 miRNAs interacting with 84 DECs were involved in ovarian functions and ovarian circadian entrainment and circadian rhythm respectively. The DEC-miRNA-DEG (differentially expressed gene, DEG) networks associated with reproduction-related signaling pathways contained 22 DECs,18 miRNAs and 7 DEGs. 22 DECs were recognized as hub circRNAs during the estrus phase of Xiang pigs.ConclusionsThe circRNAs that function as miRNA sponges could play a key role in post-transcriptional regulation of gene expression during Xiang pig’s estrus cycle.

Genome-Wide 3'-UTR Single Nucleotide Polymorphism Association Study Identifies Significant Prostate Cancer Risk-Associated Functional Loci at 8p21.2 in Chinese Population.

MicroRNAs (miRNAs) are involved in the regulation of gene expression via incomplete base pairing to sequence motifs at the three prime untranslated regions (3′‐UTRs) of mRNAs and play critical roles in the etiology of cancers. Single nucleotide polymorphisms (SNPs) in the 3′‐UTR miRNA‐binding regions may influence the miRNA affinity. However, this biological mechanism in prostate cancer (PCa) remains unclear. Here, a three‐stage genome‐wide association study of 3′‐UTR SNPs (n=33 117) is performed in 5515 Chinese men. Three genome‐wide significant variants are discovered at 8p21.2 (rs1567669, rs4872176, and rs4872177), which are all located in a linkage disequilibrium region of the NKX3‐1 gene. Phenome‐wide association analysis using the FinnGen data reveals a specific association of rs1567669 with PCa over 2,264 disease endpoints. Expression quantitative trait locus analyses based on both Chinese PCa cohort and the GTEx database show that risk alleles of these SNPs are significantly associated with low expression of NKX3‐1. Based on the MirSNP database, dual‐luciferase reporter assays show that risk alleles of these SNPs downregulate the expression of NKX3‐1 via increased miRNA binding. These results indicate that the SNPs at the 3′‐UTR of NKX3‐1 significantly downregulate NKX3‐1 expression by influencing the affinity of miRNA and increase the PCa risk.

Abstract 5205: VG2025: Transcriptional and translational dually regulated (TTDR) oncolytic virus for cancer immunotherapy

Abstract VG2025 is a genetically modified HSV-1 oncolytic virus (OV) wherein the transcription and translation of critical viral genes are regulated to enhance virus safety and promote tumor specific virus replication without attenuating the virus. The TTDR design is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor specific CEA promoter, coupled with translational control of the key neurovirulence factor ICP34.5 accomplished by introducing multiple tandem copies of miRNA binding sites for miR-124 and miR-143 which are downregulated in tumors but abundantly expressed in normal brain tissues. The anti-tumor and immune stimulatory properties of VG2025 are also enhanced by expressing a pair of cytokines (IL-12 and IL-15/IL-15Rα complex) that are linked by 2A cleavage peptides and controlled by the tumor specific promoter CXCR4. The dual regulation strategy was verified in vitro and shown to be highly selective. Growth of VG2025 virus correlated with CEA expression in a panel of cell lines including normal cells and a selection of tumor cells with varying CEA levels. miRNA-mediated regulation of ICP34.5 expression was validated by transfecting HEK293t cells with miR-124 and miR-143 precursors, followed by infection with VG2025. RT-qPCR testing revealed a significant decrease in the levels of viral ICP34.5 mRNA when the miRNA was present. VG2025 virus replication was evaluated in a variety of human tumor cell lines, and two cell lines representing NSCLC and pancreatic cancer (A549 and BxPC3, respectively) were selected for testing in a humanized mouse model, where VG2025 displayed strong in vivo anti-tumor efficacy. The immune stimulatory characteristics of VG2025, and the bioactivity of expressed cytokines, were demonstrated in a syngeneic mouse colorectal tumor model (CT26) and lymphoma model (A20) using VG2026, which is an identical mouse specific variant of VG2025 that expresses murine IL-12 in place of human IL-12. In addition to a significant reduction in the growth of injected tumors, we observed a clear abscopal effect on contralateral tumors, indicating a robust anti-tumor immune response with an immune memory effect. Due to the tendency of HSV-1 to establish latency which may lead to neurotoxicity and potential safety concerns, we performed a safety pharmacology study by subcutaneously inoculating immunocompetent DBA mice with VG2025. Absence of neurotoxicity was further verified by more aggressive routes of administration, including via the nose and by corneal scarification. The risk of VG2025 reactivation from a latent state was evaluated in a two-step study: latent infection in the CNS was first established and confirmed, then the trigeminal ganglia were removed and co-cultured with Vero cells to detect reactivation-related cytopathic effects. All studies showed that VG2025 was safe with no reactivation events detected. Citation Format: Yanal Murad, Guoyu Liu, I-Fang Lee, Zahid Delwar, Dmitry Chouljenko, Will Liu, Jun Ding, Luke Bu, Ronghua Zhao, William Jia. VG2025: Transcriptional and translational dually regulated (TTDR) oncolytic virus for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5205.

