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Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, typically affecting males in infancy. The disease causes progressive weakness and atrophy of skeletal muscles, with approximately 20,000 new cases diagnosed yearly. Currently, methods for diagnosing DMD are invasive, laborious, and unable to make accurate early detections. While there is no cure for DMD, there are limited treatments available for managing symptoms. As such, there is a crucial unmet need to develop a simple and non-invasive method for accurately detecting DMD as early as possible. Raman spectroscopy with chemometric analysis is shown to have the potential to fill this diagnostic need.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and affects boys in infancy or early childhood. Current methods for diagnosing DMD are often laborious, expensive, invasive, and typically diagnose the disease late in its progression. In an effort to improve the accuracy and ease of diagnosis, this study focused on developing a novel method for diagnosing DMD which combines Raman hyperspectroscopic analysis of blood serum with advanced statistical analysis. Partial least squares discriminant analysis was applied to the spectral dataset acquired from blood serum of a mouse model of Duchenne muscular dystrophy (mdx) and control mice. Cross-validation showed 95.2% sensitivity and 94.6% specificity for identifying diseased spectra. These results were verified via external validation, which achieved 100% successful classification accuracy at the donor level. This proof-of-concept study presents Raman hyperspectroscopic analysis of blood serum as an easy, fast, non-expensive, and minimally invasive detection method for distinguishing control and mdx model mice, with a strong potential for clinical diagnosis of DMD.

Skeletal muscle regenerates throughout the lifetime to maintain normal development, growth, and physiological function. Skeletal muscle regeneration occurs in a coordinated fashion and requires strict regulation of myogenic gene expression during the process. Numerous studies have established the critical role of microRNAs in regulating post-transcriptional gene expression in diverse biological processes including differentiation, development, and regeneration. We have revealed in an earlier study that a large number of microRNAs were differentially expressed during myoblast differentiation. Here, we report the role of one such microRNA, the miR-24-3p, in skeletal muscle differentiation and regeneration. miR-24-3p is induced during myoblast differentiation and skeletal muscle regeneration. Exogenous miR-24-3p promotes while inhibition of miR-24-3p represses myoblast differentiation. miR-24-3p promotes myoblast differentiation by directly targeting and regulating the high mobility group AT-hook 1 (HMGA1). Consistent with the finding that HMGA1 is a repressor of myogenic differentiation, the miR-24-3p-resistant form of HMGA1 devoid of 39untranslated region, inhibits myoblast differentiation. Intramuscular injection of antagomirs specific to miR-24-3p into the tibialis anterior muscle prevents HMGA1 down-regulation and impairs regeneration. These findings provide evidence for the requirement of the miR-24-3p/HMGA1 axis for skeletal muscle differentiation and regeneration.

Numerous studies have established the critical roles of microRNAs in regulating post-transcriptional gene expression in diverse biological processes. Here, we report on the role and mechanism of miR-24-3p in skeletal muscle differentiation and regeneration. miR-24-3p promotes myoblast differentiation and skeletal muscle regeneration by directly targeting high mobility group AT-hook 1 (HMGA1) and regulating it and its direct downstream target, the inhibitor of differentiation 3 (ID3). miR-24-3p knockdown in neonatal mice increases PAX7-positive proliferating muscle stem cells (MuSCs) by derepressing Hmga1 and Id3. Similarly, inhibition of miR-24-3p in the tibialis anterior muscle prevents Hmga1 and Id3 downregulation and impairs regeneration. These findings provide evidence that the miR-24-3p/HMGA1/ID3 axis is required for MuSC differentiation and skeletal muscle regeneration in vivo.

MicroRNAs are involved in the crucial processes of development and diseases and have emerged as a new class of biomarkers. The field of DNA nanotechnology has shown great promise in the creation of novel microRNA biosensors that have utility in lab-based biosensing and potential for disease diagnostics. In this Survey and Summary, we explore and review DNA nanotechnology approaches for microRNA detection, surveying the literature for microRNA detection in three main areas of DNA nanostructures: DNA tetrahedra, DNA origami, and DNA devices and motifs. We take a critical look at the reviewed approaches, advantages and disadvantages of these methods in general, and a critical comparison of specific approaches. We conclude with a brief outlook on the future of DNA nanotechnology in biosensing for microRNA and beyond.

