microRNAs Research Articles

Apoptotic vesicles (apoVs) derived from fibroblast-converted hepatocyte-like cells effectively ameliorate liver fibrosis

Liver fibrosis is a serious global health issue for which effective treatment remains elusive. Chemical-induced hepatocyte-like cells (ciHeps) have emerged as an appealing source for cell transplantation therapy, although they present several challenges such as the risk of lung thromboembolism or hemorrhage. Apoptotic vesicles (apoVs), small membrane vesicles generated during the apoptosis process, have gained attention for their role in regulating various physiological and pathological processes. In this study, we generated ciHep-derived apoVs (ciHep-apoVs) and investigated their therapeutic potential in alleviating liver fibrosis. Our findings revealed that ciHep-apoVs induced the transformation of macrophages into an anti-inflammatory phenotype, effectively suppressed the activity of activated hepatic stellate cells (aHSCs), and enhanced the survival of hepatocytes. When intravenously administered to mice with liver fibrosis, ciHep-apoVs were primarily engulfed by macrophages and myofibroblasts, leading to a reduction in liver inflammation and fibrosis. Proteomic and miRNA analyses showed that ciHep-apoVs were enriched in various functional molecules that modulate crucial cellular processes, including metabolism, signaling transduction, and ECM-receptor interactions. ciHep-apoVs effectively suppressed aHSCs activity through the synergistic inhibition of glycolysis, the PI3K/AKT/mTOR pathway, and epithelial-to-mesenchymal transition (EMT) cascades. These findings highlight the potential of ciHep-apoVs as multifunctional nanotherapeutics for liver fibrosis and provide insights into the treatment of other liver diseases and fibrosis in other organs.Graphical abstract

Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury.

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical-fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.

Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA

Given the critical role of miRNAs in regulating gene expression and their potential as biomarkers for various diseases, accurate and sensitive miRNA detection is essential for early diagnosis and monitoring of conditions such as cancer. In this study, we introduce a dimeric molecular beacon (Di-MB) based isothermal strand displacement amplification (ISDA) system (Di-MB-ISDA) for enhanced miRNA detection. The Di-MB system is composed of two monomeric MBs (Mono-MBs) connected by a double-stranded DNA linker with single-stranded sequences in the middle, facilitating binding with the flexible arms of the Mono-MBs. This design forms a compact, high-density structure, significantly improving biostability against nuclease degradation. In the absence of target miRNA, the Di-MB maintains its stable structure. When target miRNA is present, it binds to the stem-loop regions, causing the hairpin structure to unfold and expose the stem sequences. These sequences serve as templates for the built-in primers, triggering DNA replication through an intramolecular recognition mechanism. This spatial confinement effect accelerates the strand displacement reaction, allowing the target miRNA to initiate additional reaction cycles and amplify the detection signal. The Di-MB-ISDA system addresses key challenges such as poor biostability and limited sensitivity seen in traditional methods. By enhancing biostability and optimizing reaction conditions, this system demonstrates robust performance for miRNA detection with a detection limit of 100 pM. The findings highlight the potential of Di-MB-ISDA for sensitive and accurate miRNA analysis, paving the way for its application in biomedical study and disease diagnosis in complex biological samples.

CROSSTALK OF CONSERVATION OF SEQUENCES OF miRNAs AND ENZYMATIC MACHINERY OF miRNAs PRODUCTION IN AMPULLARIIDAE

BACKGROUND: The Ampullariidae family of molluscs is an emerging model for evolutionary studies due to its high diversity, ancient history and wide geographic distribution. miRNAs are essential for the development of the organism, as they help and act in the control of gene expression. The analysis of miRNAs in molluscs, especially in the family Ampullariidae, is especially important due to the low representativeness of analyzed species and the possibility of analyzing the conservation of miRNAs among ampullariids species. OBJECTIVE: Identification and characterization of miRNAs (precursors and matures) and their processing pathway genes. METHODS: Computational prediction and characterization of miRNAs and genes involved in the miRNA pathway were performed using public database of Lanistes nyassanus, Marisa cornuarietis, Pomacea canaliculata and Pomacea maculata. The in silico analysis was performed using a robust algorithm to identify and characterize miRNAs and their precursors in genome of ampullariids species. To search for the putative proteins involved in the miRNA biogenesis the putative proteomes from 4 Ampullariids species and Blastp tool were used. Characterization of conserved protein domains was performed using the PFAM and CDD. Phylogenetic analyzes were performed for the ampullariids miRNA precursors and their orthologs and also for the putative ampullariids proteins involved in the miRNA pathway and their orthologs using MEGA program. FINDINGS: 141 pre-miRNAs and 162 mature miRNAs were identified in the genome of L. nyassanus, 279 pre-miRNAs and 297 mature miRNAs in the genome of M. cornuarietis, 269 pre-miRNAs and 296 mature miRNAs in the genome of P. canaliculata and 299 pre-miRNAs and 316 mature miRNAs in the genome of P. maculata. We identified and characterized 24 putative key proteins involved in the miRNA pathway including Argonaute, DICER, DROSHA and EXPORTIN protein families in the predicted proteome of the 4 ampullariids. The data obtained in this work will support studies of phylogeny, population divergence, speciation and patterns of diversity in the Ampullariidae Family. MAIN CONCLUSIONS: The searching for novel miRNAs and their processing pathway genes in 4 species of ampullariids was able to predict new structures expanding the study of miRNAs in molluscs and in Ampullariidae family, as well as open an avenue to study the roles of miRNAs in the organisms.

