Reexamining assumptions about miRNA-guided gene silencing.

MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of several thousand papers each year connecting miRNAs to many different genes and human diseases. By contrast, relatively few papers appear that investigate the molecular mechanism used by miRNAs. There is a disconnect between rigorous understanding of mechanism and the extraordinary diversity of reported roles for miRNAs. Consequences of this disconnect include confusion about the assumptions underlying the basic science of human miRNAs and slow development of therapeutics that target miRNAs. Here, we present an overview of investigations into miRNAs and their impact on gene expression. Progress in our understanding of miRNAs would be aided by a greater focus on the mechanism of miRNAs and a higher burden of evidence on researchers who seek to link expression of a particular miRNA to a biological phenotype.

Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons.

ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3' mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.

Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

Exosomes-Coated miR-34a Displays Potent Antitumor Activity in Pancreatic Cancer Both in vitro and in vivo.

PURPOSE: MiR-34a, which acts as an important tumor suppressor gene, plays an important role in pancreatic cancer. However, the therapeutic application of miR-34a is limited by the lack of an effective delivery system. In the present study, we synthesize exosomes-coated miR-34a (exomiR-34a), and the anticancer effect of exomiR-34a was evaluated in pancreatic cancer. MATERIALS AND METHODS: An ultrasound approach was used to synthesize exomiR-34a, and its transfection efficiency was examined by confocal microscopy and flow cytometry. The level of miR-34a and its targeted gene Bcl-2 was detected by real-time quantitative PCR (qRT-PCR). MTT analysis was performed to determine the effect of exomiR-34a on the growth of pancreatic cancer cells. Annexin-V/PI double staining and Western blot analysis were carried out to determine the apoptosis of the pancreatic cancer cells. The xenograft nude mice model bearing human pancreatic cancer Panc28 cells was used to determine the antitumor effect of exomiR-34a in vivo. RESULTS: The exomiR-34a could cross the cell membrane efficiently, and downregulated the expression of the targeted gene Bcl-2. Treatment with exomiR-34a inhibited the growth of the pancreatic cancer cells significantly and the nanoparticles also induced apoptosis in cancer cells via affecting the expression of apoptotic-related genes. In vivo study using xenograft nude mice bearing Panc28 cancer cells revealed that exomiR-34a suppressed the growth of tumors significantly. CONCLUSION: ExomiR-34a can inhibit the growth of pancreatic cancer both in vitro and in vivo. Targeting miR-34a is a promising strategy for the treatment of pancreatic cancer. ExomiR-34a has the potential to be developed as a novel anticancer agent for the treatment of human pancreatic malignancy.

Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation.

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.

Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

Challenges and standardization of microRNA profiling in serum and cerebrospinal fluid in dogs suffering from non-infectious inflammatory CNS disease.

Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demonstration of inflammatory changes in the cerebrospinal fluid (CSF). However, less-invasive methods and peripheral biomarkers are desired. Changes in circulating microRNA (miRNA), which are short non-coding regulatory RNAs, may serve as biomarkers of disease. The aim of this pilot study was to investigate selected miRNAs in serum and CSF, hypothesizing that the levels of specific miRNAs in serum correlate with their presence in CSF, and that changes in serum miRNAs levels may reflect CNS disease. We profiled serum and CSF samples using quantitative real-time PCR (qPCR) searching for selected and previously profiled miRNAs in serum (let-7a, let-7c, miR-15b, miR-16, miR-21, miR-23a, miR-24, miR-26a, miR-146a, miR-155, miR-181c and miR-221-3p) and in CSF (let-7c, miR-16, miR-21, miR-24, miR-146a, miR-155, miR-181c and miR-221-3p) from 13 dogs with NII CNS disease and six control dogs. We demonstrated the presence of several miRNAs in CSF (let-7c and miR-21 dominating) and serum (miR-23a and miR-21 dominating). However, we generally failed to reproduce consistent results in CSF samples due to several reasons: unacceptable PCR efficiency, a wide variation between cDNA replicates and/or no-amplification in qPCR suggesting very low levels of the investigated miRNAs in canine CSF. Serum samples performed better, and 10 miRNAs qPCR assays were qualified for analysis. We were nevertheless unable to detect a difference in the expression of miRNA levels between cases and controls. Moreover, we could not confirm the results of recent miRNA investigations of canine CNS diseases. We believe that these disagreements highlight the significant effect of methodological/analytical variation, rather than the incapacity of circulating miRNAs as biomarkers of CNS disease. A secondary aim was therefore to communicate methodological challenges in our study and to suggest recommendations for circulating miRNA profiling, including pre-, post- and analytical methods based on our experience, in order to reach reproducible and comparable results in veterinary miRNA research.

Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications.

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.

Design, Synthesis, and Functional Analysis of Highly Specific Artificial Small RNAs with Antiviral Activity in Plants.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) that have been broadly used to confer antiviral resistance in plants. However, methods for designing, synthesizing and functionally analyzing antiviral artificial sRNAs have not been optimized for time and cost-effectiveness and high-throughput applicability since recently. Here we present a systematic methodology for the simple and fast-forward design, generation, and functional analysis of large numbers of artificial sRNA constructs engineered to induce high levels of antiviral resistance in plants. Artificial sRNA constructs are transiently expressed in Nicotiana benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each artificial sRNA construct is assessed by monitoring viral symptom appearance, and through molecular analysis of virus accumulation in plant tissues. This approach is aimed to easily identify artificial sRNAs with high antiviral activity that could be expressed in transgenic plants for highly durable antiviral resistance.

Synthetic microRNA-205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection.

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA-205 (miR-205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR-205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR-205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR-205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR-205BP/S3 is a potentially applicable treatment for canine melanoma.

Impairment of K-Ras signaling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miR-143.

Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC50 : 1.32 nmol L-1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines.

Regulation of T cell proliferation with drug-responsive microRNA switches.

As molecular and cellular therapies advance in the clinic, the role of genetic regulation is becoming increasingly important for controlling therapeutic potency and safety. The emerging field of mammalian synthetic biology provides promising tools for the construction of regulatory platforms that can intervene with endogenous pathways and control cell behavior. Recent work has highlighted the development of synthetic biological systems that integrate sensing of molecular signals to regulated therapeutic function in various disease settings. However, the toxicity and limited dosing of currently available molecular inducers have largely inhibited translation to clinical settings. In this work, we developed synthetic microRNA-based genetic systems that are controlled by the pharmaceutical drug leucovorin, which is readily available and safe for prolonged administration in clinical settings. We designed microRNA switches to target endogenous cytokine receptor subunits (IL-2Rß and γc) that mediate various signaling pathways in T cells. We demonstrate the function of these control systems by effectively regulating T cell proliferation with the drug input. Each control system produced unique functional responses, and combinatorial targeting of multiple receptor subunits exhibited greater repression of cell growth. This work highlights the potential use of drug-responsive genetic control systems to improve the management and safety of cellular therapeutics.

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

OBJECTIVE: MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. RESULTS: The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

Small Molecule Release and Activation through DNA Computing.

DNA-based logic gates can be assembled into computational devices that generate a specific output signal in response to oligonucleotide input patterns. The ability to interface with biological and chemical environments makes DNA computation a promising technology for monitoring cellular systems. However, DNA logic gate circuits typically provide a single-stranded oligonucleotide output, limiting the ability to effect biology. Here, we introduce a novel DNA logic gate design capable of yielding a small molecule output signal. Employing a Staudinger reduction as a trigger for the release and activation of a small molecule fluorophore, we constructed AND and OR logic gates that respond to synthetic microRNA (miRNA) inputs. Connecting the gates in series led to more complex DNA circuits that provided a small molecule output in response to a specific pattern of three different miRNAs. Moreover, our gate design can be readily multiplexed as demonstrated by simultaneous small molecule activation from two independent DNA circuits.

Gold-loaded nanoporous superparamagnetic nanocubes for catalytic signal amplification in detecting miRNA.

This paper reports the development of a nonenzymatic, amplification-free, and sensitive platform for the detection of microRNA based on a new class of electrocatalytically active superparamagnetic gold-loaded nanoporous iron oxide nanocubes (Au@NPFe2O3NC). The assay showed an excellent detection sensitivity down to 100 fM and specificity towards the analysis of miR-21 in cell lines and tissue samples derived from patients with oesophageal squamous-cell carcinoma (ESCC).