We report a novel method to purify individual RNA species using programmable DNA nanoswitches to facilitate RNA capture, release, and isolation. We validate sequence-based purification of microRNA, rRNA, and an mRNA fragment from total RNA, demonstrate multiplexing with two different RNA species, and show an application with downstream LC/MS analysis to identify RNA modifications. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption.Competing Interest StatementLZ, ARC, and KH have intellectual property related to DNA nanoswitches. All other authors declare no competing interests.View Full Text

Nanometer-sized features and molecular recognition properties make DNA a useful material for nanoscale construction, but degradation in biological fluids poses a considerable roadblock to biomedical applications of DNA nanotechnology. Here, we report the remarkable biostability of a multistranded motif called paranemic crossover (PX) DNA. Compared to double stranded DNA, PX DNA has dramatically enhanced (sometimes >1000 fold) resistance to degradation by four different nucleases, bovine and human serum, and human urine. We trace the cause of PX's biostability to DNA crossovers, showing a continuum of protection that scales with the number of crossovers. These results suggest that enhanced biostability can be engineered into DNA nanostructures by adopting PX-based architectures or by strategic crossover placement.

MicroRNAs are short non-coding RNAs involved in post-transcriptional gene regulation, and are increasingly considered to be biomarkers for numerous biological processes and human diseases. Current techniques used for microRNA detection can be expensive and labor-intensive, and typically require amplification, labeling, or radioactive probes. In this protocol, we describe a DNA nanoswitch-based microRNA detection assay termed "miRacles": microRNA-activated conditional looping of engineered switches. This method uses conformationally responsive DNA nanoswitches that detect the presence of specific microRNAs with a simple and unambiguous gel-shift assay that can be performed on the benchtop. The assay is low cost, minimalistic, and capable of direct detection of specific microRNAs in unprocessed total RNA samples, with no enzymatic amplification, labeling, or special equipment. The protocol for detection of microRNAs in total RNA can be completed in as little as a few hours, making this assay a compelling alternative to qPCR and Northern blotting. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of DNA nanoswitches Basic Protocol 2: Detection of microRNAs from total RNA samples Support Protocol 1: Optional nanoswitch purification by PEG precipitation Support Protocol 2: Optional nanoswitch purification by liquid chromatography.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and affects boys in infancy or early childhood. DMD is known to trigger progressive muscle weakness due to skeletal muscle degeneration and ultimately causes death. There are limited treatment regimens available that can either slow or stop the progression of DMD. An accurate and specific method for diagnosing DMD in its earliest stages is needed to prevent progressive muscle degeneration and death. Current methods for diagnosing DMD are often laborious, expensive, invasive, and typically diagnose the disease later on it is progression. In an effort to improve the accuracy and ease of diagnosis, this study focused on developing a novel method for diagnosing DMD which combines Raman hyperspectroscopic analysis of blood serum with advanced statistical analysis. Partial Least Squares Discriminant Analysis (PLS-DA), was applied to the spectral dataset acquired from control andmdxblood serum of 3- and 12-month old mice to build a diagnostic algorithm. Internal cross-validation showed 95.2% sensitivity and 94.6% specificity for identifying diseased spectra. These results were verified using external validation, which achieved 100% successful classification efficiency at the level of individual donor. This proof-of-concept study presents Raman hyperspectroscopic analysis of blood serum as a fast, non-expensive, minimally invasive and early detection method for the diagnosis of Duchenne muscular dystrophy.

