miRNA Target Research Articles

A comprehensive analysis of 3'UTRs in Caenorhabditis elegans.

3'Untranslated regions (3'UTRs) are essential portions of genes containing elements necessary for pre-mRNA 3'end processing and are involved in post-transcriptional gene regulation. Despite their importance, they remain poorly characterized in eukaryotes. Here, we have used a multi-pronged approach to extract and curate 3'UTR data from 11533 publicly available datasets, corresponding to the entire collection of Caenorhabditis elegans transcriptomes stored in the NCBI repository from 2009 to 2023. We have also performed high throughput cloning pipelines to identify and validate rare 3'UTR isoforms and incorporated and manually curated 3'UTR isoforms from previously published datasets. This updated C. elegans 3'UTRome (v3) is the most comprehensive resource in any metazoan to date, covering 97.4% of the 20362 experimentally validated protein-coding genes with refined and updated 3'UTR boundaries for 234893'UTR isoforms. We also used this novel dataset to identify and characterize sequence elements involved in pre-mRNA 3'end processing and update miRNA target predictions. This resource provides important insights into the 3'UTR formation, function, and regulation in eukaryotes.

Transcriptome and microRNAome elucidate the mechanism underlying the immunomodulatory effects of dietary CpG oligodeoxynucleotides (CpG ODNs) in Litopenaeus vannamei

Dietary supplementary of CpG oligodeoxynucleotides (CpG ODNs) could regulate the composition of gut microbiota and augment its immune function in Litopenaeus vannamei. However, the molecular mechanism of CpG ODNs remains poorly understood, particularly in the transcription level. In this study, transcriptome and microRNA sequencing were conducted to analyze the alterations of related genes and regulators in hepatopancreas of shrimp following the administration of CpG ODNs. The results revealed a total of 7820 differentially expressed genes (DEGs) and 573 differentially expressed miRNAs, indicating that the miRNAs targeted 1263 DEGs. These DEGs were found to be involved in the regulation of multiple immune-related signaling pathways, such as ubiquinone biosynthesis, folate biosynthesis, glutathione metabolism and alpha-linolenic acid metabolism, which were significantly enhanced upon stimulation with CpG ODNs. The pathway analysis of DEGs and differential miRNA target genes revealed significant alterations in the TGF-β signaling pathway, FoxO signaling pathway, mTOR signaling pathway, autophagy, lysosome function, ubiquitin-mediated proteolysis, and cytochrome P450 metabolism. It can be concluded that CpG ODNs induced activation of immune-related signaling pathways, thereby eliciting a robust immune response and augmenting overall immunity. These findings establish a robust theoretical framework for the practical implementation of CpG ODNs in the L. vannamei aquaculture industry.

Automated System for Attomolar-Level Detection of MiRNA as a Biomarker for Influenza A Virus.

We have developed an automated sensing system for the repeated detection of a specific microRNA (miRNA) of the influenza A (H1N1) virus. In this work, magnetic particles functionalized with DNAs, target miRNAs, and alkaline phosphate (ALP) enzymes formed sandwich structures. These particles were trapped on nickel (Ni) patterns of our sensor chip by an external magnetic field. Then, additional electrical signals from electrochemical markers generated by ALP enzymes were measured using the sensor, enabling the highly sensitive detection of target miRNA. The magnetic particles used on the sensor were easily removed by applying the opposite direction of external magnetic fields, which allowed us to repeat sensing measurements. As a proof of concept, we demonstrated the detection of miRNA-1254, one of the biomarkers for the H1N1 virus, with a high sensitivity down to 1 aM in real time. Moreover, our sensor could selectively detect the target from other miRNA samples. Importantly, our sensor chip showed reliable electrical signals even after six repeated miRNA sensing measurements. Furthermore, we achieved technical advances to utilize our sensor platform as part of an automated sensing system. In this regard, our reusable sensing platform could be utilized for versatile applications in the field of miRNA detection and basic research.

CsPbBr3 Perovskite Quantum Dots Encapsulated by a Polymer Matrix for Ultrasensitive Dynamic Imaging of Intracellular MicroRNA.