Circular RNA regulation of fat deposition and muscle development in cattle.

Circular RNAs (circRNAs) are important transcriptional regulatory RNA molecule that can regulate the transcription of downstream genes by competitive binding of miRNAs or coding proteins or by blocking mRNAs translation. Numerous studies have shown that circRNAs are extensively involved in cell proliferation, differentiation and apoptosis, gene transcription and signal transduction. Fat deposition and muscle development have important effects on beef traits. CircRNAs are involved in regulating bovine fat and muscle cells and are differentially expressed in the tissues composed of these cells, suggesting that circRNAs play an important role in regulating bovine fat formation and muscle development. This review describes differential expression of circRNAs in bovine fat and muscle tissues, research progress in understanding how circRNAs regulate the proliferation and differentiation of bovine fat and muscle cells through competing endogenous RNAs networks, and provide a reference for the subsequent research on the molecular mechanism of circRNAs in regulating fat deposition and muscle development in cattle.

Detection of features predictive of microRNA targets by integration of network data.

Gene activity is controlled by multiple molecular mechanisms, for instance through transcription factors or by microRNAs (miRNAs), among others. Established bioinformatics tools for the prediction of miRNA target genes face the challenge of ensuring accuracy, due to high false positive rates. Further, these tools present poor overlap. However, we demonstrated that it is possible to filter good predictions of miRNA targets from the bulk of all predictions by using information from the gene regulatory network. Here, we take advantage of this strategy that selects a large subset of predicted microRNA binding sites as more likely to possess less false-positives because of their over-representation in RE1 silencing transcription factor (REST)-regulated genes from the background of TargetScanHuman 7.2 predictions to identify useful features for the prediction of microRNA targets. These enriched miRNA families would have silencing activity for neural transcripts overlapping the repressive activity on neural genes of REST. We analyze properties of associated microRNA binding sites and contrast the outcome to the background. We found that the selected subset presents significant differences respect to the background: (i) lower GC-content in the vicinity of the predicted miRNA binding site, (ii) more target genes with multiple identical microRNA binding sites and (iii) a higher density of predicted microRNA binding sites close to the 3’ terminal end of the 3’-UTR. These results suggest that network selection of miRNA-mRNA pairs could provide useful features to improve microRNA target prediction.

Identification and functional annotation of long intergenic non-coding RNAs in Brassicaceae.

Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts that can have critical biological functions. The wealth of RNA-sequencing (RNA-seq) data available for plants provides the opportunity to implement a harmonized identification and annotation effort for lincRNAs that enables cross-species functional and genomic comparisons as well as prioritization of functional candidates. In this study, we processed >24 Tera base pairs of RNA-seq data from >16,000 experiments to identify ∼130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify conserved motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our data set with sequence or transcriptional conservation. Finally, we used guilt-by-association analyses to assign putative functions to lincRNAs within our data set. We tested this approach on a subset of lincRNAs associated with germination and seed development, observing germination defects for Arabidopsis lines harboring T-DNA insertions at these loci. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, small open reading frames, or abiotic-stress modulated expression are a few of the annotations that will guide functional analyses into this cryptic portion of the transcriptome.

Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum.

Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and modulators of fundamental biological processes including immunoregulation. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given their involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovary-enriched and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both endogenous and exogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length and endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we have previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect of ivermectin treatment on Ascaris circRNAs expression or secretion.

