MicroRNAs are small, non-coding molecules which function by silencing or degrading messenger RNA (mRNA). This is achieved via complementary base-pairing between an miRNA-protein complex and a specific seed sequence on an mRNA strand. mRNA is blocked from accessing the transcriptional machinery of the cell, or degraded completely. It is through this mechanism that miRNAs have been found to play important roles in development and the etiology of many common diseases, and it is why many researchers are increasingly looking toward this relatively new class of biological molecules for further insight into their chosen area of study.
Since their discovery almost 20 years ago, microNRAs (miRNAs) have increasingly come to the forefront of biological and medical research as important players in development and human disease. Aberrant miRNA expression has been connected in recent years to cancers, cardiac disease, endocrine disease, and bone disease. miRNA and miRNA inhibitors may also have biomedical applications in the developments of tissue engineering methods and wound healing, as they play important regulatory roles in cell proliferation and differentiation.
The team at NaturemiRI believes that researchers are only now beginning to scratch the surface of miRNA inhibitor applications, largely due to obstacles encountered by traditional miRNA inhibiting systems. NaturemiRI’s technology will allow researchers to harness the power of miRNA expression manipulation with the potential for transformative effects in several areas of miRNA-based research and therapeutic applications.
The Plasmid-based microRNA Inhibitor System (PMIS) platform uses a patented RNA-based hairpin molecule, the PMIS Inhibitor Complex, which carries an anti-sense microRNA seed sequence that effectively knocks down endogenous microRNA and mitigates their effects on messenger RNA in the cell. By using a native RNA-based molecule for miRNA knockdown, the PMIS platform avoids many of the traditional pitfalls associated with microRNA inhibition using synthetic oligonucleotides: the PMIS approach is more specific and stable, effective at a lower dose, more cost-effective, and perhaps most importantly, is non-toxic to living tissues, in contrast with many traditional miRNA inhibition strategies.
Importantly, the non-toxic nature of the PMIS molecule makes it promising platform for the delivery of miRNA inhibiting effects that could have potential as a treatment of human diseases tied to aberrant miRNA expression, something that has proven difficult for traditional oligonucleotide approaches to miRNA inhibition.
Mature miRNAs associate with a host of proteins, together called the RNA-Induced Silencing Complex (RISC). These proteins then facilitate the interaction of mature miRNAs with mRNAs, leading to translational repression. In the PMIS system, the PMIS Inhibitor Complex transiently binds to this complex containing the mature miRNA of interest. Dicer is released from the complex because it is unable to cleave the PMIS Inhibitor Complex, but the Complex retains its association with Argonaute 2, TRBP, and the mature miRNA. Through this mechanism, the PMIS system allows for the translation of a significantly increased number of mRNAs that would typically be repressed via interactions with the RISC complex factors and associated mature endogenous miRNAs.
Efficiency of the PMIS. a) PMIS-miR-inhibitors reduce specific miR expression. Real time PCR of mature endogenous miR-17 and miR-200c from 293 cells transfected with the indicated PMIS inhibitor. b) Northern blot of mature endogenous miRs and the PMIS construct from 293 cells transfected with PMIS Vector or PMIS-miR-17-18. U6 RNA is shown as a loading control. c) Western blot of Bim, a known target of miR-17 from cells transfected with the PMIS-vector, PMIS-miR-17 or PMIS-miR-200a. In addition, a Western blot of PTEN, identified as a target of miR-17. β-tubulin and GAPDH are shown as loading controls.
Genome-wide analyses of PMIS-miR-17 function. a) Experiment design for the whole genome wide analysis of mRNA change after transduction with PMIS-miR-17. b, c) Changes in abundance of mRNAs in PMIS-miR-17 expressing 293 cells were monitored by microarrays. TargetScan was used to predict miR-17 targets. We further separate miR-17 targets into two groups, one with context+ score lower than -0.30 and the other with context+ score higher than -0.30. Distributions of changes (0.1 unit bins) for mRNA UTRs containing no site in black line, site with score higher than -0.30 in red line and site with score less than -0.30 in blue line. Up-regulation of mRNAs with context+ score less than -0.30 was significantly more than that from mRNAs with no site (p<10-13, one-sided K-S test). Comparisons between mRNAs with context+ score less than -0.30 versus context+ score higher than -0.30, context+ score higher than -0.30 versus no site were also signiﬁcant (p<10-8 , p<10-3, respectively). d) 293 cells were transduced with PMIS-miR-17, total RNA harvested and randomly selected miR expression was analyzed by real time PCR using Taqman probes (N=3).
