Challenges and opportunities for circRNA identification and delivery.

Circular RNAs (circRNAs) are evolutionarily conserved noncoding RNAs with tissue-specific expression patterns, and exert unique cellular functions that have the potential to become biomarkers in therapeutic applications. Therefore, accurate and sensitive detection of circRNA with facile platforms is essential for better understanding of circRNA biological processes and circRNA-related disease diagnosis and prognosis; and precise regulation of circRNA through efficient delivery of circRNA or siRNA is critical for therapeutic purposes. Here, we reviewed the current development of circRNA identification methodologies, including overviewing the purification steps, summarizing the sequencing methods of circRNA, as well as comparing the advantages and disadvantages of traditional and new detection methods. Then, we discussed the delivery and manipulation strategies for circRNAs in both research and clinic treatment. Finally, the challenges and opportunities of analyzing circRNAs were addressed.

A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

AGO2 and its partners: a silencing complex, a chromatin modulator, and new features.

AGO2 is the only member with catalytic activity in Argonaute family and contains four functional core domains, which are N domain, PAZ domain, MID domain, and PIWI domain from N-terminal to C-terminal. In traditional view, AGO2 serves as the catalytic engine of the RNA induced silencing complex and plays an important role in small RNAs guided post transcriptional gene silencing, including mRNA degradation and translational repression. Moreover, AGO2 also plays multiple roles in gene regulation processes in nuclei, such as chromatin remodeling, transcriptional repression and activation, double-strand break repair and alternative splicing. Recent studies have also implicated AGO2 in several other cellular processes, including alternative polyadenylation, translational activation, and transposon repression.

Brain-Penetration and Neuron-Targeting DNA Nanoflowers Co-Delivering miR-124 and Rutin for Synergistic Therapy of Alzheimer's Disease.

Alzheimer disease (AD) is the leading cause of dementia that affects millions of old people. Despite significant advances in the understanding of AD pathobiology, no disease modifying treatment is available. MicroRNA-124 (miR-124) is the most abundant miRNA in the normal brain with great potency to ameliorate AD-like pathology, while it is deficient in AD brain. Herein, the authors develop a DNA nanoflowers (DFs)-based delivery system to realize exogenous supplementation of miR-124 for AD therapy. The DFs with well-controlled size and morphology are prepared, and a miR-124 chimera is attached via hybridization. The DFs are further modified with RVG29 peptide to simultaneously realize brain-blood barrier (BBB) penetration and neuron targeting. Meanwhile, Rutin, a small molecular ancillary drug, is co-loaded into the DFs structure via its intercalation into the double stranded DNA region. Interestingly, Rutin could synergize miR-124 to suppress the expression of both BACE1 and APP, thus achieving a robust inhibition of amyloid ß generation. The nanosystem could pro-long miR-124 circulation in vivo, promote its BBB penetration and neuron targeting, resulting in a significant increase of miR-124 in the hippocampus of APP/PS1 mice and robust therapeutic efficacy in vivo. Such a bio-derived therapeutic system shows promise as a biocompatible nanomedicine for AD therapy.

Traditional and new applications of the HCR in biosensing and biomedicine.

The hybridization chain reaction is a very popular isothermal nucleic acid amplification technology. A single-stranded DNA initiator triggers an alternate hybridization event between two hairpins forming a double helix polymer. Due to isothermal, enzyme-free and high amplification efficiency characteristics, the HCR is often used as a signal amplification technology for various biosensing and biomedicine fields. However, as an enzyme-free self-assembly reaction, it has some inevitable shortcomings of relatively slow kinetics, low cell internalization efficiency, weak biostability of DNA probes and uncontrollable reaction in these applications. More and more researchers use this reaction system to synthesize new materials. New materials can avoid these problems skillfully by virtue of their inherent biological characteristics, molecular recognition ability, sequence programmability and biocompatibility. Here, we summarized the traditional application of the HCR in biosensing and biomedicine in recent years, and also introduced its new application in the synthesis of new materials for biosensing and biomedicine. Finally, we summarized the development and challenges of the HCR in biosensing and biomedicine in recent years. We hope to give readers some enlightenment and help.