Retinal diseases are frequently characterized by the accumulation of excessive scar tissue found throughout the neural retina. However, the pathophysiology of retinal fibrosis remains poorly understood, and the cell types that contribute to the fibrotic response are incompletely defined. Here, we show that myofibroblast differentiation of mural cells contributes directly to retinal fibrosis. Using lineage tracing technology, we demonstrate that after chemical ocular injury, Myh11+ mural cells detach from the retinal microvasculature and differentiate into myofibroblasts to form an epiretinal membrane. Inhibition of TGFβR attenuates Myh11+ retinal mural cell myofibroblast differentiation, and diminishes the subsequent formation of scar tissue on the surface of the retina. We demonstrate retinal fibrosis within a murine model of oxygen-induced retinopathy resulting from the intravitreal injection of adipose Myh11-derived mesenchymal stem cells, with ensuing myofibroblast differentiation. In this model, inhibiting TGFβR signaling does not significantly alter myofibroblast differentiation and collagen secretion within the retina. This work shows the complexity of retinal fibrosis, where scar formation is regulated both by TGFβR and non-TGFβR dependent processes involving mural cells and derived mesenchymal stem cells. It also offers a cautionary note on the potential deleterious, pro-fibrotic effects of exogenous MSCs once intravitreally injected into clinical patients.

Inherent nanometer-sized features and molecular recognition properties make DNA a useful material in constructing nanoscale objects, with alluring applications in biosensing and drug delivery. However, DNA can be easily degraded by nucleases present in biological fluids, posing a considerable roadblock to realizing the full potential of DNA nanotechnology for biomedical applications. Here we investigated the nuclease resistance and biostability of the multi-stranded motif called paranemic crossover (PX) DNA and discovered a remarkable and previously unreported resistance to nucleases. We show that PX DNA has more than an order of magnitude increased resistance to degradation by DNase I, serum, and urine compared to double stranded DNA. We further demonstrate that the degradation resistance decreases monotonically as DNA crossovers are removed from the structure, suggesting that frequent DNA crossovers disrupt either the binding or catalysis of nucleases or both. Further, we show using mouse and human cell lines that PX DNA does not affect cell proliferation or interfere with biological processes such as myogenesis. These results have important implications for building DNA nanostructures with enhanced biostability, either by adopting PX-based architectures or by carefully engineering crossovers. We contend that such crossover-dependent nuclease resistance could potentially be used to add “tunable biostability” to the many features of DNA nanotechnology.

Cellular differentiation is a fundamental process in which one cell type changes into one or more specialized cell types. Cellular differentiation starts at the beginning of embryonic development when a simple zygote begins to transform into a complex multicellular organism composed of various cell and tissue types. This process continues into adulthood when adult stem cells differentiate into more specialized cells for normal growth, regeneration, repair, and cellular turnover. Any abnormalities associated with this fundamental process of cellular differentiation is linked to life threatening conditions including degenerative diseases and cancers. Detection of undifferentiated and different stages of differentiated cells can be used for disease diagnosis but is often challenging due to the laborious procedures, expensive tools, and specialized technical skills which are required. Here, a novel approach, called deep ultraviolet resonance Raman spectroscopy, is used to study various stages of cellular differentiation using a well-known myoblast cell line as a model system. These cells proliferate in the growth medium and spontaneously differentiate in differentiation medium into myocytes and later into myotubes and myofibers. The cellular and molecular characteristics of these cells mimic very well actual muscle tissue in vivo. We have found that undifferentiated myoblast cells and myoblast cells differentiated at three different stages are able to be easily separated using deep ultraviolet resonance Raman spectroscopy in combination with chemometric techniques. Our study has a great potential to study cellular differentiation during normal development as well as to detect abnormal cellular differentiation in human pathological conditions in future studies.

A DNA nanotechnology approach enables label-free and unamplified detection of cellular microRNAs using gel electrophoresis., MicroRNAs are short noncoding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive and often requires amplification, labeling, or radioactive probes. Here, we report a single-step, nonenzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA-activated conditional looping of engineered switches), our assay has subattomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells and multiplex our detection for several microRNAs from one biological sample. We demonstrate 1-hour detection without expensive equipment or reagents, making this assay a compelling alternative to quantitative polymerase chain reaction and Northern blotting.

