Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA

ABSTRACT

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2′-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.

Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3′-UTR: 3′-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.     

miR-185 Plays an Anti-Hypertrophic Role in the Heart via Multiple Targets in the Calcium-Signaling Pathways

MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.     

Interleukin-17 (IL-17)-induced MicroRNA 873 (miR-873) Contributes to the Pathogenesis of Experimental Autoimmune Encephalomyelitis by Targeting A20 Ubiquitin-editing Enzyme

Interleukin 17 (IL-17), produced mainly by T helper 17 (Th17) cells, is increasingly recognized as a key regulator in various autoimmune diseases, including human multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although several microRNAs (miRNAs) with aberrant expression have been shown to contribute to the pathogenesis of MS and EAE, the mechanisms underlying the regulation of abnormal miRNA expression in astrocytes upon IL-17 stimulation remain unclear. In the present study, we detected the changes of miRNA expression profiles both in the brain tissue of EAE mice and in cultured mouse primary astrocytes stimulated with IL-17 and identified miR-873 as one of the co-up-regulated miRNAs in vivo and in vitro. The overexpression of miR-873, demonstrated by targeting A20 (TNFα-induced protein 3, TNFAIP3), remarkably reduced the A20 level and promoted NF-κB activation in vivo and in vitro as well as increasing the production of inflammatory cytokines and chemokines (i.e. IL-6, TNF-α, MIP-2, and MCP-1/5). More importantly, silencing the endogenous miR-873 or A20 gene with lentiviral vector of miR-873 sponge (LV-miR-873 sponge) or short hairpin RNA (shRNA) of A20 (LV-A20 shRNA) in vivo significantly lessened or aggravated inflammation and demyelination in the central nervous system (CNS) of EAE mice, respectively. Taken together, these findings indicate that miR-873 induced by IL-17 stimulation promotes the production of inflammatory cytokines and aggravates the pathological process of EAE mice through the A20/NF-κB pathway, which provides a new insight into the mechanism of inflammatory damage in MS.     

Potent degradation of neuronal miRNAs induced by highly complementary targets

MicroRNAs (miRNAs) regulate target mRNAs by silencing them. Reciprocally, however, target mRNAs can also modulate miRNA stability. Here, we uncover a remarkable efficacy of target RNA-directed miRNA degradation (TDMD) in rodent primary neurons. Coincident with degradation, and while still bound to Argonaute, targeted miRNAs are 3′ terminally tailed and trimmed. Absolute quantification of both miRNAs and their decay-inducing targets suggests that neuronal TDMD is multiple turnover and does not involve co-degradation of the target but rather competes with miRNA-mediated decay of the target. Moreover, mRNA silencing, but not TDMD, relies on cooperativity among multiple target sites to reach high efficacy. This knowledge can be harnessed for effective depletion of abundant miRNAs. Our findings bring insight into a potent miRNA degradation pathway in primary neurons, whose TDMD activity greatly surpasses that of non-neuronal cells and established cell lines. Thus, TDMD may be particularly relevant for miRNA regulation in the nervous system.     

Non-Specific Blocking of miR-17-5p Guide Strand in Triple Negative Breast Cancer Cells by Amplifying Passenger Strand Activity

Conventional wisdom holds that only one of the two strands in a micro ribonucleic acid (miRNA) precursor duplex is selected as the active miRNA guide strand. The complementary miRNA passenger strand, however, is thought to be inactive. High levels of the oncogenic miRNA (oncomiR) guide strand called miR-17-5p is overexpressed in triple negative breast cancer (TNBC) and can inhibit ribosomal translation of tumor suppressor gene mRNAs, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, antisense knockdown of oncomiR miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human TNBC cells instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3’UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knock down miR-17-5p reduced PDCD4 and PTEN protein expression instead of raising them. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand.     

Reduction of Type IV Collagen by Upregulated miR-29 in Normal Elderly Mouse and klotho-Deficient,Senescence-Model Mouse

MicroRNA (miRNA), a small non-coding RNA that functions as a mediator in gene silencing, plays important roles in gene regulation in various vital functions and activities. Here we show that the miR-29 members are upregulated in klotho-deficient [klotho(−/−)] mice, a senescence-model animal, and also in normal elderly ICR mice relative to wild-type littermates and young ICR mice. In addition, levels of type IV collagen, a major component of basement membranes and a putative target of miR-29, were lower in klotho(−/−) and elderly ICR mice than in wild-type littermates and young ICR mice. RNA degradation mediated by miR-29 may participate in the suppression of type IV collagen, both in vivo and in vitro. Taken together, our current findings suggest that the miR-29 upregulated in aging may be involved in the downregulation of type IV collagen, leading to a possible weakening of the basal membrane in senescent tissues, and miR-29 may be a useful molecular marker of senescence.     

Identification and Validation of miRNAs as Endogenous Controls for RQ-PCR in Blood Specimens for Breast Cancer Studies

Introduction

A prerequisite to accurate interpretation of RQ-PCR data is robust data normalization. A commonly used method is to compare the cycle threshold (C_T) of target miRNAs with those of a stably expressed endogenous (EC) miRNA(s) from the same sample. Despite the large number of studies reporting miRNA expression patterns, comparatively few appropriate ECs have been reported thus far. The purpose of this study was to identify stably expressed miRNAs with which to normalize RQ-PCR data derived from human blood specimens.

Methods

MiRNA profiling of approximately 380 miRNAs was performed on RNA derived from blood specimens from 10 women with breast cancer and 10 matched controls. Analysis of mean expression values across the dataset (GME) identified stably expressed candidates. Additional candidates were selected from the literature and analyzed by the geNorm algorithm. Further validation of three candidate ECs by RQ-PCR was performed in a larger cohort (n = 40 cancer, n = 20 control) was performed, including analysis by geNorm and NormFinder algorithms.

Results

Microarray screening identified 10 candidate ECs with expression patterns closest to the global mean. Geometric averaging of candidate ECs from the literature using geNorm identified miR-425 as the most stably expressed miRNA. MiR-425 and miR-16 were the best combination, achieving the lowest V-value of 0.185. Further validation by RQ-PCR confirmed that miR-16 and miR-425 were the most stably expressed ECs overall. Their combined use to normalize expression data enabled the detection of altered target miRNA expression that reliably differentiated between cancers and controls in human blood specimens.

Conclusion

This study identified that the combined use of 2 miRNAs, (miR-16 and miR-425) to normalize RQ-PCR data generated more reliable results than using either miRNA alone, or use of U6. Further investigation into suitable ECs for use in miRNA RQ-PCR studies is warranted.