MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo. Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.
Significance Genome-wide identification of changes in the expression of large intergenic noncoding RNAs (lincRNAs) in a classical model of innate immune cell activation revealed a panel of 159 lincRNAs that were highly modulated in stimulated THP1 macrophages. One of the lincRNAs, named TNFα and heterogenous nuclear ribonucleoprotein L (hnRNPL) related immunoregulatory LincRNA (THRIL), was essential for induction of TNFα, functions through a ribonucleoprotein (RNP) complex with hnRNPL, and plays an important role as regulator of physiological and pathological inflammatory immune responses.
SUMMARY Here, we generated the first genome-scale shRNA library targeting lincRNAs in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 novel lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA), was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA–RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence and central nervous system-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington’s patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.
Small RNA-mediated regulation of iPS cell generationThe generation of induced pluripotent stem cells is limited by the low reprogramming efficiency of somatic cells. Here, three clusters of miRNAs are shown to enhance reprogramming efficiency by targeting the TGF-β and p53 pathways, which inhibit the process.
Although induced pluripotent stem cells (iPSCs) hold great promise for customized regenerative medicine, the molecular basis of reprogramming is largely unknown. Overcoming barriers that maintain cell identities is a critical step in the reprogramming of differentiated cells. Since microRNAs (miRNAs) modulate target genes tissue-specifically, we reasoned that distinct mouse embryonic fibroblast (MEF)-enriched miRNAs post-transcriptionally modulate proteins that function as reprogramming barriers. Inhibiting these miRNAs should influence cell signaling to lower those barriers. Here we show that depleting miR-21 and miR-29a enhances reprogramming efficiency in MEFs. We also show that the p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. In addition, we provide the first evidence that c-Myc enhances reprogramming partly by repressing MEF-enriched miRNAs, such as miR-21 and miR-29a. Our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in iPSC generation. These studies should facilitate development of clinically applicable reprogramming strategies.
With a dataset of more than 600 million small RNAs deeply sequenced from mouse hippocampal and staged sets of mouse cells that underwent reprogramming to induced pluripotent stem cells, we annotated the stem–loop precursors of the known miRNAs to identify isomoRs (miRNA-offset RNAs), loops, non-preferred strands, and guide strands. Products from both strands were readily detectable for most miRNAs. Changes in the dominant isomiR occurred among the cell types, as did switches of the preferred strand. The terminal nucleotide of the dominant isomiR aligned well with the dominant off-set sequence suggesting that Drosha cleavage generates most miRNA reads without terminal modification. Among the terminal modifications detected, most were non-templated mono- or di-nucleotide additions to the 3′-end. Based on the relative enrichment or depletion of specific nucleotide additions in an Ago-IP fraction there may be differential effects of these modifications on RISC loading. Sequence variation of the two strands at their cleavage sites suggested higher fidelity of Drosha than Dicer. These studies demonstrated multiple patterns of miRNA processing and considerable versatility in miRNA target selection.
Somatic cells can be reprogrammed to form embryonic stem cell-like induced pluripotent stem cells (iPSCs), but the process suffers from low efficiency and the underlying molecular mechanisms that control reprogramming remain poorly understood. Here we perform an inhibitor screen to identify kinases that enhance, or present a barrier to, reprogramming. In particular, inhibitors of p38, inositol trisphosphate 3-kinase, and Aurora A kinase potently enhance iPSC generation, and iPSCs derived from inhibitor-treated somatic cells are capable of reaching a fully reprogrammed state. Knockdown of target kinases by short interfering RNAs confirms that they function as barrier genes. We show that Aurora A kinase, which functions in centrosome activity and spindle assembly, is highly induced during reprogramming and inhibits Akt-mediated inactivation of GSK3β, resulting in compromised reprogramming efficiency. Together, our results not only identify new compounds that enhance iPSC generation but also shed new light on the function of Aurora A kinase in the reprogramming process.
Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with Kaposi's sarcoma and lymphomas. The pathogenesis of KSHV depends on the balance between two phases of the viral cycle: latency and lytic replication. In this study, we report that KSHV-encoded microRNAs (miRNAs) function as regulators by maintaining viral latency and inhibiting viral lytic replication. MiRNAs are short, noncoding, small RNAs that post-transcriptionally regulate the expression of messenger RNAs. Of the 12 viral miRNAs expressed in latent KSHV-infected cells, we observed that expression of miR-K3 can suppress both viral lytic replication and gene expression. Further experiments indicate that miR-K3 can regulate viral latency by targeting nuclear factor I/B. Nuclear factor I/B can activate the promoter of the viral immediate-early transactivator replication and transcription activator (RTA), and depletion of nuclear factor I/B by short hairpin RNAs had similar effects on the viral life cycle to those of miR-K3. Our results suggest a role for KSHV miRNAs in regulating the viral life cycle.
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