Publications by Year: 2018

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

Thousands of unique non-coding RNA (ncRNA) sequences exist within cells. Work from the past decade has altered our perception of ncRNAs from 'junk' transcriptional products to functional regulatory molecules that mediate cellular processes including chromatin remodelling, transcription, post-transcriptional modifications and signal transduction. The networks in which ncRNAs engage can influence numerous molecular targets to drive specific cell biological responses and fates. Consequently, ncRNAs act as key regulators of physiological programmes in developmental and disease contexts. Particularly relevant in cancer, ncRNAs have been identified as oncogenic drivers and tumour suppressors in every major cancer type. Thus, a deeper understanding of the complex networks of interactions that ncRNAs coordinate would provide a unique opportunity to design better therapeutic interventions.

Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models.Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors.Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars.Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734-47. ©2018 AACR.

MicroRNAs (miRNAs) have recently emerged as promising biomarkers for the profiling of diseases. Translation of miRNA biomarkers to clinical practice, however, remains a challenge due to the lack of analysis platforms for sensitive, quantitative, and multiplex miRNA assays that have simple and robust workflows suitable for translation. The platform we present here utilizes functionalized hydrogel posts contained within isolated nanoliter well reactors for quantitative and multiplex assays directly from unprocessed cell samples without the need of prior nucleic acid extraction. Simultaneous reactor isolation and delivery of miRNA extraction reagents is achieved by sealing an array of wells containing the functionalized hydrogel posts and cells against another array of wells containing lysis and extraction reagents. The nanoliter well array platform features >100× better sensitivity compared to previous technology utilizing hydrogel particles without relying on signal amplification and enables >100 parallel assays in a single device. These advances provided by this platform lay the groundwork for translatable and robust analysis technologies for miRNA expression profiling in samples with small populations of cells and in precious, material-limited samples.

The past decade has witnessed enormous progress, and has seen the noncoding RNAs (ncRNAs) turn from the so-called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer.

MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m6A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.

MicroRNAs (miRNA) are a class of small non-coding RNA that play a major role in various cellular processes by negatively regulating gene expression. In the past decade, miRNA dysregulation has been reported to be closely linked to inflammatory diseases. The immune response modulates cancer initiation and progression; miRNAs including let-7 family members have been shown to act as key regulators of the immune responses in various diseases and cancers. Notably, the let-7 miRNA has been reported to be closely associated with immunity, specifically with Toll-like receptors that mediate cytokine expression during pathogen infection and with the regulation of various other immune effectors. Areas covered: In this review, the authors describe the discovery of let-7 as the starting point of the RNA revolution and highlight let-7 as an efficient tool for cancer and immune therapy. Expert opinion: let-7 miRNA has emerged as a key player in cancer therapy and immune responses and it has potential role as a new immunotherapeutic target. However, while there are challenges regarding miRNA delivery, the exciting emergence of personalized medicine for cancer and immunotherapy could be beneficial for the development of let-7 therapeutics.