Number Of miRNA Binding Sites Research Articles

Multiple Copies of microRNA Binding Sites in Long 3'UTR Variants Regulate Axonal Translation.

Rapid responses to changes within subcellular compartments of highly polarized cells, such as neuron axons, depend on local translation and post-transcriptional regulation. The mechanism by which microRNAs (miRNAs) regulate this process is not fully understood. Here, using live cell imaging and RNA sequencing analysis, we demonstrated how miRNAs can differentially control hundreds of transcripts at the subcellular level. We demonstrated that the seed match length of the miRNA target-sequence regulates both mRNA stability and protein translation rates. While longer seed matches have an increased inhibitory effect, transcriptome analysis did not reveal differences in seed match length between axonal and somata mRNAs of motor neurons. However, mRNA variants with longer 3'UTR are enriched in axons and contain multiple repeats of specific miRNA target sequences. Finally, we demonstrated that the long 3'UTR mRNA variant of the motor protein Kif5b is enriched explicitly in motor neuron axons and contains multiple sequence repeats for binding miR-129-5p. This subsequently results in the differential post-transcriptional regulation of kif5b and its synthesis in axons. Thus, we suggest that the number of miRNA binding sites at the 3'UTR of the mRNA, rather than the miRNA seed match length, regulates the axonal transcriptome.

Enhancer Function of MicroRNA-3681 Derived from Long Terminal Repeats Represses the Activity of Variable Number Tandem Repeats in the 3' UTR of SHISA7.

microRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. miRNAs inhibit gene expression by binding to the 3′ untranslated region (UTR) of their target gene. miRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p share the binding sites in the 3’ UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p to inhibit the activity of VNTR. In conclusion, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p acts as a repressor of VNTR activity in the 3′ UTR of SHISA7.

Characteristics of miRNA interaction with mRNA of candidate genes of the non-small cell lung cancer

Lung cancer is one of the most common malignant diseases. It is expected to involve more than 200 candidate genes in the development of the non-small cell lung cancer (NSCLC). The present study aimed to study the interaction characteristics of the 137 NSCLC candidate genes and miRNAs. The data obtained show that a significant number of genes are targets of two or more mRNAs. The mRNA of some genes contain binding sites for several miRNAs with overlapping nucleotide sequences, which have been termed clusters. The properties of clusters of non-small cell lung cancer genes, containing binding sites of several miRNAs, were studied for the first time. The properties of clusters with a large number of miRNA binding sites in the 5'UTR are considered. The mRNA of CHKA, E2F1, HMGA2, PTEN, HTRA2, ING1, MTA3, SMARCA4, NFATC2 genes contain clusters of binding sites for the largest number of miRNAs. Several clusters are available in the CDS mRNA of CEBPA, NOTCH3, TRIO, LATS2, RB1 and ZBTB7A genes. 32 genes are targets of 49 miRNAs, binding sites of which are localized in the 3'UTR. The CDK6 and IRS1 genes have clusters with the largest number of miRNA binding sites. The AKT1, EMP and HMGA2 genes have clusters of 34 nt, 40 nt and 41 nt in length, respectively, of the binding sites of three miRNAs. Each mRNA of BCAR1, ITGA11, MYLK, PTK6, PXN, SOX2 and SSX2 genes has clusters of the binding sites of two miRNAs. It is assumed that the expression of the examined genes will depend more on the miRNA interacting with mRNA with a higher free binding energy. Key words: miRNA, mRNA, Non-Small Cell Lung Cancer, target genes.

Endogenous Cellular MicroRNAs Mediate Antiviral Defense against Influenza A Virus.