Tandem DNAzyme for double digestion: a new tool for circRNA suppression.

Circular RNA (circRNA) play a crucial role in many biological processes and have been proved as potential biomarkers and therapeutic targets in many diseases. Manipulation of their expression is a critical task. In this study, we developed a new strategy for circRNA suppression with DNAzyme. Data showed single-digestion DNAzymes cleaved circRNA efficiently in vitro but not in cell culture. However, tandem DNAzymes for double digestion showed higher cleavage efficacy both in vitro and in cell culture. Functional study demonstrated that double-digestion DNAzymes suppressed the miRNA sponge function of circRNA and changed the proliferation and migration rates of HCC cells.

Super enhancers at the miR-146a and miR-155 genes contribute to self-regulation of inflammation.

Inflammatory response is essential to host defense and repair, and requires tight regulation as excessive and constant inflammatory response is deleterious. We recently identified that one of the general but key mechanisms for inflammatory gene transcription regulation is controlled by the formation of super enhancers mediated by NF-κB, and bromodomain and extraterminal (BET) proteins. Given that microRNA transcription shares a similar mechanism to mRNA, we assume that the inflammatory microRNAs transcription could be NF-κB and BET bromodomain dependent. In the present study, we confirmed that inflammatory stimuli changed human umbilical vein endothelial cells (HUVEC) microRNA profile. Among these microRNAs, miR-146a and miR-155, two well-established inflammatory microRNAs, are both downregulated at transcriptional level by NF-κB and BET bromodomain inhibition. To pursue this mechanism, we analyzed the ChIP-seq data and found that NF-κB, BRD4 and RNA POL II were rapidly distributed at the upstream regions of miR-146a and miR-155, and more importantly mediated the formation of the super enhancers that drive miR-146a and miR-155 transcription. These microRNAs transcription driven by super enhancers in turn downregulate both in vitro and in vivo canonical inflammatory genes expression through targeting inflammatory mediators. This novel finding demonstrated how the host self-regulates inflammatory genes expression at both transcriptional and post-transcriptional level to ensure the appropriate level of the host inflammatory response.

Simotinib as a modulator of P-glycoprotein: substrate, inhibitor, or inducer?

As a new antitumor drug, simotinib hydrochloride is prescribed for prolonged periods, often to patients with comorbidities. Therefore, the risk for developing drug resistance and drug-drug interactions between simotinib and other agents has to be taken into consideration. As P-glycoprotein (P-gp) is an efflux transporter, which plays a significant role in drug resistance and influences the pharmacological properties and toxicities of the drugs it interacts with, the interactions between simotinib and P-gp were investigated. Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular drug concentrations were detected by high-performance liquid chromatography, fluorescence-activated cell sorting and using a fluorescence reader. P-gp ATPase activity was measured using the Pgp-Glo assay, and intracellular pH was assessed using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl. The expression and transcription of P-gp were detected by western blotting and the luciferase assay. Simotinib has no cross-resistance to P-gp substrates, and its efflux rate was independent of either the P-gp expression or the coadministered P-gp substrate. Simotinib reversed chemotherapeutic agent resistance in a short time by increasing the intracellular concentration of the chemotherapeutic agent and blocked rhodamine 123 efflux. Further studies demonstrated that simotinib inhibited P-gp activity by modulating its ATPase activity and the intracellular pH. Although simotinib induced P-gp expression after extended treatment, the induced expression of P-gp had little impact on drug resistance. Simotinib is not a substrate of P-gp. As a modulator, it functions mainly as an inhibitor of P-gp by modulating the intracellular pH and ATPase activity, although it also induces P-gp expression after extended treatment.

MiR-183/-96/-182 cluster is up-regulated in most breast cancers and increases cell proliferation and migration.