Herein, CsPbBr3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Target-Triggered Self-Assembly of Single-Band Red Emission of Upconversion Nanoparticle-Based Dual-Signal Fluorescent Probes for Sensitive and Accurate Determination of microRNAs

MicroRNAs (miRNAs) are small molecules with significant regulatory functions that have served as biomarkers for the early diagnosis of disease. Accordingly, it is purposeful to develop a rapid, targeted assay for the sensitive, selective, and determination of target miRNAs. Single-band red emission of upconversion nanoparticles (UCNPs) offer high photostability, large anti-Stokes shifts, and low background. Additionally, improving the accuracy via dual-signal fluorescence is also effective for reducing false positive signals induced by background. Herein, taking advantage of the single-band red emission of UCNPs and DNA complementary nanotechnologies, a target-triggered self-assembled dual-signal response biosensor via a strand displacement cascade is reported. The reported approach was used for the determination of miRNA-222 (a model analyte) in serum and the results were similar to those obtained by the polymerase chain reaction (PCR). The spiked recoveries were from 92.5 to 108.4%, indicating that this biosensor has potential for accurate and sensitive determination of miRNAs in biological samples.

A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employedto mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5pM. Furthermore, we successfully applied this approach to determinemiRNA-21 in cell lysatesandhuman serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

Photonic crystal-enhanced fluorescence biosensor with logic gate operation based on one-pot cascade amplification DNA circuit for enzyme-free and ultrasensitive analysis of two microRNAs

The development of sensitive and efficient analytical methods for multiple biomarkers is crucial for cancer screening at early stage. MicroRNAs (miRNAs) are a kind of biomarkers with diagnostic potential for cancer. However, the ultrasensitive and logical analysis of multiple miRNAs with simple operation still faces some challenges. Herein, a photonic crystal (PC)-enhanced fluorescence biosensor with logic gate operation based on one-pot cascade amplification DNA circuit was developed for enzyme-free and ultrasensitive analysis of two cancer-related miRNAs. The fluorescence biosensor was performed by biochemical recognition amplification module (BCRAM) and physical enhancement module (PEM) to achieve logical and sensitive detection. In the BCRAM, one-pot cascade amplification circuit consisted of the upstream parallel entropy-driven circuit (EDC) and the downstream shared catalytic hairpin assembly (CHA). The input of target miRNA would trigger each corresponding EDC, and the parallel EDCs released the same R strand for triggering subsequent CHA; thus, the OR logic gate was obtained with minimization of design and operation. In the PEM, photonic crystal (PC) array was prepared easily for specifically enhancing the fluorescence output from BCRAM by the optical modulation capabilities; meanwhile, the high-throughput signal readout was achieved by microplate analyzer. Benefiting from the integrated advantages of two modules, the proposed biosensor achieved ultrasensitive detection of two miRNAs with easy logic gate operation, obtaining the LODs of 8.6 fM and 6.7 fM under isothermal and enzyme-free conditions. Hence, the biosensor has the advantages of high sensitivity, easy operation, multiplex and high-throughput analysis, showing great potential for cancer screening at early stage.

