Enoxacin extends lifespan of C. elegans by inhibiting miR-34-5p and promoting mitohormesis.
ABSTRACT: Alterations in microRNA (miRNA) processing have been previously linked to aging. Here we used the small molecule enoxacin to pharmacologically interfere with miRNA biogenesis and study how it affects aging in C. elegans. Enoxacin extended worm lifespan and promoted survival under normal and oxidative stress conditions. Enoxacin-induced longevity required the transcription factor SKN-1/Nrf2 and was blunted by the antioxidant N-acetyl-cysteine, suggesting a prooxidant-mediated mitohormetic response. The longevity effects of enoxacin were also dependent on the miRNA pathway, consistent with changes in miRNA expression elicited by the drug. Among these differentially expressed miRNAs, the widely conserved miR-34-5p was found to play an important role in enoxacin-mediated longevity. Enoxacin treatment down-regulated miR-34-5p and did not further extend lifespan of long-lived mir-34 mutants. Moreover, N-acetyl-cysteine abrogated mir-34(gk437)-induced longevity. Evidence also points to double-stranded RNA-specific adenosine deaminases (ADARs) as new targets of enoxacin since ADAR loss-of-function abrogates enoxacin-induced lifespan extension. Thus, enoxacin increases lifespan by reducing miR-34-5p levels, interfering with the redox balance and promoting healthspan.
Project description:MicroRNAs (miRNAs) have been implicated in oxidative metabolism and brown/beige adipocyte identity. Here, we tested whether widespread changes in miRNA expression promoted by treatment with the small-molecule enoxacin cause browning and prevent obesity. Enoxacin mitigated diet-induced obesity in mice, and this was associated with increased energy expenditure. Consistently, subcutaneous white and brown adipose tissues and skeletal muscle of enoxacin-treated mice had higher levels of markers associated with thermogenesis and oxidative metabolism. These effects were cell autonomous since they were recapitulated in vitro in murine and human cell models. In preadipocytes, enoxacin led to a reduction of <i>miR-34a-5p</i> expression and up-regulation of its target genes (e.g., <i>Fgfr1</i>, <i>Klb</i>, and <i>Sirt1</i>), thus increasing FGF21 signaling and promoting beige adipogenesis. Our data demonstrate that enoxacin counteracts obesity by promoting thermogenic signaling and inducing oxidative metabolism in adipose tissue and skeletal muscle in a mechanism that involves, at least in part, miRNA-mediated regulation.
Project description:Prostate cancer (PCa) is one of the most incident malignancies worldwide. Although efficient therapy is available for early-stage PCa, treatment of advanced disease is mainly ineffective and remains a clinical challenge. microRNA (miRNA) dysregulation is associated with PCa development and progression. In fact, several studies have reported a widespread downregulation of miRNAs in PCa, which highlights the importance of studying compounds capable of restoring the global miRNA expression. The main aim of this study was to define the usefulness of enoxacin as an anti-tumoral agent in PCa, due to its ability to induce miRNA biogenesis in a TRBP-mediated manner. Using a panel of five PCa cell lines, we observed that all of them were wild type for the TARBP2 gene and expressed TRBP protein. Furthermore, primary prostate carcinomas displayed normal levels of TRBP protein. Remarkably, enoxacin was able to decrease cell viability, induce apoptosis, cause cell cycle arrest, and inhibit the invasiveness of cell lines. Enoxacin was also effective in restoring the global expression of miRNAs. This study is the first to show that PCa cells are highly responsive to the anti-tumoral effects of enoxacin. Therefore, enoxacin constitutes a promising therapeutic agent for PCa.
Project description:Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 ?M) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 ?M enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.