MicroRNAs are short non-coding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive, and often requires amplification, labeling, or radioactive probes. Here we report a single-step, non-enzymatic detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNAactivatedconditionallooping ofengineeredswitches), our assay has sub-attomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells, and demonstrate multiplexed detection of several microRNAs from one biological sample. We demonstrate one-hour detection without expensive equipment or reagents, making this assay a compelling alternative to qPCR and Northern blotting.Significance statementDetection of microRNAs play a key role in biological research and medical diagnostics, and current detection methods are expensive and require sophisticated processes. We presentmicroRNAactivatedconditionallooping ofengineeredswitches (miRacles), a mix-and-read strategy that is based on conformational changes of DNA nanoswitches upon binding a target microRNA. MiRacles has a sensitivity of ∼4 copies/cell and specificity of a single nucleotide, and can be performed in one hour at a fraction of the cost of traditional microRNA detection techniques. Our method can also be multiplexed to detect multiple microRNAs from one biological sample. The minimalistic miRacles assay has immediate application in biomedical research and longer term potential as a clinical tool.

Regulated expression of the H19 long noncoding RNA gene has been well characterized as a paradigm for genomic imprinting, but the H19 RNA's biological function remains largely unclear. H19 is abundantly expressed maternally in embryonic tissues but is strongly repressed after birth, and significant transcription persists only in skeletal muscle. Thus, we examined the role of the H19 RNA in skeletal muscle differentiation and regeneration. Knockdown of H19 RNA in myoblast cells and H19 knockout mouse satellite cells decreases differentiation. H19 exon1 encodes two conserved microRNAs, miR-675-3p and miR-675-5p, both of which are induced during skeletal muscle differentiation. The inhibition of myogenesis by H19 depletion during myoblast differentiation is rescued by exogenous expression of miR-675-3p and miR-675-5p. H19-deficient mice display abnormal skeletal muscle regeneration after injury, which is rectified by reintroduction of miR-675-3p and miR-675-5p. miR-675-3p and miR-675-5p function by directly targeting and down-regulating the anti-differentiation Smad transcription factors critical for the bone morphogenetic protein (BMP) pathway and the DNA replication initiation factor Cdc6. Therefore, the H19 long noncoding RNA has a critical trans-regulatory function in skeletal muscle differentiation and regeneration that is mediated by the microRNAs encoded within H19.

The genetics of skeletal muscle lineage commitment are deceptively complicated. MyoD overexpression is sufficient to convert fibroblasts into skeletal muscle myotubes. In vivo, there are a number of different steps of differentiation that require a large network of transcription factors that control differentiation and homeostasis of skeletal muscle progenitors. Each transcription factor has been shown to have the ability to promote the next factor in the cascade, but the mechanisms regulating the transitions remain incomplete. Recently, microRNAs have been shown to be important for a large number of developmental and oncogenic processes. In this review, we will discuss recent advances in the understanding of how microRNA is critical for skeletal muscle development by interacting with protein-coding genes that had previously been shown to be important for myogenesis.

MicroRNAs play important roles in many cell processes, including the differentiation process in several different lineages. For example, microRNAs can promote differentiation by repressing negative regulators of transcriptional activity. These regulated transcription factors can further up-regulate levels of the microRNA in a feed-forward mechanism. Here we show that MyoD up-regulates miR-378 during myogenic differentiation in C2C12 cells. ChIP and high throughput sequencing analysis shows that MyoD binds in close proximity to the miR-378 gene and causes both transactivation and chromatin remodeling. Overexpression of miR-378 increases the transcriptional activity of MyoD, in part by repressing an antagonist, MyoR. The 3' untranslated region of MyoR contains a direct binding site for miR-378. The presence of this binding site significantly reduces the ability of MyoR to prevent the MyoD-driven transdifferentiation of fibroblasts. MyoR and miR-378 were anticorrelated during cardiotoxin-induced adult muscle regeneration in mice. Taken together, this shows a feed-forward loop where MyoD indirectly down-regulates MyoR via miR-378.

