During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic ...capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migration
and
in male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135-KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration.
Axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135-KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.
Reversing epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, but a limited number of broadly ...comprehensive investigations of miRNAs involved in this process have been conducted. In this study, we screened a library of 1120 miRNA for their ability to transcriptionally activate the E-cadherin gene
in a promoter reporter assay as a measure of EMT reversal. By this approach, we defined miR-520f as a novel EMT-reversing miRNA. miR-520f expression was sufficient to restore endogenous levels of E-cadherin in cancer cell lines exhibiting strong or intermediate mesenchymal phenotypes. In parallel, miR-520f inhibited invasive behavior in multiple cancer cell systems and reduced metastasis in an experimental mouse model of lung metastasis. Mechanistically, miR-520f inhibited tumor cell invasion by directly targeting
, the TGFβ receptor
and the EMT inducers
, and the snail transcriptional repressor
, each crucial factors in mediating EMT. Collectively, our results show that miR-520f exerts anti-invasive and antimetastatic effects
and
, warranting further study in clinical settings.
.
Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of ...patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that
is selectively upregulated within neurons in epileptic brain and report that targeting
using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of
, including
Mef2 proteins are key regulators of excitatory synapse density. Mef2a and
show reciprocal expression regulation in human (of both sexes) and experimental TLE, and
regulates dendritic spine number and type through Mef2. Together, our data show that
is target for reducing seizure activity in chronic epilepsy, and that deregulation of
in epilepsy may alter
expression and thereby affect synaptic function and plasticity.
miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA
in both experimental and human mesial temporal lobe epilepsy. Silencing
in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of
, including members of the Mef2 protein family, are identified that begin to explain how deregulation of
may contribute to epilepsy.
Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) ...are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic ...angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.
Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of ...miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.
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1B3 is a mimic of the naturally occurring miRNA-193a-3p, a gene regulator that has potent tumor suppressor functions. We characterized the effect of 1B3 on gene expression in six human cancer cell lines. 1B3 was shown to strongly activate tumor suppressor pathways while inhibiting oncogenic cell signaling.
MicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to ...prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs.
To systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs.
With the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.
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Angiogenesis is one of the hallmarks of cancer which renders it an attractive target for therapy of malignancies. Tumor growth suppression can be achieved by inhibiting angiogenesis ...since it would deprive tumor cells of oxygen and vital nutrients. Activation of endothelial cells of tumor vasculature is the first step in angiogenesis which is mediated by various factors. One of the major triggers in this process is vascular endothelial growth factor (VEGF) which binds to VEGF receptors on endothelial cells of tumor vessels. This induces a series of signaling cascades leading to activation of cellular processes involved in angiogenesis, and therefore down-regulation of VEGF receptor-2 (VEGFR-2) expression seems a viable option to inhibit angiogenesis. In our investigations, this aim has been pursued by using siRNA interfering with the expression of VEGFR-2. Since the discovery of RNA interference (RNAi) as a gene regulation process, successful delivery of small non-coding RNA has presented itself as a major challenge. In the current study, we have characterized a galectin-1 targeted anginex-coupled lipoplex (Angiplex) containing siRNA against the gene of VEGFR-2 as an angiostatic therapeutic. Angiplex particles had a size of approximately 120nm with a net negative charge and were stable in vitro. These particles were internalized in a specific manner by HUVECs compared to a non-targeted lipoplex system, and their uptake was higher than Lipofectamine 2000. Gene silencing efficiency of Angiplex was shown to be 61%.
During renal injury, activation of p38 mitogen-activated protein kinase (MAPK) in proximal tubular cells plays an important role in the inflammatory events that eventually lead to renal fibrosis. We ...hypothesized that local inhibition of p38 within these cells may be an interesting approach for the treatment of renal fibrosis. To effectuate this, we developed a renal-specific conjugate of the p38 inhibitor SB202190 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole and the carrier lysozyme. First, we demonstrated that SB202190 inhibited the expression of albumin-induced proinflammatory (monocyte chemoattractant protein-1) and transforming growth factor (TGF)-beta1-induced profibrotic (procollagen-Ialpha1) genes over 50% in renal tubular cells (normal rat kidney-52E). Next, we conjugated SB202190 via a carbamate linkage to lysozyme. However, this conjugate rapidly released the drug upon incubation in serum. Therefore, we applied a new platinum(II)-based linker approach, the so-called universal linkage system (ULS), which forms a coordinative bond with SB202190. The SB202190-ULS-lysozyme remained stable in serum but released the drug in kidney homogenates. SB202190-ULS-lysozyme accumulated efficiently in renal tubular cells and provided a local drug reservoir during a period of 3 days after a single intravenous injection. Treatment with SB202190-ULS-lysozyme inhibited TGF-beta1-induced gene expression for procollagen-Ialpha1 by 64% in HK-2 cells. Lastly, we evaluated the efficacy of a single dose of the conjugate in the unilateral renal ischemia-reperfusion rat model. A reduction of intrarenal p38 phosphorylation and alpha-smooth muscle actin protein expression was observed 4 days after the ischemia-reperfusion injury. In conclusion, we have developed a novel strategy for local delivery of the p38 MAPK inhibitor SB202190, which may be of use in the treatment of renal fibrosis.
As cancer is a multifactorial disease, the multimodal action of microRNAs makes them an attractive tool for novel therapeutic approaches. The tumor suppressive miR-7-5p has been shown to act on many ...aspects of oncogenesis, including cell proliferation, migration and angiogenesis, by targeting a spectrum of key genes. We developed a synthetic chemically modified miR-7-5p mimic, 5A2, and performed a comprehensive functional characterization in a panel of human cancer cell lines. 5A2 reduced cell proliferation in most cell lines by inducing cell cycle arrest. To enable systemic delivery of 5A2 to tumors, it was formulated in a novel lipid nanoparticle (INT-5A2) and we demonstrated the anti-tumor activity of INT-5A2 in an experimental human liver tumor-bearing mouse model. Next, RNA-sequencing was used to gain more insight into the molecular mechanism of action of 5A2 and demonstrated a broad repression of target mRNAs. Interestingly, Ingenuity Pathway Analysis revealed a new role for 5A2 in metabolic pathways. Validation experiments
in vitro
showed that 5A2 reduced the expression of key glycolysis and glutaminolysis enzymes, leading to a decrease in glycolysis, lactate secretion and intracellular glutamate availability. Taken together, these data strongly suggest that miR-7-5p/5A2 is a potent tumor suppressor that targets various key cellular pathways across cancer types. Therefore, 5A2 may represent a promising novel treatment strategy in oncology.
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