Project description:Chiroptera, the bats, are the only order of mammals capable of true self-powered flight. Bats exhibit a number of other exceptional traits such as echolocation, viral tolerance and, perhaps most puzzlingly, extreme longevity given their body size. Little is known about the molecular mechanisms driving their extended longevity particularly at the levels of gene expression and post-transcriptional regulation. To elucidate the molecular mechanisms that may underlie their unusual longevity, we have deep sequenced 246.5 million small RNA reads from whole blood of the long-lived greater mouse-eared bats, Myotis myotis, and conducted a series of genome-wide comparative analyses between bat and non-bat mammals (human, pig and cow) in both blood miRNomes and transcriptomes, for the first time.We identified 539 miRNA gene candidates from bats, of which 468 unique mature miRNA were obtained. More than half of these miRNA (65.1 %) were regarded as bat-specific, regulating genes involved in the immune, ageing and tumorigenesis pathways. We have also developed a stringent pipeline for genome-wide miRNome comparisons across species, and identified 37 orthologous miRNA groups shared with bat, human, pig and cow, 6 of which were differentially expressed. For bats, 3 out of 4 up-regulated miRNA (miR-101-3p, miR-16-5p, miR-143-3p) likely function as tumor suppressors against various kinds of cancers, while one down-regulated miRNA (miR-221-5p) acts as a tumorigenesis promoter in human breast and pancreatic cancers. Additionally, a genome-wide comparison of mRNA transcriptomes across species also revealed specific gene expression patterns in bats. 127 up-regulated genes were enriched mainly in mitotic cell cycle and DNA repair mechanisms, while 364 down-regulated genes were involved primarily in mitochondrial activity.Our comprehensive and integrative analyses revealed bat-specific and differentially expressed miRNA and mRNA that function in key longevity pathways, producing a distinct bat gene expression pattern. For the first time, we show that bats may possess unique regulatory mechanisms for resisting tumorigenesis, repairing cellular damage and preventing oxidative stresses, all of which likely contribute to the extraordinary lifespan of Myotis myotis.
Project description:<h4>Unlabelled</h4>Enoxacin inhibits binding between the B-subunit of vacuolar H(+)-ATPase (V-ATPase) and microfilaments, and also between osteoclast formation and bone resorption in vitro. We hypothesized that a bisphosphonate derivative of enoxacin, bis-enoxacin (BE), which was previously studied as a bone-directed antibiotic, might have similar activities. BE shared a number of characteristics with enoxacin: It blocked binding between the recombinant B-subunit and microfilaments and inhibited osteoclastogenesis in cell culture with IC50s of about 10 µM in each case. BE did not alter the relative expression levels of various osteoclast-specific proteins. Even though tartrate-resistant acid phosphatase 5b was expressed, proteolytic activation of the latent pro-enzyme was inhibited. However, unlike enoxacin, BE stimulated caspase-3 activity. BE bound to bone slices and inhibited bone resorption by osteoclasts on BE-coated bone slices in cell culture. BE reduced the amount of orthodontic tooth movement achieved in rats after 28 days. Analysis of these data suggests that BE is a novel anti-resorptive molecule that is active both in vitro and in vivo and may have clinical uses.<h4>Abbreviations</h4>BE, bis-enoxacin; V-ATPase, vacuolar H(+)-ATPase; TRAP, tartrate-resistant acid phosphatase; ?MEM D10, minimal essential media, alpha modification with 10% fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RANKL, receptor activator of nuclear factor kappa B-ligand; NFATc1, nuclear factor of activated T-cells; ADAM, a disintegrin and metalloprotease domain; OTM, orthodontic tooth movement.
Project description:The aging process starts directly after birth and lasts for the entire lifespan; it manifests itself with a decline in an organism's ability to adapt and is linked to the development of age-related diseases that eventually lead to premature death. This review aims to explore how microRNAs (miRNAs) are involved in skin functioning and aging. Recent evidence has suggested that miRNAs regulate all aspects of cutaneous biogenesis, functionality, and aging. It has been noted that some miRNAs were down-regulated in long-lived individuals, such as let-7, miR-17, and miR-34 (known as longevity-related miRNAs). They are conserved in humans and presumably promote lifespan prolongation; conversely, they are up-regulated in age-related diseases, like cancers. The analysis of the age-associated cutaneous miRNAs revealed the increased expression of miR-130, miR-138, and miR-181a/b in keratinocytes during replicative senescence. These miRNAs affected cell proliferation pathways via targeting the p63 and Sirtuin 1 mRNAs. Notably, miR-181a was also implicated in skin immunosenescence, represented by the Langerhans cells. Dermal fibroblasts also expressed increased the levels of the biomarkers of aging that affect telomere maintenance and all phases of the cellular life cycle, such as let-7, miR-23a-3p, 34a-5p, miR-125a, miR-181a-5p, and miR-221/222-3p. Among them, the miR-34 family, stimulated by ultraviolet B irradiation, deteriorates collagen in the extracellular matrix due to the activation of the matrix metalloproteinases and thereby potentiates wrinkle formation. In addition to the pro-aging effects of miRNAs, the plausible antiaging activity of miR-146a that antagonized the UVA-induced inhibition of proliferation and suppressed aging-related genes (e.g., p21WAF-1, p16, and p53) through targeting Smad4 has also been noticed. Nevertheless, the role of miRNAs in skin aging is still not fully elucidated and needs to be further discovered and explained.