INTRODUCTION: The miR-183/-96/-182 cluster is a conserved polycistronic microRNA (miRNA) cluster which is highly expressed in most breast cancers. Although there are some sporadic reports which demonstrate the importance of each miRNA in this cluster in breast cancer, the biological roles of this cluster as a whole and its regulation mechanisms in breast cancer are still unclear. We compared the expression of this cluster in different cancer types, analyzed the regulation mechanism of this cluster, identified new target genes, and examined the impact of this cluster on breast cancer cells. METHODS: The miRNA level was detected by LNA-based northern blot and Real-time PCR, and was also analyzed from TCGA dataset. Bioinformatics research and luciferase assay were applied to find the promoter regions and transcription factors. To investigate the biological effects of the miR-183/-96 /-182 cluster in breast cancer, we generated miR-96, miR-182 and miR-183 overexpression stable cell lines to check the overdose effects; we also used miR-Down™ antagomir for each miRNA as well as miR-183/-96 /-182 cluster sponge lentivirus to check the knockdown effects. Growth, migration, cell cycle profile and survival of these cells was then monitored by colony formation assay, MTT assay, cell wound healing assay, flow cytometry and microscopy. The target gene was validated by Real-time PCR, luciferase assay, Western blot and Phalloidin/DAPI counterstaining. RESULTS: The miR-183/-96/-182 cluster was highly expressed in most breast cancers, and its transcription is disordered in breast cancer. The miR-183/-96/-182 cluster was transcribed in the same pri-miRNA and its transcription was regulated by ZEB1 and HSF2. It increased breast cell growth by promoting more rapid completion of mitosis, promoted cell migration and was essential for cell survival. MiR-183 targeted the RAB21 mRNA directly in breast cancer. CONCLUSION: The miR-183/-96/-182 cluster is up-regulated in most breast cancer. It functions as an oncogene in breast cancer as it increases cell proliferation and migration.

New advances in signal amplification strategies for DNA methylation detection in vitro.

5-methylcytosine (5 mC) DNA methylation is a prominent epigenetic modification ubiquitous in the genome. It plays a critical role in the regulation of gene expression, maintenance of genome stability, and disease control. The potential of 5 mC DNA methylation for disease detection, prognostic information, and prediction of response to therapy is enormous. However, the quantification of DNA methylation from clinical samples remains a considerable challenge due to its low abundance (only 1% of total bases). To overcome this challenge, scientists have recently developed various signal amplification strategies to enhance the sensitivity of DNA methylation biosensors. These strategies include isothermal nucleic acid amplification and enzyme-assisted target cycling amplification, among others. This review summarizes the applications, advantages, and limitations of these signal amplification strategies over the past six years (2018-2023). Our goal is to provide new insights into the selection and establishment of DNA methylation analysis. We hope that this review will offer valuable insights to researchers in the field and facilitate further advancements in this area.

Quantitative measurement of acute myocardial infarction cardiac biomarkers by "All-in-One" immune microfluidic chip for early diagnosis of myocardial infarction.

Acute myocardial infarction (AMI) is a life-threatening condition with a narrow treatment window, necessitating rapid and accurate diagnostic methods. We present an "all-in-one" convenient and rapid immunoassay system that combines microfluidic technology with a colloidal gold immunoassay. A degassing-driven chip replaces a bulky external pump, resulting in a user-friendly and easy-to-operate immunoassay system. The chip comprises four units: an inlet reservoir, an immunoreaction channel, a waste pool, and an immunocomplex collection chamber, allowing single-channel flow for rapid and accurate AMI biomarker detection. In this study, we focused on cardiac troponin I (cTnI). With a minimal sample of just 4 µL and a total detection time of under 3 min, the chip enabled a quantitative visual analysis of cTnI concentration within a range of 0.5 âˆ¼ 60.0 ng mL-1. This all-in-one integrated microfluidic chip with colloidal gold immunoassay offers a promising solution for rapid AMI diagnosis. The system's portability, small sample requirement, and quantitative visual detection capabilities make it a valuable tool for AMI diagnostics.