BIOM-14. UTILITY OF MIRNA-BASED LIQUID BIOPSY AND RADIOGRAPHIC IMAGING IN PEDIATRIC BRAIN TUMOR PATIENTS

Abstract BACKGROUND MRI is the current gold standard imaging technique for diagnostic evaluation and monitoring of pediatric CNS tumors; however, MRI measures can only provide inference into tumor biology and molecular stratification. miRNAs are an abundant and stable nucleic acid found circulating in blood and cerebrospinal fluid (CSF) and can be utilized as a tumor biomarker. METHODS We hypothesized that correlating miRNA biomarkers with radiological tumor measurements may provide improved diagnostic and monitoring for patients with pediatric brain tumors. Using a cohort of 54 pediatric brain tumors including low grade glioma, ependymoma, germ cell tumor, medulloblastoma, atypical teratoid rhabdoid tumor and high-grade glioma we attempted to combine MRI findings and circulating miRNA data. The miRNA expression was profiled in 33 CSF and 52 plasma samples using the HTG EdgeSeq platform. Clinically acquired, multi-parametric MRI scans at time-points close to liquid biopsy collection were collected retrospectively and used to generate volumetric tumor segmentations. Machine learning methods were implemented to evaluate combinatory features. RESULTS We identified unique miRNA targets that significantly correlated with MRI features, clinical findings, and patient outcomes. In both CSF and plasma, miRNA expression was found to correlate with diagnosis and clinical features including tumor grade and disease burden (p < 0.05). In CSF, miRNA expression was correlated with MRI features including cystic core presence, edema, leptomeningeal disease and tumor location (p <0.05). In plasma, differences in miRNA expression were related to MRI measurements including cystic core volume, location, and leptomeningeal disease (p < 0.05). Combination of miRNA targets for plasma or CSF with imaging-based features significantly improved histology specific multimodal diagnosis predictions. CONCLUSIONS These results demonstrate the potential utility of miRNAs as a pediatric brain tumor biomarker which when combined with imaging features can improve minimally and non-invasive diagnostics and the subsequent management of pediatric brain tumor patients.

Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

MiR-540-3p partially recovers the locomotor function of spinal cord injury mice by targeting SIX4/Yap1 and inactivation of astrocytes

ABSTRACT Background Spinal cord injury (SCI) lacks therapeutic reagents. miRNAs are responsible for mesenchymal stem cells (MSCs) therapy in spinal cord injury. Purpose To discover the underlying therapeutic miRNA target and its mechanism for the treatment of SCI. Method Two RNA sequence datasets were retrieved from the GEO Datasets database which was accessed on 30 December 2023. The targets of the top 2 ranked miRNAs (miR-540-3p and miR-433-5p) were analyzed using online databases (miRDB, miRMap, TargetScan and STRING database) and both miRNAs were screened by cell counting kit-8 (CCK-8) assay. Then, transfection and local injection of miR-540-3p were performed to examine the capacity of secretion of astrocytes and the locomotor function of SCI mice. Results The significantly high levels of miR-540-3p/433-5p were revealed. Transfection of miR-540-3p conferred inactivation of reactive astrocytes and weakened the capacity of secreting inflammatory cytokines of astrocytes. miR-433-5p was proven to not impact the proliferation of astrocytes. Co-culture of culture supernate from astrocytes transfected with miR-540-3p and neurons demonstrated the significantly preserved neurite length and decreased apoptotic level of neurons. Meanwhile, sine oculis homeobox (SIX4)/Yap1, as the target of miR-540-3p, is critical for abrogating inflammatory damage of neurons in vivo and in vitro, decreasing glial scar, and recovering locomotor function of spinal cord injury mice. Furthermore, SCI mice receiving a local injection of miR-540-3p showed smaller and lighter bladder volume and higher limb strength, but the period from urinary retention to autonomous urination of SCI mice showed no significance. Conclusions Conclusively, miR-540 discovered from hypoxia-treated exosomes suppresses the inflammatory cytokines secreted by reactive astrocytes, partially preserves the neuronal function of spinal cord injury mice, through the SIX4/Yap1 signalling pathway.

Palindrome sequence mediated target recycling integrating with self-priming assisted signal reaction for sensitive miRNA detection

The development of a sensitive and isothermal technique with a greatly enhanced miRNA detection signal is still technically problematic due to the low abundance of miRNA and high sequence similarities with homologous miRNAs. Herein, we propose a novel fluorescence approach for sensitive and reliable miRNA detection by integrating the palindrome sequence mediated target recycling with self-priming assisted signal reaction. In this method, a dual toehold DNA nano-probe (HT) with two functional arms is designed to mediate specific target recognition and signal amplification. In the presence of target miRNA, it binds to the recognition module of HT probe, releasing the “2” sequence to initiate strand displacement amplification (SDA) and a self-priming-induced signal reaction. Based on the elegant design, the proposed method exhibits a wide linear response range exceeding five orders of magnitude and a low limit of detection of 0.96 fM according to the 3δ rule. The non-specific signal is below 5 % for non-target miRNA detection. Taking the merits of excellent sensitivity, desirable specificity, and superior anti-interference ability, the proposed approach shows a promising prospect for detecting miRNAs in complicated biological environments and early diagnosis of diseases.