Integrated transcriptome and miRNA analysis provides insight into the floral buds dormancy in Prunus armeniaca

Integrated transcriptome and miRNA analysis provides insight into the floral buds dormancy in Prunus armeniaca

Transcriptomic analysis of mRNA and miRNA reveals new insights into the regulatory mechanisms of Anadara granosa responses to heat stress

Temperature fluctuations resulting from climate change and global warming pose significant threats to various species. The blood clam, Anadara granosa, a commercially important marine bivalve, predominantly inhabits intertidal mudflats that are especially susceptible to elevated temperatures. This vulnerability has led to noticeable declines in the survival rates of A. granosa larvae, accompanied by an increase in malformations. Despite these observable trends, there is a lack of comprehensive research on the regulatory mechanisms underlying A. granosa's responses to heat stress. In this study, we examined the survival rates of A. granosa under varying high temperature conditions, selecting 34 °C as heat stress temperature. Enzyme activity assays have shed light on A. granosa's adaptive response to heat stress, revealing its ability to maintain redox balance and transition from aerobic to anaerobic metabolic pathways. Subsequently, mRNA and miRNA transcriptome analyses were conducted, elucidating several key responses of A. granosa to heat stress. These responses include the upregulation of transcription and protein synthesis, downregulation of proteasome activity, and metabolic pattern adjustments. Furthermore, we identified miRNA-mRNA networks implicated in heat stress responses, potentially serving as valuable candidate markers for A. granosa's heat stress response. Notably, we validated the involvement of agr-miR-3199 in A. granosa's heat stress response through its regulation of the target gene Foxj1. These findings not only deepen our understanding of the molecular mechanisms underlying the blood clam's response to heat stress but also offer valuable insights for enhancing heat stress resilience in the blood clam aquaculture industry. Moreover, they contribute to improved cultivation strategies for molluscs in the face of global warming and have significant implications for the conservation of marine resources and the preservation of ecological balance.

Dumbbell probe initiated multi-rolling circle amplification assisted CRISPR/Cas12a for highly sensitive detection of clinical microRNA

A novel miRNA detection technique named Dumbbell probe initiated multi-Rolling Circle Amplification assisted CRISPR/Cas12a (DBmRCA) was developed relying on the ligation-free dumbbell probe and the high-sensitivity CRISPR/Cas12a signal out strategy. This DBmRCA assay streamlines miRNA quantification within a mere 30-min timeframe and with exceptional analytical precision. The efficacy of this method was validated by assessing miRNA levels in clinical samples, revealing distinct expression panel of miR-200a and miR-126 in lung cancer/adjacent/normal tissue specimens. Moreover, a predictive model was established to classify benign and malignant tumor. Due to its time efficiency, enhanced sensitivity, and streamlined workflow, this assay would be a reliable tool for miRNA analysis in clinical settings, offering potential guidance for early diagnosis and treatment of lung cancer.

The Untapped Biomarker Potential of MicroRNAs for Health Risk-Benefit Analysis of Vaping vs. Smoking.

Despite the popularity of electronic cigarettes (e-cigs) among adolescent never-smokers and adult smokers seeking a less pernicious substitute for tobacco cigarettes, the long-term health impact of vaping is largely unknown. Like cigarette smoke, e-cig vapor contains harmful and potentially harmful compounds, although in fewer numbers and at substantially lower concentrations. Many of the same constituents of e-cig vapor and cigarette smoke induce epigenetic changes that can lead to the dysregulation of disease-related genes. MicroRNAs (MiRNAs) are key regulators of gene expression in health and disease states. Extensive research has shown that miRNAs play a prominent role in the regulation of genes involved in the pathogenesis of smoking-related diseases. However, the use of miRNAs for investigating the disease-causing potential of vaping has not been fully explored. This review article provides an overview of e-cigs as a highly consequential electronic nicotine delivery system, describes trends in e-cig use among adolescents and adults, and discusses the ongoing debate on the public health impact of vaping. Highlighting the significance of miRNAs in cell biology and disease, it summarizes the published and ongoing research on miRNAs in relation to gene regulation and disease pathogenesis in e-cig users and in vitro experimental settings. It identifies gaps in knowledge and priorities for future research while underscoring the need for empirical evidence that can inform the regulation of tobacco products to protect youth and promote public health.