This article describes a novel role of Cdc25A down-regulation during differentiation of proliferating myoblasts., Induction of a G1 phase cell cycle arrest, caused primarily by the inhibition of cyclin-dependent-kinase 2 (cdk2), is a critical step in the differentiation of myoblasts into myotubes. Here, we report that two microRNAs, miR-322/424 and miR-503, are induced and promote cdk2 inhibition during myogenesis. These microRNAs down-regulate Cdc25A, the phosphatase responsible for removing inhibitory phosphorylation of cdk2, both in myoblasts differentiating into myotubes and in nonmuscle cells. Cdc25A is down-regulated during muscle differentiation by multiple pathways: action of these two microRNAs, proteasomal degradation of Cdc25A protein and transcriptional repression. Overexpression of Cdc25A or of cdk2 with mutations on T14 and Y15 (cdk2-AF), so that it cannot be inhibited by phosphorylation, decreases differentiation and differentiation-induced cell cycle quiescence. Introduction of miR-322/424 and miR-503 in heterologous cancer cells induces G1 arrest, which is also attenuated by overexpression of the cdk2-AF mutant. Until now Cdc25A and the inhibitory phosphorylation on T14 and Y15 of cdk2 have only been implicated in the intra-S phase checkpoint pathway after DNA damage. Our results reveal an unexpected role of Cdc25A down-regulation and the inhibitory phosphorylation of cdk2 T14 and Y15 in cell cycle quiescence during muscle differentiation and implicate two muscle differentiation-induced microRNAs in the process.

Following publication of the article, the author named as “B Dey”, wished to point out that his full name is “Bijan K. Dey”. This was not reflected in the typesetting of the article, and as a consequence the article is not visible on Pub Med when a search is conducted on his full name.

We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

An in silico screen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRR(eRNA)), is encoded 5 kb upstream of the transcription start site of MyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5' end overlaps with the cis-acting distal regulatory region (DRR) of MyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenous MyoD, Myogenin, and Myh3 (myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act in trans to promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNC in vivo impaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classic cis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboring MyoD gene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression.

The Drosophila disconnected (disco) gene encodes a transcription factor containing two C2H2 zinc-fingers that functions in the development of both embryonic and adult structures. The disco gene was initially identified from a mutation in which adult photoreceptor axons fail to connect with their target cells in the larval optic lobe (Steller et al., 1987). The optic lobes of disco mutants are poorly developed and lack optic lobe neurons, including the lateral neurons. Abnormal circadian rhythmicity has been attributed to the absence or aberrant differentiation of these lateral neurons (Hardin et al., 1992; Helfrich-Forster et al., 1998). Additionally, disco mutants show defects in thoracic and abdominal sensory axon development (Glossop and Shepherd, 1998). More recently, disco was found to have a broader role in embryonic pattern formation, functioning as a cofactor for the Hox proteins Deformed (Dfd) and Sex Combs Reduced (Scr) in the gnathocephalic segments (mandibular, maxillary, and labial); this more extensive role was masked by the partially redundant gene, disco-related (disco-r) (Mahaffey et al., 2001). Lack of both Disco and Disco-r causes embryonic lethality and the absence of some larval mouth structures arising from the gnathocephalic segments. The disco gene is also expressed in the antennal and leg imaginal discs and likely has a role in the development of these structures (Lee et al., 1991). The disco gene is located in region 14B of the X chromosome and consists of two exons separated by a single intron. The first exon is untranslated and comprises the majority of the disco 5 UTR. The second exon contains the complete open reading frame (ORF) encoding the 568 amino acid protein. The two C2H2 zinc finger regions, defining the DNA binding domain, are located between amino acids 89 and 149 (Heilig et al., 1991). As tools for further study of Disco function, we generated three UAS-disco constructs—two encoding wildtype proteins and one producing an altered protein like that encoded by the disco null allele. The UAS-disco wild-type construct was generated by the sequential insertion of two disco fragments into the Drosophila transformation vector pUAST (Brand and Perrimon, 1993). A 1,270-bp BglII-XbaI fragment representing the majority of the disco ORF (a subclone from 542.2; Surdej et al., 1990) was inserted into BglII-XbaI digested pUAST. Next, a 473-bp fragment, with an artificial EcoRI site immediately upstream of the initiation codon, was obtained by an EcoRI-BglII digestion of a 1,276-bp PCR product generated with the following primers: Forward primer, 5 GGA TCC GAATTC ATG GAG CAC ATA ATG 3 ; Reverse Primer, 5 ATG TAG GCA CTG TCG CTA TCC 3 . This fragment was inserted into the EcoRI-BglII sites of the initial BglII-XbaI pUAST construct. The final construct includes a 1,743-bp EcoRI-XbaI insert encoding the entire disco ORF with some 3 UTR. The second wild-type construct (further referred to as UAS-disco) and the mutant construct (further referred to as UAS-disco) were generated by PCR amplification of genomic DNA. A 1,704-bp fragment containing the complete disco ORF was generated using primers containing EcoRI sites: Forward primer, 5 CCC ACC ACA GAATTC ATG GAG CAC 3 ; Reverse Primer, 5 CCA TGG ATC GAATTC TGG ACA TTA 3 . After EcoRI digestion, the resulting PCR product was inserted into an EcoRI digested pUAST. For the UAS-disco construct, Oregon R genomic DNA was used as a template, while homozygous disco genomic DNA was used as a template for the mutant UAS-disco construct. This form of the protein contains a single amino acid change— Cysteine 127 is substituted with a Serine—which disrupts the first zinc finger. Prior analysis indicated that this mutant form of the Disco protein is nonfunctional (Heilig et al., 1991; Mahaffey et al., 2001). The sequence and reading frame of all constructs were verified by DNA sequence analysis. Integration of UAS-disco constructs into the Drosophila genome was carried out using P-element-mediated germ-line transformation essentially as described by Rubin and Spradling (1982). Two UAS-disco lines were obtained—a heterozygous line carrying UAS-disco on the second chromosome balanced over CyO (UAS-disco(II)), and a homozygous line with the insert on the