Nucleostemin inhibits TRF1 dimerization and shortens its dynamic association with the telomere.

TRF1 is a key component of the telomere-capping complex and binds double-strand telomeric DNA as homodimers. So far, it is not clear whether TRF1 dimerization coincides with its telomere binding or is actively controlled before it binds the telomere, and in the latter case, how this event might affect its telomere association. We previously found that TRF1 dimerization and its telomere binding can be increased by GNL3L, which is the vertebrate paralogue of nucleostemin (NS). Here, we show that NS and GNL3L bind TRF1 directly but competitively through two separate domains of TRF1. In contrast to GNL3L, NS prevents TRF1 dimerization through a mechanism not determined by its ability to displace TRF1-bound GNL3L. Furthermore, NS is capable of shortening the dynamic association of TRF1 with the telomere in normal and TRF2(ΔBΔM)-induced telomere-damaged cells without affecting the amount of telomere-bound TRF1 proteins in vivo. Importantly, NS displays a protective function against the formation of telomere-dysfunction-induced foci. This work demonstrates that TRF1 dimerization is actively and oppositely regulated by NS and GNL3L extrachromosomally. Changing the relative amount of TRF1 monomers versus dimers in the nucleoplasm might affect the dynamic association of TRF1 with the telomere and the repair of damaged telomeres.

Circular RNAs: Emerging roles and new insights in human cancers.

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules formed by mRNA exon back-splicing. Although the circRNA functions remain largely unknown, their currently known biological activities include: acting as competing endogenous RNA (ceRNA) to adsorb microRNA (miRNA), binding proteins, regulating transcription or splicing, and ability to be translated into proteins or peptides. A growing number of studies have found that many circRNAs are abnormally expressed in various cancers, and their dysregulation is highly correlated with tumor progression. Therefore, diagnosis and treatment using circRNAs as biomarkers and therapeutic targets, respectively, has gradually become an attractive research topic. In this review, we introduced the canonical biogenesis pathways and degradation mechanisms of circRNAs. In addition, we examined the biological functions of circRNAs in vivo. Finally, we discussed the current clinical applications and challenges faced by circRNA, and proposed future directions for this promising research field.

Application of intelligent responsive DNA self-assembling nanomaterials in drug delivery.

Smart nanomaterials are nano-scaled materials that respond in a controllable and reversible way to external physical or chemical stimuli. DNA self-assembly is an effective way to construct smart nanomaterials with precise structure, diverse functions and wide applications. Among them, static structures such as DNA polyhedron, DNA nanocages and DNA hydrogels, as well as dynamic reactions such as catalytic hairpin reaction, hybridization chain reaction and rolling circle amplification, can serve as the basis for building smart nanomaterials. Due to the advantages of DNA, such as good biocompatibility, simple synthesis, rational design, and good stability, these materials have attracted increasing attention in the fields of pharmaceuticals and biology. Based on their specific response design, DNA self-assembled smart nanomaterials can deliver a variety of drugs, including small molecules, nucleic acids, proteins and other drugs; and they play important roles in enhancing cellular uptake, resisting enzymatic degradation, controlling drug release, and so on. This review focuses on different assembly methods of DNA self-assembled smart nanomaterials, therapeutic strategies based on various intelligent responses, and their applications in drug delivery. Finally, the opportunities and challenges of smart nanomaterials based on DNA self-assembly are summarized.

Biosensors based on functional nucleic acids and isothermal amplification techniques.

In the past few years, with the in-depth research of functional nucleic acids and isothermal amplification techniques, their applications in the field of biosensing have attracted great interest. Since functional nucleic acids have excellent flexibility and convenience in their structural design, they have significant advantages as recognition elements in biosensing. At the same time, isothermal amplification techniques have higher amplification efficiency, so the combination of functional nucleic acids and isothermal amplification techniques can greatly promote the widespread application of biosensors. For the purpose of further improving the performance of biosensors, this review introduces several widely used functional nucleic acids and isothermal amplification techniques, as well as their classification, basic principles, application characteristics, and summarizes their important applications in the field of biosensing. We hope to provide some references for the design and construction of new tactics to enhance the detection sensitivity and detection range of biosensing.