Novel Role of Long Non-Coding RNA ASAP1-IT1 in Progression of Hepatocellular Carcinoma.

The long non-coding RNA (lncRNA) ASAP1-IT1 has been recently shown to aberrantly increase in ovarian and bladder cancer, while its role in other malignancies remains unexplored. This study was to characterize the expression and assess the potential role of ASAP1-IT1 in hepatocellular carcinoma (HCC). Fifty-four paired HCC and histologically normal tissues were obtained from HCC patients. Human HCC cell lines (HepG2, Huh7, SMMC-7721, and BEL-7402) and a normal liver cell line (LO2) were used for in vitro studies. ASAP1-IT1-specific siRNAs were used to silence ASAP1-IT1 expression, while the pcDNA-ASAP1-IT1 vector was constructed to up-regulate its expression. In situ hybridization and qRT-PCR were performed to characterize subcellular localization and expression of ASAP1-IT1. Cell proliferation and migration assays were conducted to examine the role of ASAP1-IT1 in the progression of HCC. In silico analysis was conducted to predict putative miRNA binding sites, which were validated by luciferase reporter assays. ASAP1-IT1 levels were significantly increased in HCC tissues and cells compared with controls. Notably, higher ASAP1-IT1 levels were significantly associated with poorer prognosis of HCC patients. In situ hybridization analysis revealed that ASAP1-IT1 was mainly localized in the nucleus of hepatoma cells and differentially expressed in trabecular, compact, and pseudoglandular forms of liver cancer. Furthermore, knockdown of ASAP1-IT1 significantly suppressed cell proliferation and migration, while its overexpression significantly promoted cell proliferation and migration of HCC cells. Mechanistically, ASAP1-IT1 might exert its role in HCC progression, at least in part, by directly interacting with miR-221-3p. In conclusion, ASAP1-IT1 is abnormally elevated in HCC, and higher levels are correlated with poorer prognosis. An underlying mechanism has been proposed for ASAP1-IT1-associated promotion of proliferation and migration in HCC cells. These findings have provided evidence supporting the oncogenic role of ASAP1-IT1 in HCC.

Transcriptional regulation of nuclear miRNAs in tumorigenesis (Review).

MicroRNAs (miRNAs/miRs) are a type of endogenous non‑coding small RNA that regulates gene expression. miRNAs regulate gene expression at the post‑transcriptional level by targeting the 3'‑untranslated region (3'UTR) of cytoplasmic messenger RNAs (mRNAs). Recent research has confirmed the presence of mature miRNAs in the nucleus, which bind nascent RNA transcripts, gene promoter or enhancer regions, and regulate gene expression via epigenetic pathways. Some miRNAs have been shown to function as oncogenes or tumor suppressor genes by modulating molecular pathways involved in human cancers. Notably, a novel molecular mechanism underlying the dysregulation of miRNA expression in cancer has recently been discovered, indicating that miRNAs may be involved in tumorigenesis via a nuclear function that influences gene transcription and epigenetic states, elucidating their potential therapeutic implications. The present review article discusses the import of nuclear miRNAs, nucleus‑cytoplasm transport mechanisms and the nuclear functions of miRNAs in cancer. In addition, some software tools for predicting miRNA binding sites are also discussed. Nuclear miRNAs supplement miRNA regulatory networks in cancer as a non‑canonical aspect of miRNA action. Further research into this aspect may be critical for understanding the role of nuclear miRNAs in the development of human cancers.