Comprehensive analysis of clinical and biological value of ING family genes in liver cancer.

Liver cancer (LIHC) is a malignant tumor that occurs in the liver and has a high mortality in cancer. The ING family genes were identified as tumor suppressor genes. Dysregulated expression of these genes can lead to cell cycle arrest, senescence and/or apoptosis. ING family genes are promising targets for anticancer therapy. However, their role in LIHC is still not well understood. To have a better understanding of the important roles of ING family members in LIHC. A series of bioinformatics approaches (including gene expression analysis, genetic alteration analysis, survival analysis, immune infiltration analysis, prediction of upstream microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) of ING1, and ING1-related gene functional enrichment analysis) was applied to study the expression profile, clinical relationship, prognostic significance and immune infiltration of ING in LIHC. The relationship between ING family genes expression and tumor associated immune checkpoints was investigated in LIHC. The molecular mechanism of ING1 mediated hepatocarcinogenesis was preliminarily discussed. mRNA/protein expression of different ING family genes in LIHC was analyzed in different databases, showing that ING family genes were highly expressed in LIHC. In 47 samples from 366 LIHC patients, the ING family genes were altered at a rate of 13%. By comprehensively analyzing the expression, clinical pathological parameters and prognostic value of ING family genes, ING1/5 was identified. ING1/5 was related to poor prognosis of LIHC, suggesting that they may play key roles in LIHC tumorigenesis and progression. One of the target miRNAs of ING1 was identified as hsa-miR-214-3p. Two upstream lncRNAs of hsa-miR-214-3p, U91328.1, and HCG17, were identified. At the same time, we found that the expression of ING family genes was correlated with immune cell infiltration and immune checkpoint genes. This study lays a foundation for further research on the potential mechanism and clinical value of ING family genes in the treatment and prognosis of LIHC.

Computational Discovery of AngiomiRs and Evaluating the Synergistic Effects of Target miRNA with Tranexamic Acid in Colorectal Cancer.

microRNA (miRNA) levels are dysregulated in many cancers, suggesting that miRNA-based therapy may be effective. The molecular pathways of colorectal cancer (CRC) development are unknown. Understanding miRNAs implicated in CRC formation may reveal new diagnostic and therapeutic targets. Angiogenesis is a key mechanism in tumor growth. CRC treatment may involve inhibiting angiogenesis, but existing drugs can cause negative effects. Tranexamic acid, an FDA-approved medication, may reduce the adverse effects of angiogenesis inhibitors. This work examined miRNAs implicated in CRC angiogenesis and how miR-16 and tranexamic acid may synergistically decrease CRC cell migration and angiogenesis. We identified miRNAs targeting CRC angiogenesis genes using bioinformatic databases. Proteins were docked with tranexamic acid utilizing the PyRx software. Quantitative Real-time PCR was used to analyze the effects of overexpressed miRNA and tranexamic acid on the expression of target genes. Scratch, transwell migration, and Chicken Chorioallantoic Membrane (CAM) assays were used to evaluate the effect of selected miRNA and tranexamic acid on the invasion and angiogenesis of CRC cells. in silico studies identified hsa-miR-16-5p, -101-3p, and 34a-5p as possible CRC angiogenesis modulators. The study found that miR-16 and tranexamic acid influence the expression of VEGFA, ANGPT2, MMP9, and HIF1A. miR-16 and tranexamic acid influenced CRC cell movement in scratch tests and transwell migration assays. Furthermore, the CAM assay results demonstrated that miR-16 and tranexamic acid can alter angiogenesis in CRC. These findings highlight the potential of miR-16 and tranexamic acid as combination therapeutic agents for CRC, with the ability to simultaneously target tumorigenesis and angiogenesis.