Trehalose in Biomedical Cryopreservation-Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems.

Cells and tissues are the foundation of translational medicine. At present, one of the main technological obstacles is their preservation for long-term usage while maintaining adequate viability and function. Optimized storage techniques must be developed to make them safer to use in the clinic. Cryopreservation is the most common long-term preservation method to maintain the vitality and function of cells and tissues. But, the formation of ice crystals in cells and tissues is considered to be the main mechanism that could harm cells and tissues during freezing and thawing. To reduce the formation of ice crystals, cryoprotective agents (CPAs) must be added to the cells and tissues to achieve the cryoprotective effect. However, conventional cryopreservation of cells and tissues often needs to use toxic organic solvents as CPAs. As a result, cryopreserved cells and tissues may need to go through a time-consuming washing process to remove CPAs for further applications in translational medicine, and multiple valuable cells are potentially lost or killed. Currently, trehalose has been researched as a nontoxic CPA due to its cryoprotective ability and stability during cryopreservation. Nevertheless, trehalose is a nonpermeable CPA, and the lack of an effective intracellular trehalose delivery method has become the main obstacle to its use in cryopreservation. This article illustrated the properties, mechanisms, delivery methods, and applications of trehalose, summarized the benefits and limits of trehalose, and summed up the findings and research direction of trehalose in biomedical cryopreservation.

CPSF3 Promotes Pre-mRNA Splicing and Prevents CircRNA Cyclization in Hepatocellular Carcinoma.

CircRNAs are crucial in tumorigenesis and metastasis, and are comprehensively downregulated in hepatocellular carcinoma (HCC). Previous studies demonstrated that the back-splicing of circRNAs was closely related to 3'-end splicing. As a core executor of 3'-end cleavage, we hypothesized that CPSF3 modulated circRNA circularization. Clinical data were analyzed to establish the prognostic correlations. Cytological experiments were performed to determine the role of CPSF3 in HCC. A fluorescent reporter was employed to explore the back-splicing mechanism. The circRNAs regulated by CPSF3 were screened by RNA-seq and validated by PCR, and changes in downstream pathways were explored by molecular experiments. Finally, the safety and efficacy of the CPSF3 inhibitor JTE-607 were verified both in vitro and in vivo. The results showed that CPSF3 was highly expressed in HCC cells, promoting their proliferation and migration, and that a high CPSF3 level was predictive of a poor prognosis. A mechanistic study revealed that CPSF3 enhanced RNA cleavage, thereby reducing circRNAs, and increasing linear mRNAs. Furthermore, inhibition of CPSF3 by JET-607 suppressed the proliferation of HCC cells. Our findings indicate that the increase of CPSF3 in HCC promotes the shift of pre-mRNA from circRNA to linear mRNA, leading to uncontrolled cell proliferation. JTE-607 exerted a therapeutic effect on HCC by blocking CPSF3.

Rational design of nonlinear hybridization immunosensor chain reactions for simultaneous ultrasensitive detection of two tumor marker proteins.