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose-derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose-derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high-throughput enzyme-linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy. Significance This work addresses the therapeutic potential of stem cells that are obtained from diabetic donors, and the clinical focus of our work is diabetic retinopathy. This work examined how injected murine adipose-derived stem cells (ASCs) affect the retinal microvasculature in a mouse model of diabetic retinopathy. Results show that ASCs obtained from healthy mice secrete angiogenic growth factors and promote retinal vascular stability when they are injected intravitreally. The findings further suggest that ASCs obtained from diabetic mice have a diminished ability to support the retinal vasculature in this mouse model of retinal vasculopathy.

Receptor tyrosine kinases (RTKs) are co-deregulated in a majority of glioblastoma (GBM), the most common and most deadly brain tumor. We show that the RTKs MET, EGFR, and PDGFR regulate microRNA-134 (miR-134) in GBM. We find that miR-134 is downregulated in human tumors and cancer stem cells and that its expression inversely correlates with the activation of MET, EGFR, and PDGFR. We demonstrate that miR-134 inhibits cancer cell and stem-cell proliferation, survival, and xenograft growth, as well as cancer stem-cell self-renewal and stemness. We identify KRAS and STAT5B as targets of miR-134, and establish molecular and functional links between RTKs, miR-134, KRAS/STAT5B and malignancy in vitro and in vivo. We show that miR-134 induction is required for the anti-tumor effects of RTK inhibitors. We also uncover the molecular pathways through which RTKs regulate miR-134 expression and demonstrate the involvement of MAPK signaling and the KLF4 transcription factor. We therefore identify miR-134 as a novel RTK-regulated tumor-suppressive hub that mediates RTK and RTK-inhibitor effects on GBM malignancy by controlling KRAS and STAT5B.

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3'UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.

The advent of ultra–high-throughput sequencing has led to the discovery of a large group of small, noncoding RNAs that are not microRNAs. The functional relevance of microRNAs has been well established over the last decade. In this Perspective, we focus on the non-micro-short RNAs that comprise a variety of functional classes and range from 16–40 nucleotides in size. We will highlight how some of these non-micro-short RNAs were discovered, as well as their biogenesis, potential mechanisms of action, and role in diverse biological processes, development, and disease. Finally, we will describe what must be done to further our understanding of these enigmatic molecules.