Fig. 1. PMIS-miR-17-18 and PMIS-miR-17-92 mice are postnatal lethal with distinct clefting phenotypes. A) The location and organization of the miR clusters are shown in the left panel. B) The seed sequence similarities (color coded) and differences between the miRs. PMIS-miR-17–18 were derived from miR-17-5p and miR-18a-5p, respectively. PMIS-miR-17-92 were derived from all four clusters (Cao, et al., 2016). C-E) Whole mount view of the ventral maxilla in P0 mice shows the different stages of palatogenesis arrest in the transgenic mice. Dashed lines outline the cleft region, if present. C) Wild type (WT) mouse shows complete fusion of the palatal shelves at both primary and secondary palate. D) In the PMIS-miR-17-18 mice the palate shelves fail to elevate, leaving large clefts in the palate. E) In the PMIS-miR-17-92 mice the palate shelves elevate but fail to extend to the midline and fuse. Abbreviations: PP, primary palate; SP, secondary palate; NS, nasal septum.
Fig. 2. Histological analyses shows distinct patterns of clefting at E18.5 in PMIS-miR-17-18 and PMIS-miR-17-92 embryos. H&E staining of E18.5 embryos shows differences in cleft palate defects observed in PMIS-miR-17-18 and PMIS-miR-17-92 embryos. A-C) Anterior, middle and posterior coronal sections of wild type embryos show complete fusion of the palate. D-F) Coronal sections of the PMIS-miR-17-92 embryos shows arrest in palatogenesis occurs before elevation of the palatal shelves. (G-I) Coronal sections of PMIS-miR-17-92 embryos shows arrest in palatogenesis occurs after elevation of the palate shelves but before extension and fusion at the midline. Scale bar= 100um. Abbreviations: NS, nasal septum; PS, palatal shelves; Tg, tongue.
Fig. 3. miRs within the miR-17-92 cluster differentially regulate craniofacial development. WT, PMIS-miR-17-18, PMIS-miR-19-92 and PMIS-miR-17-92, 3 week-old heads were analyzed by μCT. In-depth measurements were obtained for different aspects of craniofacial growth. Quantitative measurements of total cranial length and breadth of the PMIS transgenic mice are shown compared to WT, N=3.
PMIS-miR-17 attenuates endogenous miR-17 activity much more effectively on the PTEN 3’UTR in a luciferase construct relative to other miR-17 inhibitors.
Inhibition of the miR-17-92 Cluster Separates Stages of Palatogenesis.
The role that noncoding regions of the genome play in the etiology of cleft palate is not well studied. A novel method of microRNA (miR) inhibition that allows for specific miR knockdown in vivo has been developed by our laboratory. To further understand the role of miRs in palatogenesis, we used a new mouse model to inhibit specific miRs within the miR-17-92 cluster. Transgenic mice expressing inhibitory complexes for miR-17 and miR-18 manifested a clefting phenotype that was distinct from that observed in mice carrying inhibitory complexes for miR-17, miR-18, miR-19, and miR-92. An in silico candidate gene analysis and bioinformatics review led us to identify TGFBR2 as a likely target of miR-17 and miR-19 family members. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TGFBR1 and TGFBR2 expression levels were elevated in the palates of these miR transgenic embryos at embryonic day 15.5. RT-PCR data also showed that the expression of mature miRs from the miR-17-92 cluster was significantly decreased in the transgenic embryos. Decreased expression of TGFB pathway signaling ligands was also observed. Experiments in cells showed that inhibition of miR-17 and miR-18 was sufficient to induce increases in expression of TGFB receptors, while a concomitant decrease in TGFB signaling ligands was not observed. RT-PCR of mature miR-17-92 in cells demonstrated the selectivity and specificity of inhibitory complexes. While this study builds on previous studies that have implicated miR-17-92 in the regulation of important molecular components of the TGFB signaling pathway, it is likely that interactions remain to be elucidated between miR-17-92 and as-of-yet unidentified molecules important for the control of palatogenesis. The differential regulation of palatogenesis by members of the miR-17-92 cluster indicates that several gene combinations regulate palate elevation and extension during development.
J. Dent. Res. 2017, 96:1257-1264; Special issue on Craniofacial Development