Hsa_circ_0064636 regulates voltage dependent anion channel 1/ubiquitination factor E4A through miR‑326/miR‑503‑5 in osteosarcoma.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs that are important for the regulation of gene expression in eukaryotic organisms. CircRNAs exert various regulatory roles in cancer progression. However, the role of hsa_circ_0064636 in osteosarcoma (OS) remains poorly understood. In the present study, the expression of hsa_circ_0064636 in OS cell lines was measured by reverse transcription-quantitative PCR (RT-qPCR). Differentially expressed mRNAs and microRNAs (miRNA or miRs) were screened using mRNA(GSE16088) and miRNA(GSE65071) expression datasets for OS. miRNAs that can potentially interact with hsa_circ_0064636 were predicted using RNAhybrid, TargetScan and miRanda. Subsequently, RNAhybrid, TargetScan, miRanda, miRWalk, miRMap and miRNAMap were used for target gene prediction based on the overlapping miRNAs to construct a circ/miRNA/mRNA interaction network. Target genes were subjected to survival analysis using PROGgeneV2, resulting in a circRNA/miRNA/mRNA interaction sub-network with prognostic significance. miRNA and circRNA in the subnetwork may also have survival significance, but relevant data are lacking and needs to be further proved. RT-qPCR demonstrated that hsa_circ_0064636 expression was significantly increased in OS cell lines. miR-326 and miR-503-5p were identified to be target miRNAs of hsa_circ_0064636. Among the target genes obtained from the miR-326 and miR-503-5p screens, ubiquitination factor E4A (UBE4A) and voltage dependent anion channel 1 (VDAC1) were respectively identified to significantly affect prognosis; only miR-326 targets UBE4A and only miR-503 targets VDAC1. To conclude, these aforementioned findings suggest that hsa_circ_0064636 may be involved in the development of OS by sponging miR-503-5p and miR-326to inhibit their effects, thereby regulating the expression of VDAC1 and UBE4A.

Alteration of circulating ACE2-network related microRNAs in patients with COVID-19

Angiotensin converting enzyme 2 (ACE2) serves as the primary receptor for the SARS-CoV-2 virus and has implications for the functioning of the cardiovascular system. Based on our previously published bioinformatic analysis, in this study we aimed to analyze the diagnostic and predictive utility of miRNAs (miR-10b-5p, miR-124-3p, miR-200b-3p, miR-26b-5p, miR-302c-5p) identified as top regulators of ACE2 network with potential to affect cardiomyocytes and cardiovascular system in patients with COVID-19. The expression of miRNAs was determined through qRT-PCR in a cohort of 79 hospitalized COVID-19 patients as well as 32 healthy volunteers. Blood samples and clinical data of COVID-19 patients were collected at admission, 7-days and 21-days after admission. We also performed SHAP analysis of clinical data and miRNAs target predictions and advanced enrichment analyses. Low expression of miR-200b-3p at the seventh day of admission is indicative of predictive value in determining the length of hospital stay and/or the likelihood of mortality, as shown in ROC curve analysis with an AUC of 0.730 and a p-value of 0.002. MiR-26b-5p expression levels in COVID-19 patients were lower at the baseline, 7 and 21-days of admission compared to the healthy controls (P < 0.0001). Similarly, miR-10b-5p expression levels were lower at the baseline and 21-days post admission (P = 0.001). The opposite situation was observed in miR-124-3p and miR-302c-5p. Enrichment analysis showed influence of analyzed miRNAs on IL-2 signaling pathway and multiple cardiovascular diseases through COVID-19-related targets. Moreover, the COVID-19-related genes regulated by miR-200b-3p were linked to T cell protein tyrosine phosphatase and the HIF-1 transcriptional activity in hypoxia. Analysis focused on COVID-19 associated genes showed that all analyzed miRNAs are strongly affecting disease pathways related to CVDs which could be explained by their strong interaction with the ACE2 network.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Quantification of multiple microRNAs by microchip electrophoresis assisted by strand displacement amplification

MicroRNAs (miRNAs) are increasingly recognized as potential biomarkers for the early diagnosis of cancer. However, the concurrent detection of multiple miRNAs in biological samples presents a significant challenge due to their high homogeneity and low abundance. This study introduced a novel approach combining strand displacement amplification (SDA) with microchip electrophoresis (MCE) for the simultaneous quantitation of trace levels of three miRNAs associated with cancer: miRNA-21, miRNA-145, and miRNA-221. Specifically designed probes were utilized to selectively capture the target miRNAs, thereby initiating the SDA process in a single solution without cross-interference. Under optimized conditions, the SDA-MCE method achieved the limit of detection (LOD) as low as 0.02 fM (S/N = 3) and the limit of quantitation (LOQ) as low as 0.1 fM across a broad linear range spanning from 0.1 fM to 1 pM. The SDA reaction was completed in approximately 1.5 h, and all target products were separated within 135 s through MCE. Application of this method for the simultaneous detection of these three miRNAs in human lung cancer cell samples yielded satisfactory results. Featuring high sensitivity, rapid analysis, minimal reagent consumption, and straightforward operation, the proposed MCE-SDA strategy holds considerable promise for multi-miRNAs detection applications.