Project description:MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.
Project description:The global downregulation of microRNAs (miRNAs) is emerging as a common hallmark of cancer. However, the mechanisms underlying this phenomenon are not well known. We identified that the oncogenic miR-146b-5p attenuates miRNA biosynthesis by targeting DICER1 and reducing its expression. DICER1 overexpression inhibited all the miR-146b-induced aggressive phenotypes in thyroid cells. Systemic injection of an anti-miR-146b in mice with orthotopic thyroid tumors suppressed tumor growth and recovered DICER1 levels. Notably, DICER1 downregulation promoted proliferation, migration, invasion, and epithelial-mesenchymal transition through miRNA downregulation. Our analysis of The Cancer Genome Atlas revealed a general decrease in DICER1 expression in thyroid cancer that was associated with a worse clinical outcome. Administration of the small-molecule enoxacin to promote DICER1 complex activity reduced tumor aggressiveness both in vitro and in vivo. Overall, our data confirm DICER1 as a tumor suppressor and show that oncogenic miR-146b contributes to its downregulation. Moreover, our results highlight a potential therapeutic application of RNA-based therapies including miRNA inhibitors and restoration of the biogenesis machinery, which may provide treatments for thyroid and other cancers.
Project description:MicroRNAs (miRNAs) are small non-coding RNA species that have been shown to have roles in multiple processes that occur in higher eukaryotes. They act by binding to specific sequences in the 3' untranslated region of their target genes and causing the transcripts to be degraded by the RNA-induced silencing complex (RISC). MicroRNAs have previously been reported to demonstrate altered expression in several aging phenotypes such as cellular senescence and age itself. Here, we have measured the expression levels of 521 small regulatory microRNAs (miRNAs) in spleen tissue from young and old animals of 6 mouse strains with different median strain lifespans by quantitative real-time PCR. Expression levels of 3 microRNAs were robustly associated with strain lifespan, after correction for multiple statistical testing (miR-203-3p [?-coefficient?=?-0.6447, p?=?4.8?×?10-11], miR-664-3p [?-coefficient?=?0.5552, p?=?5.1?×?10-8] and miR-708-5p [?-coefficient?=?0.4986, p?=?1.6?×?10-6]). Pathway analysis of binding sites for these three microRNAs revealed enrichment of target genes involved in key aging and longevity pathways including mTOR, FOXO and MAPK, most of which also demonstrated associations with longevity. Our results suggests that miR-203-3p, miR-664-3p and miR-708-5p may be implicated in pathways determining lifespan in mammals.
Project description:Angiosarcoma is a rare malignant mesenchymal tumor with poor prognosis. We aimed to identify malignancy-associated miRNAs and their target genes, and explore biological functions of miRNA and its target in angiosarcoma. By miRNA microarrays and reverse transcription polymerase chain reaction, we identified 1 up-regulated miRNA (miR-222-3p) and 3 down-regulated miRNAs (miR-497-5p, miR-378-3p and miR-483-5p) in human angiosarcomas compared with human capillary hemangiomas. The intermediate-conductance calcium activated potassium channel KCa3.1 was one of the putative target genes of miR-497-5p, and marked up-regulation of KCa3.1 was detected in angiosarcoma biopsy specimens by immunohistochemistry. The inverse correlation of miR-497-5p and KCa3.1 also was observed in the ISO-HAS angiosarcoma cell line at the mRNA and protein levels. The direct targeting of KCa3.1 by miR-497-5p was evidenced by reduced luciferase activity due to complementary binding of miR-497-5p to KCa3.1 mRNA 3' untranslated region. For the functional role of miR-497-5p/KCa3.1 pair, we showed that application of TRAM-34, a specific KCa3.1 channel blocker, or transfection of ISO-HAS cells with KCa3.1 siRNA or miR-497-5p mimics inhibited cell proliferation, cell cycle progression, and invasion by down-regulating cell-cycle related proteins including cyclin D1, surviving and P53 and down-regulating matrix metallopeptidase 9. In an in vivo angiosarcoma xenograft model, TRAM-34 or miR-497-5p mimics both inhibited tumor growth. In conclusion, the tumor suppressor miR-497-5p down-regulates KCa3.1 expression and contributes to the inhibition of angiosarcoma malignancy development. The miR-497-5p or KCa3.1 might be potential new targets for angiosarcoma treatment.