Circulating microRNAs as biomarkers for stratifying different phases of liver cancer progression and response to therapy

Hepatocellular carcinoma (HCC) is the most common liver cancer and is among the leading causes of cancer-related death worldwide. There is no reliable biomarker for the early diagnosis of HCC. Circulating microRNAs (miRNAs) have attracted attention as potential biomarkers of disease. By small-RNA next-generation sequencing, the analysis of serum miRNAs led to the identification of molecular signatures able to discriminate advanced HCC from early HCC (n = 246); advanced HCC from CIRRHOSIS (n = 299); advanced HCC from HEALTHY (n = 320); HEALTHY from early HCC (n = 343); and HEALTHY from CIRRHOSIS (n = 414). Cirrhotic patients and early HCC patients exhibited similar serum miRNA profiles, yet a small number of miRNAs (n = 57) were able to distinguish these two classes of patients. A second objective of the study was to identify serum miRNAs capable of predicting the response to therapy in patients with advanced HCC. All patients were treated with sorafenib as first-line therapy: 24 were nonresponsive and 24 responsive. Analysis of circulating miRNAs revealed a 54 miRNAs signature able to separate the two subgroups. This study suggested that circulating miRNAs could be useful biomarkers for monitoring patients with liver diseases ranging from cirrhosis to advanced HCC and possibly predicting susceptibility to first-line treatment based on sorafenib.

Computational Analyses Reveal Deregulated Clock Genes Associated with Breast Cancer Development in Night Shift Workers.

Breast cancer (BC) is the leading cause of cancer death among women worldwide. Women employed in shift jobs face heightened BC risk due to prolonged exposure to night shift work (NSW), classified as potentially carcinogenic by the International Agency for Research on Cancer (IARC). This risk is linked to disruptions in circadian rhythms governed by clock genes at the cellular level. However, the molecular mechanisms are unclear. This study aimed to assess clock genes as potential BC biomarkers among women exposed to long-term NSW. Clock gene expression was analysed in paired BC and normal breast tissues within Nurses' Health Studies I and II GEO datasets. Validation was performed on additional gene expression datasets from healthy night shift workers and women with varying BC susceptibility, as well as single-cell sequencing datasets. Post-transcriptional regulators of clock genes were identified through miRNA analyses. Significant alterations in clock gene expression in BC compared to normal tissues were found. BHLHE40, CIART, CLOCK, PDPK1, and TIMELESS were over-expressed, while HLF, NFIL3, NPAS3, PER1, PER3, SIM1, and TEF were under-expressed. The downregulation of PER1 and TEF and upregulation of CLOCK correlated with increased BC risk in healthy women. Also, twenty-six miRNAs, including miR-10a, miR-21, miR-107, and miR-34, were identified as potential post-transcriptional regulators influenced by NSW. In conclusion, a panel of clock genes and circadian miRNAs are suggested as BC susceptibility biomarkers among night shift workers, supporting implications for risk stratification and early detection strategies.

Mix-and-Read Digital MicroRNA Analysis Based on Flow Cytometric Counting of Target-Clicked Nanobead Dimer.

A one-step, enzyme-free, and highly sensitive digital microRNA (miRNA) assay is rationally devised based on flow cytometric counting of target miRNA-clicked nanobead dimers via a facile mix-and-read manner. In this strategy, highly efficient miRNA-sandwiched click chemical ligation of two DNA probes may remarkably stabilize and boost the dimer formation between two kinds of fluorescence-coded nanobeads, and the number of as-produced bead dimers will be target dose-responsive, particularly when the trace number of miRNA is much less than that of employed nanobeads. Finally, each fluorescence-coded bead dimer can be easily identified and digitally counted by a powerful flow cytometer (FCM) and accordingly, the amount of target miRNA can be accurately quantified in a digital way. This new digital miRNA assay can be accomplished with a facile mix-and-read operation just by simply mixing the target miRNA with two kinds of preprepared DNA probe-functionalized nanobeads, which do not require any nucleic acid amplification, purification, and complex operation procedures. In spite of the extremely simple one-step operation, benefiting from the low-background but high target-mediated click ligation efficiency, and the powerfully digital statistical capability of FCM, this strategy achieves high sensitivity with a quite low detection limit of 5.2 fM target miRNA as well as high specificity and good generality for miRNA analysis, pioneering a new direction for fabricating digital bioassays.

From sequencing to validation: NGS-based exploration of plasma miRNA in papillary thyroid carcinoma.