Notch3 is expressed in myogenic precursors, but its function is not well known.Notch3 represses the activity of Mef2c and is in turn inhibited by the microRNAs-1 and -206.Notch3 serves as a regulator for preventing premature myogenic differentiation.Understanding how precocious differentiation is prevented is critical for designing therapy for skeletal muscle regeneration. The Notch signaling pathway is a well known regulator of skeletal muscle stem cells known as satellite cells. Loss of Notch1 signaling leads to spontaneous myogenic differentiation. Notch1, normally expressed in satellite cells, is targeted for proteasomal degradation by Numb during differentiation. A homolog of Notch1, Notch3, is also expressed in these cells but is not inhibited by Numb. We find that Notch3 is paradoxically up-regulated during the early stages of differentiation by an enhancer that requires both MyoD and activated Notch1. Notch3 itself strongly inhibits the myogenic transcription factor Mef2c, most likely by increasing the p38 phosphatase Mkp1, which inhibits the Mef2c activator p38 MAP kinase. Active Notch3 decreases differentiation. Mef2c, however, induces microRNAs miR-1 and miR-206, which directly down-regulate Notch3 and allow differentiation to proceed. Thus, the myogenic differentiation-induced microRNAs miR-1 and -206 are important for differentiation at least partly because they turn off Notch3. We suggest that the transient expression of Notch3 early in differentiation generates a temporal lag between myoblast activation by MyoD and terminal differentiation into myotubes directed by Mef2c.

Multiple microRNAs are known to be induced during the differentiation of myoblasts to myotubes. Yet, experiments in animals have not provided clear evidence for the requirement of most of these microRNAs in myogenic differentiation in vivo. miR-26a is induced during skeletal muscle differentiation and is predicted to target a well-known inhibitor of differentiation, the transforming growth factor β/bone morphogenetic protein (TGF-β/BMP) signaling pathway. Here we show that exogenous miR-26a promotes differentiation of myoblasts, while inhibition of miR-26a by antisense oligonucleotides or by Tough-Decoys delays differentiation. miR-26a targets the transcription factors Smad1 and Smad4, critical for the TGF-β/BMP pathway, and expression of microRNA-resistant forms of these transcription factors inhibits differentiation. Injection of antagomirs specific to miR-26a into neonatal mice derepressed both Smad expression and activity and consequently inhibited skeletal muscle differentiation. In addition, miR-26a is induced during skeletal muscle regeneration after injury. Inhibiting miR-26a in the tibialis anterior muscles through the injection of adeno-associated virus expressing a Tough-Decoy targeting miR-26a prevents Smad down-regulation and delays regeneration. These findings provide evidence for the requirement of miR-26a for skeletal muscle differentiation and regeneration in vivo.

The Drosophila disconnected (disco) gene encodes a C(2)H(2)-type zinc finger transcription factor required for the development of the central and peripheral nervous systems. We report that disco participates in a positive feedback loop with the Dll gene, a master regulator of ventral appendage development. Dll function is not only required for proper disco expression in antenna and leg discs, but is also sufficient for ectopic expression of disco in the developing retina and wing imaginal discs. Conversely, disco gene function is required for the maintenance of Dll expression. We show that Dll phenotypes are partially rescued by the up-regulation of disco expression in the Dll domain. Reduction in disco gene function disrupts antenna and leg development, and the phenotypes closely resemble that produced by Dll alleles. These observations demonstrate that disco plays a fundamental role in the Dll-dependent patterning of antenna and leg, perhaps as a regulator of Dll gene expression.