Vector-delivered artificial miRNA effectively inhibited replication of Chikungunya virus.

Chikungunya virus (CHIKV) has emerged as one of the most significant arboviral threats in many parts of the world. In spite of large scale morbidity, and long lasting polyarthralgia, no licensed vaccine or antivirals are available for the clinical management of CHIKV infection. In this study, a novel RNA interference based strategy has been adopted for effective inhibition of CHIKV. Four artificial microRNAs (amiRNAs) were designed to target different regions of CHIKV genome. These amiRNAs significantly inhibited CHIKV replication in Vero cells at both RNA and protein levels as assessed by qRT-PCR, immunoblotting and immunofluorescence techniques. Further inhibition of the infectious CHIKV up to 99.8% was demonstrated by plaque reduction assay. Concatemerization of amiRNA resulted in higher inhibition of CHIKV than individual amiRNAs. In addition, we studied the effect of combination of RNAi based therapy with other classical antivirals like chloroquine, ribavirin and mycophenolic acid, that helped in understanding the rational selection of RNAi based combination therapy. These findings provide a promising avenue for the development of novel amiRNA or combination based therapeutics against emerging CHIKV.

High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.

Argonaute 2-dependent Regulation of Gene Expression by Single-stranded miRNA Mimics.

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression. Aberrant expression of miRNAs can affect development of cancer and other diseases. Synthetic miRNA mimics can modulate gene expression and offer an approach to therapy. Inside cells, mature miRNAs are produced as double-stranded RNAs and miRNA mimics typically retain both strands. This need for two strands has the potential to complicate drug development. Recently, synthetic chemically modified single-stranded silencing RNAs (ss-siRNA) have been shown to function through the RNAi pathway to induce gene silencing in cell culture and animals. Here, we test the hypothesis that single-stranded miRNA (ss-miRNA) can also mimic the function of miRNAs. We show that ss-miRNAs can act as miRNA mimics to silence the expression of target genes. Gene silencing requires expression of argonaute 2 (AGO2) protein and involves recruitment of AGO2 to the target transcripts. Chemically modified ss-miRNAs function effectively inside cells through endogenous RNAi pathways and broaden the options for miRNA-based oligonucleotide drug development.

AmiRNA Designer - new method of artificial miRNA design.

MicroRNAs (miRNAs) are small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner. MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. Involvement of miRNAs in a wide range of biological processes makes them excellent candidates for studying gene function or for therapeutic applications. For this purpose, different RNA-based gene silencing techniques have been developed. Artificially transformed miRNAs (amiRNAs) targeting one or several genes of interest represent one of such techniques being a potential tool in functional genomics. Here, we present a new approach to amiRNA*design, implemented as AmiRNA Designer software. Our method is based on the thermodynamic analysis of the native miRNA/miRNA* and miRNA/target duplexes. In contrast to the available automated tools, our program allows the user to perform analysis of natural miRNAs for the organism of interest and to create customized constraints for the design stage. It also provides filtering of the amiRNA candidates for the potential off-targets. AmiRNA Designer is freely available at http://www.cs.put.poznan.pl/arybarczyk/AmiRNA/.

[MicroRNA regulates animal adipocyte differentiation].

Adipose tissues play a critical role in the regulation of energy metabolism and homeostasis, and is also an important endocrine organ. Adipocyte differentiation is a complicated physiological process during which mesenchymal stem cells differentiate into adipocytes. This process is synergistically regulated by a large number of transcription factors, hormones and signaling pathway molecules. As a class of endogenous non-coding RNA (ncRNA), microRNAs (miRNAs) regulate gene expression mainly through post-transcriptional translational repression. In recent years, numerous studies have demonstrated that miRNA could have an impact on adipocyte differentiation and adipogenesis by modulating the expression levels of several adipogenic transcription factors and key signaling molecules. In this review, we summarize the mechanism of miRNA in regulating the differentiation of white/brown/beige adipocytes and the relevant signaling pathways and key factors, in the hope of providing theoretical guidance and new thoughts for treating obesity and other metabolic diseases.