The reciprocal interaction between influenza virus and host microRNAs (miRNAs) has been implicated in the regulation of viral replication and host tropism. However, the global roles of the cellular miRNA repertoire and the mechanisms of miRNA-mediated antiviral defense await further elucidation. In this study, we systematically screened 297 cellular miRNAs from human and mouse epithelial cells and identified five inhibitory miRNAs that efficiently inhibited influenza virus replication in vitro and in vivo. Among these miRNAs, hsa-mir-127-3p, hsa-mir-486-5p, hsa-mir-593-5p, and mmu-mir-487b-5p were found to target at least one viral gene segment of both the human seasonal influenza H3N2 and the attenuated PR8 (H1N1) virus, whereas hsa-miR-1-3p inhibited viral replication by targeting the supportive host factor ATP6V1A. Moreover, the number of miRNA binding sites in viral RNA segments was positively associated with the activity of host miRNA-induced antiviral defense. Treatment with a combination of the five miRNAs through agomir delivery pronouncedly suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice. These data suggest that the highly expressed miRNAs in respiratory epithelial cells elicit effective antiviral defenses against influenza A viruses and will be useful for designing miRNA-based therapies against viral infection.

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID50. No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells.

Tissue elasticity regulated tumor gene expression: implication for diagnostic biomarkers of primitive neuroectodermal tumor.

BackgroundThe tumor microenvironment consists of both physical and chemical factors. Tissue elasticity is one physical factor contributing to the microenvironment of tumor cells. To test the importance of tissue elasticity in cell culture, primitive neuroectodermal tumor (PNET) stem cells were cultured on soft polyacrylamide (PAA) hydrogel plates that mimics the elasticity of brain tissue compared with PNET on standard polystyrene (PS) plates. We report the molecular profiles of PNET grown on either PAA or PS.Methodology/Principal FindingsA whole-genome microarray profile of transcriptional expression between the two culture conditions was performed as a way to probe effects of substrate on cell behavior in culture. The results showed more genes downregulated on PAA compared to PS. This led us to propose microRNA (miRNA) silencing as a potential mechanism for downregulation. Bioinformatic analysis predicted a greater number of miRNA binding sites from the 3' UTR of downregulated genes and identified as specific miRNA binding sites that were enriched when cells were grown on PAA—this supports the hypothesis that tissue elasticity plays a role in influencing miRNA expression. Thus, Dicer was examined to determine if miRNA processing was affected by tissue elasticity. Dicer genes were downregulated on PAA and had multiple predicted miRNA binding sites in its 3' UTR that matched the miRNA binding sites found enriched on PAA. Many differentially regulated genes were found to be present on PS but downregulated on PAA were mapped onto intron sequences. This suggests expression of alternative polyadenylation sites within intron regions that provide alternative 3' UTRs and alternative miRNA binding sites. This results in tissue specific transcriptional downregulation of mRNA in humans by miRNA. We propose a mechanism, driven by the physical characteristics of the microenvironment by which downregulation of genes occur. We found that tissue elasticity-mediated cytokines (TGFβ2 and TNFα) signaling affect expression of ECM proteins.ConclusionsOur results suggest that tissue elasticity plays important roles in miRNA expression, which, in turn, regulate tumor growth or tumorigenicity.

Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs.

Elucidation of microRNA (miRNA) function through a loss-of-function approach has proven difficult due to extensive genetic redundancy among most plant and animal miRNA families. Consequently, miRNA decoy technologies such as target MIMICs (MIMs) and short tandem target MIMICs (STTMs) in plants or molecular SPONGEs (SPs) in animals have been developed to generate loss-of-function phenotypes by perturbing endogenous miRNA activity. To test whether SPs can inhibit plant miRNA activity, synthetic SP transgenes containing multiple miRNA binding sites targeting different Arabidopsis miRNA families were generated. Additionally, their silencing efficacies were compared to the corresponding MIM and STTM transgenes via scoring the frequency and severity of phenotypic abnormalities elicited by each transgene. While SPs with wild-type miRNA binding sites have no apparent impact, SPs containing miRNA binding sites with two central mismatches (cmSPs) can generate strong loss-of-function phenotypes. However, their efficacy varied dramatically, from inducing strong loss-of-function phenotypes to failing to produce any phenotypic impact. Variability was also observed when MIMs and STTMs were compared to cmSPs. While cmSP165/166 and STTM165/166 showed a stronger efficacy than MIM165/166, MIM159 was stronger than cmSP159 and STTM159. Although increasing the number of miRNA binding sites or strengthening the free energy of the miRNA binding site interaction can improve decoy efficacy, clearly additional unknown overriding factors are at play. In conclusion, we demonstrate that no one approach guarantees the strongest miRNA inhibition, but rather distinct miRNA families respond differently to the various approaches, suggesting that multiple approaches may need to be taken to generate the desired loss-of-function outcome.