The histone demethylase lysine demethylase 5b (KDM5b) specifically demethylates lysine 4 of histone H3 (meH3K4), thereby repressing gene transcription. KDM5b regulates cell cycle control genes in cancer and is expressed in the early epiblast. This suggests that KDM5b plays a developmental role by maintaining uncommitted progenitors. Here we show that transient overexpression of KDM5b in embryonic stem cells decreases the expression of at least three different modulators of cell fate decisions, Egr1, p27(KIP1), and BMI1, by demethylation of their promoters. Constitutively increased KDM5b expression results in an increased mitotic rate and a decreased global 3meH3K4 but no change in cell identity. Results of two separate differentiation assays, neural differentiation and embryoid body EB (EB) formation, showed that KDM5b reduced the terminally differentiated cells and increased proliferating progenitors. These were achieved by two mechanisms, blocking of the upregulation of cell lineage markers and maintenance of cyclins, that allowed cells to escape differentiation and remain uncommitted. Additionally, EBs maintain high levels of Oct4 and Nanog and can be dissociated to reestablish highly proliferative cultures. The persistence of uncommitted progenitors may be due to the direct regulation of the Tcf/Lef family member mTcf3/hTcf7L1, an upstream regulator of Nanog expression. These findings demonstrate a role for KDM5b in the choice between proliferation and differentiation during development.

Receptor tyrosine kinases (RTKs) are co-deregulated in a majority of glioblastoma (GBM), the most common and most deadly brain tumor. In this study, we uncovered microRNA-134 (miR-134) as a novel tumor suppressive hub that is co-regulated by multiple RTKs via MAPK and KLF4 and that acts via regulation of KRAS and STAT5B expressions. Using microRNA microarrays and qRT-PCR, we screened for microRNAs that are co-regulated by multiple RTKs in GBM. We found that miR-134 was one such microRNA that is downregulated by the activation of MET, EGFR, and PDGFR, the three most commonly deregulated RTKs in GBM. Using a combination of pharmacology, promoter analysis, transcription factor functional and rescue approaches and ChIP/qPCR, we uncovered the MAPK pathway and the transcriptional repressor KLF4 as mediators of the effects of RTKs on miR-134 repression. We then measured the expression of miR-134 and found that it was downregulated in human GBM tumors and GBM stem cells (GSCs) and that it inversely correlated with the activation levels of MET, EGFR, and PDGFR in the tumors. We demonstrated that miR-134 inhibits GBM cell and GSC proliferation, survival, and in vivo xenograft growth, as well as GSC self-renewal and stemness. We uncovered the oncogenes KRAS and STAT5B as direct targets of miR-134 with immunoblots and 3′UTR reporter assays, and showed with rescue experiments that they mediate miR-134 tumor suppressive effects. Thereafter, using a series of molecular and functional assays we established direct links and dependencies between RTKs, KLF4, miR-134, KRAS/STAT5B and malignancy in vitro and in vivo. Importantly, we also found that miR-134 induction is required for the anti-tumor effects of small molecule RTK inhibitors. Altogether, our extensive study identified miR-134 as a novel RTK-regulated tumor-suppressive hub that mediates RTK and RTK-inhibitor effects on GBM malignancy by controlling KRAS and STAT5B. These findings shed new light onto the molecular and functional interactions between RTKs and microRNAs and uncover opportunities for exploiting these interactions for therapeutic purposes. Citation Format: Ying Zhang, Jungeun Kim, Adam C. Mueller, Bijan Dey, Yanzhi Yang, Daehee Lee, Jan Hachmann, Sanjo Finderle, Deric M. Park, James Christensen, David Schiff, Benjamin Purow, Anindya Dutta, Roger Abounader. Identification and characterization of microRNA-134 as a novel receptor tyrosine kinase-regulated tumor suppressive hub in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2014-1909

We report here the cloning of a novel form of the murine glial cell line-derived neurotrophic factor (GDNF) receptor. Northern blot analyses of various mouse tissues, including whole brain, demonstrated the existence of multiple transcripts of GDNF receptor. Screening of an adult mouse liver cDNA library yielded two isoforms of the receptor. One of the forms (alpha) shows a high degree of homology with other mammalian GDNFR-alpha and the other novel form (beta) is identical to the alpha form except for a deletion of five amino acids. These two forms do not share high sequence homologies with the recently isolated neurturin receptor. Both the alpha and beta forms are expressed in various murine tissues but not in muscle.