Distinct small RNAs are expressed at different stages of Phytophthora capsici and play important roles in development and pathogenesis.

Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici (P. capsici), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25-26nt in HY, SP, and ZO but distributed at 18-34nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici.

Uncovering periodontitis-associated markers through the aggregation of transcriptomics information from diverse sources.

Periodontitis, a common chronic inflammatory disease, significantly impacted oral health. To provide novel biological indicators for the diagnosis and treatment of periodontitis, we analyzed public microarray datasets to identify biomarkers associated with periodontitis. The Gene Expression Omnibus (GEO) datasets GSE16134 and GSE106090 were downloaded. We performed differential analysis and robust rank aggregation (RRA) to obtain a list of differential genes. To obtain the core modules and core genes related to periodontitis, we evaluated differential genes through enrichment analysis, correlation analysis, protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network analysis. Potential biomarkers for periodontitis were identified through comparative analysis of dual networks (PPI network and ceRNA network). PPI network analysis was performed in STRING. The ceRNA network consisted of RRA differentially expressed messenger RNAs (RRA_DEmRNAs) and RRA differentially expressed long non-coding RNAs (RRA_DElncRNAs), which regulated each other's expression by sharing microRNA (miRNA) target sites. RRA_DEmRNAs were significantly enriched in inflammation-related biological processes, osteoblast differentiation, inflammatory response pathways and immunomodulatory pathways. Comparing the core ceRNA module and the core PPI module, C1QA, CENPK, CENPU and BST2 were found to be the common genes of the two core modules, and C1QA was highly correlated with inflammatory functionality. C1QA and BST2 were significantly enriched in immune-regulatory pathways. Meanwhile, LINC01133 played a significant role in regulating the expression of the core genes during the pathogenesis of periodontitis. The identified biomarkers C1QA, CENPK, CENPU, BST2 and LINC01133 provided valuable insight into periodontitis pathology.

Real-Time Detection of Multiple Intracellular MicroRNAs using an Ultrasound-Propelled Nanomotor-Based Dynamic Fluorescent Probe.

Multiple intracellular microRNA (miRNA) detection is essential for disease diagnosis and management. Nonetheless, the real-time detection of multiple intracellular miRNAs has remained challenging. Herein, we have developed an ultrasound (US)-powered nanomotor-based dynamic fluorescent probe for the real-time OFF-ON fluorescent determination of multiple intracellular miRNAs. The new probe relies on the utilization of multicolored quantum dot (QD)-labeled single-stranded DNA (ssDNA)/graphene oxide (GO)-coated US-powered gold nanowire (AuNW) nanomotors. The fluorescence of QDs is quenched due to π-π interactions with the GO. Upon binding to target miRNAs, the QDs-ssDNA is now distant from the AuNWs, resulting in effective OFF-ON QD fluorescence switching. Compared with conventional passive probes, the dynamic fluorescent probe enhances probe-target interactions by using the US-propelled nanomotor, resulting in exceptionally efficient and prompt hybridization. Simultaneous quantitative analysis of miR-10b and miR-21 in vitro can be achieved within 15 min with high sensitivity and specificity. Additionally, multicolor QDs provide strong signal intensity and multiplexed detection, enabling one-step real-time discrimination between cancer cells (A549) and normal cells (L02). The obtained results are in good agreement with those from qRT-PCR. This dynamic fluorescent probe based on a nanomotor and QDs enables rapid "on the move" specific detection of multiple intracellular miRNAs in intact cells, facilitating real-time monitoring of diverse intracellular miRNA expression, and it could pave the way for novel applications of nanomotors in biodetection.