CNVs-microRNAs Interactions Demonstrate Unique Characteristics in the Human Genome. An Interspecies in silico Analysis

MicroRNAs (miRNAs) and copy number variations (CNVs) represent two classes of newly discovered genomic elements that were shown to contribute to genome plasticity and evolution. Recent studies demonstrated that miRNAs and CNVs must have co-evolved and interacted in an attempt to maintain the balance of the dosage sensitive genes and at the same time increase the diversity of dosage non-sensitive genes, contributing to species evolution. It has been previously demonstrated that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA binding sites are significantly higher than those of genes found in non-CNV regions. These findings raise the possibility that miRNAs may have been created under evolutionary pressure, as a mechanism for increasing the tolerance to genome plasticity. In the current study, we aimed in exploring the differences of miRNAs-CNV functional interactions between human and seven others species. By performing in silico whole genome analysis in eight different species (human, chimpanzee, macaque, mouse, rat, chicken, dog and cow), we demonstrate that miRNAs targeting genes located within CNV regions in humans have special functional characteristics that provide an insight into the differences between humans and other species.

Evidence for Positive Selection on a Number of MicroRNA Regulatory Interactions during Recent Human Evolution

MicroRNA (miRNA)–mediated gene regulation is of critical functional importance in animals and is thought to be largely constrained during evolution. However, little is known regarding evolutionary changes of the miRNA network and their role in human evolution. Here we show that a number of miRNA binding sites display high levels of population differentiation in humans and thus are likely targets of local adaptation. In a subset we demonstrate that allelic differences modulate miRNA regulation in mammalian cells, including an interaction between miR-155 and TYRP1, an important melanosomal enzyme associated with human pigmentary differences. We identify alternate alleles of TYRP1 that induce or disrupt miR-155 regulation and demonstrate that these alleles are selected with different modes among human populations, causing a strong negative correlation between the frequency of miR-155 regulation of TYRP1 in human populations and their latitude of residence. We propose that local adaptation of microRNA regulation acts as a rheostat to optimize TYRP1 expression in response to differential UV radiation. Our findings illustrate the evolutionary plasticity of the microRNA regulatory network in recent human evolution.

An Optimized Sponge for microRNA miR-9 Affects Spinal Motor Neuron Development in vivo

The specification of motor neuron (MN) subtypes and columnar organization in developing spinal cord is controlled by multiple transcription factors. FoxP1 drives specification of lateral motor neuron (LMN) subtypes, and we demonstrated in our previous work that FoxP1 expression levels are regulated by the microRNA miR-9. Here we show that ectopic FoxP1 expression in the chick spinal cord can rescue Lhx3 and Hb9 expression in MNs altered by miR-9 over-expression, demonstrating that FoxP1 is a critical functional interaction partner for miR-9 in LMN development. Moreover, we have optimized a technique called a miRNA sponge in vitro, to permit easy discovery of the role of individual miRNA in vivo using a loss-of-function approach. We here show that narrow spacing between binding sites, inclusion of a coding gene, and optimizing the number of miRNA binding sites can significantly increase the blocking ability of a sponge. We go on to show that a miR-9 sponge reduces detectable miR-9 in the ventral horn, preventing miR-9 silencing of FoxP1 in vivo, and in turn modifies MN subtypes in the spinal cord. Our designs for optimized sponges provide a knockdown tool that is ready to be used to study the function of miRNA in vivo, and in particular for generating transgenic animal models.

Increased Number of MicroRNA Target Sites in Genes Encoded in CNV Regions. Evidence for an Evolutionary Genomic Interaction

MicroRNAs (miRNAs) and copy number variations (CNVs) are two newly discovered genetic elements that have revolutionized the field of molecular biology and genetics. By performing in silico whole genome analysis, we demonstrate that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA-binding sites are significantly higher than those of genes found in non-CNV regions. This suggests that miRNAs may have acted as equilibrators of gene expression during evolution in an attempt to regulate aberrant gene expression and to increase the tolerance to genome plasticity.