TLR-2 Germ Line Variants as a Risk for Obesity in Local Pakistani Population

Obesity is increasing rapidly affecting half billion adult's population. Pathophysiology of obesity involves low grade inflammation sustained by Toll like receptor 2 (TLR-2) the innate immune adapters. This study was conducted for detection and association of TLR-2 gene mutations with obesity. In this case-control study 228 individuals with obesity and 228 controls were enrolled based on Body Mass Index (BMI) ≥25 and 18-24 kg/m2 respectively. The variations in TLR-2 gene were detected by Sanger sequencing. These identified TLR-2 variants were further analyzed in silico for change in miRNA binding and mRNA strucutre. Four novel single base substitutions (153688371 T >C, 153702295 T >C, 153703504 T >C and 153705074 C >A) were identified in exon 3 and 4 of TLR-2 gene affecting splice site and poly-A tail. The genotypic and allelic frequencies of the variants were strongly associated with increasing obesity susceptibility. Only variant 153703504 T >C was significantly associated with preobesity. Despite variations in gene sequence, no change in miRNA binding except for variant 153688371 T >C of Exon 3 where a novel binding site for hsa-miR-4523 was created. Furthermore, mRNA stability and secondary structure were also compromised in identified variants. All detected variants of TLR-2 gene were significantly associated with and posed risk for development of obesity. Furthermore, in silico analysis revealed generation of new miRNA (hsa-miR-4523) binding site and change in mRNA structure/stability which needs to be further investigated for possible role in altering TLR-2 gene regulation/expression in obesity.

In silico study predicts a key role of RNA-binding domains 3 and 4 in nucleolin-miRNA interactions.

RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules. RBPs are critical for posttranscriptional mechanisms keeping gene regulation in a fine equilibrium. Conversely, dysregulation of RBPs and RNA metabolism pathways is an established hallmark of tumorigenesis. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). Particularly, NCL interacts directly with microRNAs (miRNAs) and is involved in their aberrant processing linked with many cancers, including breast cancer. Nonetheless, molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize how NCL targets miRNAs and whether this specificity is imposed by a definite RBD-interface. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL-miRNA interactions generated using docking algorithms. Our study suggests a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL-substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. Moreover, the insights from this study can ultimately support the design of novel antineoplastic drugs aimed at regulating NCL-dependent biological pathways with a causal role in tumorigenesis.

Identification of functionally important miRNA targeted genes associated with child obesity trait in genome-wide association studies

BackgroundGenome-wide association studies (GWAS) have uncovered thousands of genetic variants that are associated with complex human traits and diseases. miRNAs are single-stranded non-coding RNAs. In particular, genetic variants located in the 3’UTR region of mRNAs may play an important role in gene regulation through their interaction with miRNAs. Existing studies have not been thoroughly conducted to elucidate 3’UTR variants discovered through GWAS. The goal of this study is to analyze patterns of GWAS functional variants located in 3’UTRs about their relevance in the network between hosting genes and targeting miRNAs, and elucidate the association between the genes harboring these variants and genetic traits.MethodsWe employed MIGWAS, ANNOVAR, MEME, and DAVID software packages to annotate the variants obtained from GWAS for 31 traits and elucidate the association between their harboring genes and their related traits. We identified variants that occurred in the motif regions that may be functionally important in affecting miRNA binding. We also conducted pathway analysis and functional annotation on miRNA targeted genes harboring 3’UTR variants for a trait with the highest percentage of 3’UTR variants occurring.ResultsThe Child Obesity trait has the highest percentage of 3’UTR variants (75%). Of the 16 genes related to the Child Obesity trait, 5 genes (ETV7, GMEB1, NFIX, ZNF566, ZBTB40) had a significant association with the term DNA-Binding (p < 0.05). EQTL analysis revealed 2 relevant tissues and 10 targeted genes associated with the Child Obesity trait.In addition, Red Blood Cells (RBC), Hemoglobin (HB), and Package Cell Volume (PCV) have overlapping variants. In particular, the PIM1 variant occurred inside the HB Motif region 37,174,641–37,174,660, and LUC7L3 variant occurred inside RBC Motif region 50,753,918–50,753,937.ConclusionVariants located in 3’UTR can alter the binding affinity of miRNA and impact gene regulation, thus warranting further annotation and analysis. We have developed a bioinformatics bash pipeline to automatically annotate variants, determine the number of variants in different categories for each given trait, and check common variants across different traits. This is a valuable tool to annotate a large number of GWAS result files.

LINC00152 Drives a Competing Endogenous RNA Network in Human Hepatocellular Carcinoma.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.

