Directly reprogramming fibroblasts into cardiomyocytes improves cardiac function in the infarcted heart. However, the low efficacy of this approach hinders clinical applications. Unlike the adult ...mammalian heart, the neonatal heart has an intrinsic regenerative capacity. Consequently, we hypothesized that birth imposes fundamental changes in cardiac fibroblasts which limit their regenerative capabilities. In support, we found that reprogramming efficacy in vitro was markedly lower with fibroblasts derived from adult mice versus those derived from neonatal mice. Notably, fibroblasts derived from adult mice expressed significantly higher levels of pro-angiogenic genes. Moreover, under conditions that promote angiogenesis, only fibroblasts derived from adult mice differentiated into tube-like structures. Targeted knockdown screening studies suggested a possible role for the transcription factor Epas1. Epas1 expression was higher in fibroblasts derived from adult mice, and Epas1 knockdown improved reprogramming efficacy in cultured adult cardiac fibroblasts. Promoter activity assays indicated that Epas1 functions as both a transcription repressor and an activator, inhibiting cardiomyocyte genes while activating angiogenic genes. Finally, the addition of an Epas1 targeting siRNA to the reprogramming cocktail markedly improved reprogramming efficacy in vivo with both the number of reprogramming events and cardiac function being markedly improved. Collectively, our results highlight differences between neonatal and adult cardiac fibroblasts and the dual transcriptional activities of Epas1 related to reprogramming efficacy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MicroRNA (miRNA) is a pivotal biomarker in the diagnosis of various cancers, including bladder cancer (BCa). Despite their significance, the low abundance of miRNA presents a substantial challenge ...for sensitive and reliable detection. We introduce an innovative, highly sensitive assay for miRNA expression quantification that is both enzyme-free and portable. This method leverages the synergy of target recycling and entropy-driven assembly (EDA) for enhanced sensitivity and specificity. The proposed method possesses several advantages, including i) dual signal amplification through target recycling and EDA, which significantly boosts sensitivity with a lower limit of detection of 2.54 fM; ii) elimination of enzyme requirements, resulting in a cost-effective and stable signal amplification process; and iii) utilization of a personal glucose meter (PGM) for signal recording, rendering the method portable and adaptable to diverse settings. In summary, this PGM-based approach holds promising potential for clinical molecular diagnostics, offering a practical and efficient solution for miRNA analysis in cancer detection.
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•We proposed an enzyme-free and portable biosensor for ultrasensitive detection of miRNA.•The method showed a high sensitivity and a low limit of detection of 2.54 fM.•No enzymes are required for the signal amplification.•A PGM is used to record the signals, which has a wide range of applicable.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Couch's spadefoot toad (Scaphiopus couchii) spends most of the year underground in a hypometabolic state known as estivation. During this time, they overcome significant dehydration and lack of food ...through many mechanisms including employing metabolic rate depression (MRD), increasing urea concentration, switching to lipid oxidation as the primary energy source, and decreasing their breathing and heart rate. MicroRNA (miRNA) are known to regulate translation by targeting messenger RNA (mRNA) for degradation or temporary storage, with several studies having reported that miRNA is differentially expressed during MRD, including estivation. Thus, we hypothesized that miRNA would be involved in gene regulation during estivation in S. couchii heart. Next-generation sequencing and bioinformatic analyses were used to assess changes in miRNA expression in response to two-month estivation and to predict the downstream effects of this expression. KEGG and GO analyses indicated that ribosome and cardiac muscle contraction are among the pathways predicted to be upregulated, whereas cell signaling and fatty acid metabolism were predicted to be downregulated. Together these results suggest that miRNAs contribute to the regulation of gene expression related to cardiac muscle physiology and energy metabolism during estivation.
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•Spadefoot toads undergo estivation for ∼10 months in response to drought conditions.•4 miRNAs increase and 11 miRNAs decrease in S. couchii heart during estivation.•miRNAs predict upregulation of ribosome and cardiac muscle contraction.•miRNAs predict downregulation of cell signaling and fatty acid metabolism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Breast cancer is one of the most prevalent cancers in women. Triple‐negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several ...causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA‐MB‐231 and MCF‐7 cells and characterized. Overexpression of the miRNAs of MDA‐MB‐231 cells and their exosomes were analyzed using quantitative Real‐time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF‐7 cells were treated with MDA‐MB‐231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR‐9 and miR‐155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and
DUSP14 tumor suppressor genes. Quantitative Real‐time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target
PTEN and
DUSP14. Treatment of MCF‐7 cells with MDA‐MB‐231 cells’ exosomes resulted in target genes downregulation in MCF‐7 cells. We found that miR‐9 and miR‐155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.
Exosomes can transfer microRNAs (miRNAs) to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. Exosomes were extracted from MDA‐MB‐231 and MCF‐7 cells and characterized. Overexpression of miR‐9 and miR‐155 of MDA‐MB‐231 cells and their exosomes were analyzed using quantitative Real‐time PCR (RT‐qPCR). Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF‐7 cells with MDA‐MB‐231 cells’ exosomes resulted in target genes downregulation in MCF‐7 cells.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be ...protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs.
OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves.
A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC.
