Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

Jin, Y.; Chen, Zujian; Liu, Xiqiang; Zhou, Xiaofeng

doi:10.1007/978-1-62703-083-0_10

Cited by 139 publications

(105 citation statements)

References 17 publications

Supporting

Mentioning

102

Contrasting

Order By: Relevance

“…Among these, Eip74EF has previously been shown to be an miR-34 target that functions in the regulation of neurodegeneration7, and all three genes are annotated as having transcription factor and DNA binding activities. We confirmed that the 3′ UTRs of Eip74EF, Hr4, and yem contain functional miR-34 target sites using a previously described luciferase reporter gene assay26 (Supplemental Figure 4b). However, RNA interference (RNAi) experiments in which the expression of each transcript was knocked down separately, defective γ lobe pruning in MB neurons was not observed (Supplemental Figure 4c–f). These results indicate that neither Eip74EF, Hr4, nor yem alone is crucial for γ axon pruning in MB γ neurons.…”

Section: Resultssupporting

confidence: 82%

Drosophila microRNA-34 Impairs Axon Pruning of Mushroom Body γ Neurons by Downregulating the Expression of Ecdysone Receptor

Lai

Chu

Wei

et al. 2016

Sci Rep

View full text Add to dashboard Cite

MicroRNA-34 (miR-34) is crucial for preventing chronic large-scale neurite degeneration in the aged brain of Drosophila melanogaster. Here we investigated the role of miR-34 in two other types of large-scale axon degeneration in Drosophila: axotomy-induced axon degeneration in olfactory sensory neurons (OSNs) and developmentally related axon pruning in mushroom body (MB) neurons. Ectopically overexpressed miR-34 did not inhibit axon degeneration in OSNs following axotomy, whereas ectopically overexpressed miR-34 in differentiated MB neurons impaired γ axon pruning. Intriguingly, the miR-34-induced γ axon pruning defect resulted from downregulating the expression of ecdysone receptor B1 (EcR-B1) in differentiated MB γ neurons. Notably, the separate overexpression of EcR-B1 or a transforming growth factor- β receptor Baboon, whose activation can upregulate the EcR-B1 expression, in MB neurons rescued the miR-34-induced γ axon pruning phenotype. Future investigations of miR-34 targets that regulate the expression of EcR-B1 in MB γ neurons are warranted to elucidate pathways that regulate axon pruning, and to provide insight into mechanisms that control large-scale axon degeneration in the nervous system.

show abstract

Section: Resultssupporting

confidence: 82%

Drosophila microRNA-34 Impairs Axon Pruning of Mushroom Body γ Neurons by Downregulating the Expression of Ecdysone Receptor

Lai

Chu

Wei

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The luciferase reporter gene construct containing 2 adjacent miR-99 family targeting sites from the 3′-UTR of AKT1 mRNA was created by cloning an 81-bp fragment into the XbaI site on the 3′-UTR of the luciferase gene in the pGL3-Control firefly luciferase reporter vector (Promega) as described previously [26]. The corresponding mutant constructs were created by replacing the seed regions (positions 2–8) of the miR-99 family binding sites with 5′-TTTTTTT-3′.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing

et al. 2013

Self Cite

View full text Add to dashboard Cite

Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3′-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling.

show abstract

“…For the purpose of building a positive dataset to train our classifier, we reviewed miRNA target identification studies related to CRC with an emphasis on experimental data from Luciferase reporter assays which is one of the most reliable methods for target identification 29 . More specifically, just those miRNA-mRNA interactions for which the exact binding sites were characterized by site-directed mutagenesis were considered in the positive dataset.…”

Section: Training Datamentioning

confidence: 99%

Naïve Bayes classifier predicts functional microRNA target interactions in colorectal cancer

et al. 2015

View full text Add to dashboard Cite

Alterations in the expression of miRNAs have been extensively characterized in several cancers, including human colorectal cancer (CRC). Recent publications provide evidence for tissue-specific miRNA target recognition. Several computational methods have been developed to predict miRNA targets; however, all of these methods assume a general pattern underlying these interactions and therefore tolerate reduced prediction accuracy and a significant number of false predictions. The motivation underlying the presented work was to unravel the relationship between miRNAs and their target mRNAs in CRC. We developed a novel computational algorithm for miRNA-target prediction in CRC using a Naïve Bayes classifier. The algorithm, which is referred to as CRCmiRTar, was trained with data from validated miRNA target interactions in CRC and other cancer entities. Furthermore, we identified a set of position-based, sequence, structural, and thermodynamic features that identify CRC-specific miRNA target interactions. Evaluation of the algorithm showed a significant improvement of performance with respect to AUC, and sensitivity, compared to other widely used algorithms based on machine learning. Based on miRNA and gene expression profiles in CRC tissues with similar clinical and pathological features, our classifier predicted 204 functional interactions, which involve 11 miRNAs and 41 mRNAs in this cancer entity. While the approach is here validated for CRC, the implementation of disease-specific miRNA target prediction algorithms can be easily adopted for other applications too. The identification of disease-specific miRNA target interactions may also facilitate the identification of potential drug targets.

show abstract

Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

Cited by 139 publications

References 17 publications

Drosophila microRNA-34 Impairs Axon Pruning of Mushroom Body γ Neurons by Downregulating the Expression of Ecdysone Receptor

Drosophila microRNA-34 Impairs Axon Pruning of Mushroom Body γ Neurons by Downregulating the Expression of Ecdysone Receptor

MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing

Naïve Bayes classifier predicts functional microRNA target interactions in colorectal cancer

Contact Info

Product

Resources

About