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. The nonlinear hybridization chain reaction (NHCR) is widely used as an output signal amplification technique for biosensor platforms. A novel hairpin-free NHCR was developed in this study as a flow cytometric immunoassay to detect alpha-fetoprotein (AFP) and prostate specific antigen (PSA). First, the target AFP is captured on magnetic beads (MBs) that are modified with capture antibodies. Then, the prepared biotin-streptavidin-biotin (B-S-B) system, which links biotinylated detection antibodies and biotinylated trigger DNA together through the high affinity between biotin-streptavidin interaction, is added to label the target AFP, forming a sandwich complex with three trigger DNA chains. Each trigger DNA chain grows a dendritic DNA nanostructure following a nonlinear hybridization chain reaction. As the substrate flue chains are labeled with fluorophores, the self-assembly process of dendritic DNA is accompanied by the continuous release of fluorophores. Dendrites with strong fluorescence then form on the surface of MBs. Finally, the target AFP is quantified by analyzing the fluorescent MBs using flow cytometry. The proposed immunoassay has a high selectivity along with isothermal, enzyme-free, and exponential amplification efficiency. It shows a limit of detection (LOD) of 1.74 pg mL-1. The proposed biosensor was also successfully used to quantitatively detect AFP in serum samples. It may be utilized to detect multiple tumor markers simultaneously by changing the size of MBs and antibody-antigen pairs. Most tumor markers are only related to tumor diagnosis but without specificity, so the combined detection of multiple tumor markers can improve the accuracy of early tumor diagnoses.

Sponge transgenic mouse model reveals important roles for the microRNA-183 (miR-183)/96/182 cluster in postmitotic photoreceptors of the retina.

MicroRNA-183 (miR-183), miR-96, and miR-182 comprising the miR-183/96/182 cluster are highly expressed in photoreceptor cells. Although in vitro data have indicated an important role for this cluster in the retina, details of its in vivo biological activity are still unknown. To observe the impact of the miR-183/96/182 cluster on retinal maintenance and light adaptation, we generated a sponge transgenic mouse model that disrupted the activities of the three-component microRNAs simultaneously and selectively in the retina. Although our morphological and functional studies showed no differences between transgenic and wild type mice under normal laboratory lighting conditions, sponge transgenic mice displayed severe retinal degeneration after 30 min of exposure to 10,000 lux light. Histological studies showed that the outer nuclear layer thickness was dramatically reduced in the superior retina of transgenic mice. Real time PCR experiments in both the sponge transgenic mouse model and different microRNA stable cell lines identified Arrdc3, Neurod4, and caspase-2 (Casp2) as probable downstream targets of this cluster, a result also supported by luciferase assay and immunoblotting analyses. Further studies indicated that expression of both the cluster and Casp2 increased in response to light exposure. Importantly, Casp2 expression was enhanced in transgenic mice, and inhibition of Casp2 partially rescued their light-induced retinal degeneration. By connecting the microRNA and apoptotic pathways, these findings imply an important role for the miR-183/96/182 cluster in acute light-induced retinal degeneration of mice. This study demonstrates a clear involvement of miRs in the physiology of postmitotic cells in vivo.

Mulberry leaf polyphenols alleviated high-fat diet-induced obesity in mice.

Mulberry leaf is an important medicinal food plant, which is rich in polyphenol compounds. Mulberry leaf polyphenols (MLP) possess significant lipid-lowering and antioxidant effects, and healthcare functions. In this study, the polyphenol content of mulberry leaf ethanol extract was measured using HPLC. The analysis of mulberry leaf extract resulted in the identification of 14 compounds, of which Chlorogenic acid and Quercitrin were the highest. A high-fat diet (HFD)-induced obese mouse model was developed and treated with MLP for 12 weeks to explore their effect on lipid metabolism in HFD-induced obese mice. The results showed that the MLP could inhibit the weight gain and fat cell volume increase in the HFD-induced obese mice in a dose-dependent manner. Further analysis revealed that the MLP decelerated the fatty acid composition in the adipose tissues of HFD-induced obese mice, and significantly increased the polyunsaturated-to-saturated fatty acid (PUFA/SFA) ratio. The real-time quantitative PCR (RT-qPCR) results indicated that the MLP significantly inhibited the down regulation of uncoupling protein (UCP) 1 (UCP1), UCP3, and PR domain zinc finger protein 16 (PRDM16) caused by the HFD. These beneficial effects of MLP on HFD-induced obese mice might be attributed to their ability to change the fatty acid composition of adipose tissue and increase the expression of thermogenesis genes. Overall, the study results suggested that the MLP could serve as potential lipid-lowering and weight-loss functional food and healthcare products.