In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
The repair of a fractured bone is critical to the well‐being of humans. Failure of the repair process to proceed normally can lead to complicated fractures, exemplified by either a delay in ...union or a complete nonunion. Both of these conditions lead to pain, the possibility of additional surgery, and impairment of life quality. Additionally, work productivity decreases, income is reduced, and treatment costs increase, resulting in financial hardship. Thus, developing effective treatments for these difficult fractures or even accelerating the normal physiological repair process is warranted. Accumulating evidence shows that microRNAs (miRNAs), small noncoding RNAs, can serve as key regulatory molecules of fracture repair. In this review, a brief description of the fracture repair process and miRNA biogenesis is presented, as well as a summary of our current knowledge of the involvement of miRNAs in physiological fracture repair, osteoporotic fractures, and bone defect healing. Further, miRNA polymorphisms associated with fractures, miRNA presence in exosomes, and miRNAs as potential therapeutic orthobiologics are also discussed. This is a timely review as several miRNA‐based therapeutics have recently entered clinical trials for nonskeletal applications and thus it is incumbent upon bone researchers to explore whether miRNAs can become the next class of orthobiologics for the treatment of skeletal fractures.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The great majority of cancer deaths are due to metastasis, which remains a poorly understood pathological process. The formation of a metastasis reflects a succession of complex steps leading to the ...macroscopic outgrowth of disseminated tumor cells at the secondary site. In the past 5 years, certain microRNAs (miRNAs) have been shown to regulate either a single step or multiple steps of metastasis, doing so by downregulating the expression of their target genes. In this review, we discuss recent studies on the functions and molecular mechanisms of miRNAs in regulating epithelial–mesenchymal transition (EMT) and cancer metastasis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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RNA interference (RNAi) based therapeutics are considered an endogenous mechanism for modulating gene expression. In addition, microRNAs (miRNAs) may be tractable targets for the ...treatment of Chronic Obstructive Pulmonary Disease (COPD). In this study miR146a was adsorbed onto poly (glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, nanoparticles (NPs) to reduce target gene IRAK1 expression. NPs were prepared using an oil-in-water single emulsion solvent evaporation method incorporating cationic lipid dioleoyltrimethylammoniumpropane (DOTAP). This resulted in NPs of 244.80 ± 4.40 nm at 15% DOTAP concentration, zeta potential (ZP) of +14.8 ± 0.26 mV and miR-146a (40 µg/ml) maximum adsorption onto 15% DOTAP NPs was 36.25 ± 0.35 µg per 10 mg NP following 24 h incubation. Using the MTT assay, it was observed that over 75% at 0.312 mg/ml of A549 cells remained viable after 18 h exposure to cationic NPs at a concentration of 1.25 mg/ml. Furthermore, the in vitro release profile of miR-146a from loaded NPs showed a continuous release up to 77% after 24 h. Internalization of miR-146a loaded cationic NPs was observed in A549 cell lines using fluorescence and confocal microscopy. The miR146a delivered as miR-146a-NPs had a dose dependent effect of highest NPs concentrations 0.321 and 0.625 mg/ml and reduced target gene IRAK1 expression to 40%. In addition, IL-8 promoter reporter output (GFP) was dampened by miR-146a-NPs. In conclusion, miR-146a was successfully adsorbed onto PGA-co-PDL-DOTAP NPs and the miR-146a retained biological activity. Therefore, these results demonstrate the potential of PGA-co-PDL NPs as a delivery system for miR-146a to treat COPD.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have previously reported 27 differentially expressed microRNAs (miRNAs) during human monocyte differentiation into immature dendritic cells (imDCs) and mature DCs (mDCs). However, their roles in ...DC differentiation and function remain largely elusive. Here, we report that microRNA (miR)-146a and miR-146b modulate DC apoptosis and cytokine production. Expression of miR-146a and miR-146b was significantly increased upon monocyte differentiation into imDCs and mDCs. Silencing of miR-146a and/or miR-146b in imDCs and mDCs significantly prevented DC apoptosis, whereas overexpressing miR-146a and/or miR-146b increased DC apoptosis. miR-146a and miR-146b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 expression. Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs enhanced DC apoptosis. By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival. Moreover, silencing of miR-146a and miR-146b expression had little effect on DC maturation but enhanced IL-12p70, IL-6, and TNF-α production as well as IFN-γ production by IL-12p70-mediated activation of natural killer cells, whereas miR-146a and miR-146b overexpression in mDCs reduced cytokine production. Silencing of miR-146a and miR-146b in DCs also down-regulated NF-κB inhibitor IκBα and increased Bcl-2 expression. Our results identify a new negative feedback mechanism involving the miR-146a/b-TRAF6/IRAK1-NF-κB axis in promoting DC apoptosis.
Background: MicroRNA (miR)-146a and miR-146b (miR-146a/b) expression is induced upon human monocyte differentiation into dendritic cells (DCs).
Results: miR-146a/b negatively regulates DC apoptosis and cytokine production.
Conclusion: The miR-146a/b-TRAF6/IRAK1-NF-κB axis is responsible for DC apoptosis.
Significance: Our data reveal a novel negative feedback regulation in DCs and may have implications in the pathogenesis and treatment of autoimmune diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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