Investigating the miRNA regulatory landscape of OGT and OGA via the 3’UTR and 5’UTR regions utilizing the miRFluR high‐throughput platform

The dynamic post‐translational modification of serine or threonine residues by O‐linked N‐acetyl‐beta‐D‐glucosamine (O‐GlcNAc) contributes to diverse cellular processes including epigenetic modifications, transcription, metabolism, and cell signaling that play significant roles in development and normal physiology. O‐GlcNAcylation is catalyzed by O‐GlcNAc transferase and the modification is removed by O‐GlcNAcase (OGA). These genes are highly regulated at multiple levels, but little is known about their regulation by microRNAs (miRs). miRs are small non‐coding RNAs that fine‐tune protein expression through binding to messenger RNA (mRNA). In this work, we built a comprehensive dataset of OGT and OGA regulation via both their 3’UTR and 5’UTRs. Downregulation was almost exclusively mediated through binding to the 3’UTR. We observed independent regulation of OGT and OGA by the majority of regulatory miRs, which did not overlap. However, we did see significant co‐regulation of OGT and OGA by a subset of miRs. This is in keeping with the known transcriptional regulation of these genes.In summary, this work provides a better understanding of OGT and OGA regulation through miRNA binding via both the 3’UTR and 5’UTR regions.

Bioinformatics Analysis of the Interaction of miRNAs and piRNAs with Human mRNA Genes Having di- and Trinucleotide Repeats.

The variability of nucleotide repeats is considered one of the causes of diseases, but their biological function is not understood. In recent years, the interaction of miRNAs and piRNAs with the mRNAs of genes responsible for developing neurodegenerative and oncological diseases and diabetes have been actively studied. We explored candidate genes with nucleotide repeats to predict associations with miRNAs and piRNAs. The parameters of miRNAs and piRNA binding sites with mRNAs of human genes having nucleotide repeats were determined using the MirTarget program. This program defines the start of the initiation of miRNA and piRNA binding to mRNAs, the localization of miRNA and piRNA binding sites in the 5′-untranslated region (5′UTR), coding sequence (CDS) and 3′-untranslated region (3′UTR); the free energy of binding; and the schemes of nucleotide interactions of miRNAs and piRNAs with mRNAs. The characteristics of miRNAs and piRNA binding sites with mRNAs of 73 human genes were determined. The 5′UTR, 3′UTR and CDS of the mRNAs of genes are involved in the development of neurodegenerative, oncological and diabetes diseases with GU, AC dinucleotide and CCG, CAG, GCC, CGG, CGC trinucleotide repeats. The associations of miRNAs, piRNAs and candidate target genes could be recommended for developing methods for diagnosing diseases, including neurodegenerative diseases, oncological diseases and diabetes.

Comprehensive Genome-Wide Identification, Characterization, and Expression Analysis of CCHC-Type Zinc Finger Gene Family in Wheat (Triticum aestivum L.).

The CCHC-type zinc finger proteins (CCHC-ZFPs) play versatile roles in plant growth, development and adaptation to the environment. However, little is known about functions of CCHC-ZFP gene family memebers in Triticum aestivum. In the present study, we identified a total of 50 TaCCHC-ZFP genes from the 21 wheat chromosomes, which were phylogenetically classified into eight groups based on their specific motifs and gene structures. The 43 segmentally duplicated TaCCHC-ZFP genes were retrieved, which formed 36 segmental duplication gene pairs. The collinearity analyses among wheat and other eight mono/dicots revealed that no gene pairs were found between wheat and the three dicots. The promoter analyses of the TaCCHC-ZFP genes showed that 636 environmental stress-responsive and phytohormone-responsive cis-elements. The gene ontology enrichment analysis indicated that all the TaCCHC-ZFP genes were annotated under nucleic acid binding and metal ion binding. A total of 91 MicroRNA (miRNA) binding sites were identified in 34 TaCCHC-ZFP genes according to the miRNA target analysis. Based on the public transcriptome data, the 38 TaCCHC-ZFP genes were identified as differentially expressed gene. The expression profiles of 15 TaCCHC-ZFP genes were verified by the quantitative real-time PCR assays, and the results showed that these genes were responsive to drought or heat treatments. Our work systematically investigated the gene structures, evolutionary features, and potential functions of TaCCHC-ZFP genes. It lays a foundation for further research and application of TaCCHC-ZFP genes in genetic improvement of T. aestivum.

RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants.

Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and ‘G–U wobbles’ on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.

A microRNA binding site polymorphism in the 3\u2032 UTR region of VEGF-A gene modifies colorectal cancer risk based on ethnicity: a meta-analysis

BackgroundVascular endothelial growth factor A (VEGF-A) plays an integral role in angiogenesis by contributing to growth, development, and metastasis of solid tumors. Recently, a single-nucleotide polymorphism +936C/T located in the VEGF-A 3′ untranslated region (UTR) facilitated the susceptibility of colorectal cancer. The association between VEGF-A gene polymorphism +936C/T and colorectal cancer risk has been widely studied in the last decade, but presently, the results furnished remain enigmatic. Hence, the study aimed to investigate the association between VEGF-A +936C/T miRNA binding site polymorphism and the risk of developing colorectal cancer.MethodsThis meta-analysis included 13 published case-control studies covering 3465 cases (colorectal cancer) and 3476 healthy controls. Publication bias was examined by means of Begg’s funnel plots and Egger’s regression tests. The quality of the studies included was evaluated using Newcastle-Ottawa scale. Subgroup analyses were performed in accordance to the various ethnicities of the study subjects and the study quality.ResultsFrom the data obtained, it is implied that VEGF-A +936C/T polymorphism did not correlate with elevated colorectal cancer risk in all genetic models. But the results acquired from the subgroup analysis in over dominant model (CT vs. CC + TT: OR = 1.5047, 95% CI = 1.19–1.90) suggest that VEGF-A +936C/T polymorphism leads to the raise in the risk of developing CRC among the East Asian population. No association was observed in Caucasian and South Asian population.ConclusionsOur results indicate that VEGF-A +936C/T polymorphism is not a risk factor for developing CRC in Caucasian and South Asian population. However, the East Asian population was related to an increased risk of developing colorectal cancer due to the presence of the minor allele.

Circ-0007707/miR-429/PDGFD Pathway Regulates the Progression of Gastric Cancer by Modulating the Immune-Gene Signature.

Background Immunotherapy is an important treatment modality for gastric cancer, therefore, it is crucial to understand the regulators of the tumor microenvironment in gastric cancer. Numerous studies have shown that noncoding RNAs have a critical status in the tumor progression, and the influence of competing endogenous RNA (ceRNA) networks on gastric adenocarcinoma has been widely discussed over the years, but the connection between ceRNA networks and the immune microenvironment of cancer is unclear. This study was aimed at exploring how ceRNA networks influence the prognosis of patients with gastric cancer by modulating the tumor microenvironment. Methods The Gene Expression Omnibus was analyzed to obtain differential expression matrixes of the noncoding RNAs (circular RNAs (circRNAs), microRNAs (miRNAs)), and mRNAs. The Circular RNA Interactome web tool and TargetScan were applied to determine the miRNA binding sites of the circRNAs and miRNA target genes. The Cancer Genome Atlas provided prognostic genes for gastric cancer, and Cytoscape created the ceRNA networks. Real-time quantitative reverse transcription polymerase chain reaction and western blot assay were adopted to find out how the ceRNA network regulates the expression of the hub gene. Additionally, the TISIDB and TIMER databases were used to assess the link between the hub gene and immunotherapy, with TISIDB providing the immune genes that are coexpressed with the hub gene. Furthermore, the immune-gene signature was constructed by using Cox regression analysis. Moreover, the nomogram, which could predict the prognostic role of gastric cancer patients was created on the basis of the immune-gene signature. Results In gastric cancer, the circ-0007707/miR-429/PDGFD pathway had a differential expression. The results demonstrated that the pathway could regulate the progression and immune microenvironment of gastric cancer by modulating the immune-gene signature, which included two immune genes (TAB1 and CXCR4). Moreover, the low-risk group patients had better survival. Conclusion The circ-0007707/miR-429/PDGFD pathway may play a regulatory role in the progression and prognosis of gastric cancer by interfering with the tumor microenvironment, and the PDGFD-related immune-gene signature could be considered a moderator of prognostic factor for gastric cancer and to guide immunotherapy programs.