A non-invasive method using plasma microRNAs provides new insights into thyroid cancer diagnosis. The objective of this study was to discover potential circulating biomarkers of papillary thyroid carcinoma (PTC) through the analysis of plasma miRNAs using next-generation sequencing (NGS). Plasma miRNAs were isolated from peripheral blood samples collected from healthy individuals, patients diagnosed with PTC, and those with benign thyroid nodules. The Illumina NovaSeq 6000 platform was employed to establish the miRNA expression profiles. Candidate miRNAs for diagnostic purposes were identified utilizing the Random Forest (RF) algorithm. The selected miRNAs were subsequently validated in an independent validation set using RT-qPCR. NGS results revealed consistent plasma miRNA expression patterns among healthy individuals and patients with benign thyroid nodules in the discovery set (6 healthy cases, 17 benign cases), while differing significantly from those observed in the PTC group (17 PTC cases). Seven miRNAs exhibiting significant expression differences were identified and utilized to construct an RF classifier. Receiver operating characteristic (ROC) analysis for PTC diagnosis, and the area under the curve (AUC) was 0.978. Subsequent KEGG and GO analyses of the target genes associated with these 7 miRNAs highlighted pathways relevant to tumors and the cell cycle. Independent validation through RT-qPCR in a separate cohort (15 CONTROL, 15 PTC groups) underscored hsa-miR-301a-3p and hsa-miR-195-5p as promising candidates for PTC diagnosis. In conclusion, our study established a seven-miRNA panel in plasma by Random Forest algorithm with significant performance in discriminating PTC from healthy or benign group. hsa-miR-301a-3p, hsa-miR-195-5p in plasma have potential for further study in the diagnosis of PTC in Asian ethnic.

Combined Analysis of Multi-Study miRNA and mRNA Expression Data Shows Overlap of Selected miRNAs Involved in West Nile Virus Infections.

The emerging zoonotic West Nile virus (WNV) has serious impact on public health. Thus, understanding the molecular basis of WNV infections in mammalian hosts is important to develop improved diagnostic and treatment strategies. In this context, the role of microRNAs (miRNAs) has been analyzed by several studies under different conditions and with different outcomes. A systematic comparison is therefore necessary. Furthermore, additional information from mRNA target expression data has rarely been taken into account to understand miRNA expression profiles under WNV infections. We conducted a meta-analysis of publicly available miRNA expression data from multiple independent studies, and analyzed them in a harmonized way to increase comparability. In addition, we used gene-set tests on mRNA target expression data to further gain evidence about differentially expressed miRNAs. For this purpose, we also studied the use of target information from different databases. We detected a substantial number of miRNA that emerged as differentially expressed from several miRNA datasets, and from the mRNA target data analysis as well. When using mRNA target data, we found that the targetscan databases provided the most useful information. We demonstrated improved miRNA detection through research synthesis of multiple independent miRNA datasets coupled with mRNA target set testing, leading to the discovery of multiple miRNAs which should be taken into account for further research on the molecular mechanism of WNV infections.

Comparative Transcriptome Analysis of mRNA and miRNA during the Development of Longissimus Dorsi Muscle of Gannan Yak and Tianzhu White Yak.

The Gannan yak, a superior livestock breed found on the Tibetan Plateau, exhibits significantly enhanced body size, weight, and growth performance in comparison to the Tianzhu white yak. MiRNAs play a pivotal role in regulating muscle growth by negatively modulating target genes. In this study, we found the average diameter, area, and length of myofibers in Gannan yaks were significantly higher than those of Tianzhu white yaks. Further, we focused on analyzing the longissimus dorsi muscle from both Gannan yaks and Tianzhu white yaks through transcriptome sequencing to identify differentially expressed (DE)miRNAs that influence skeletal muscle development. A total of 254 DE miRNAs were identified, of which 126 miRNAs were up-regulated and 128 miRNAs were down-regulated. GO and KEGG enrichment analysis showed that the target genes of these DE miRNAs were significantly enriched in signaling pathways associated with muscle growth and development. By constructing a DE miRNA- DE mRNA interaction network, we screened 18 key miRNAs, and notably, four of the candidates (novel-m0143-3p, novel-m0024-3p, novel-m0128-5p, and novel-m0026-3p) targeted six genes associated with muscle growth and development (DDIT4, ADAMTS1, CRY2, AKIRIN2, SIX1, and FOXO1). These findings may provide theoretical references for further studies on the role of miRNAs in muscle growth and development in Gannan yaks.

A DNA-peptide probe coupled mass spectrometry-based method for the detection of LncRNA in hepatocyte and serum samples

Liver cancer, a prominent contributor to cancer-related deaths, necessitates timely identification for successful intervention. In recent years, long non-coding RNAs (lncRNAs) have gained attention as potential biomarkers for liver cancer, and accurate quantification of lncRNA is essential for early diagnosis of liver cancer. However, existing methods for lncRNA detection rely on amplification and labeling techniques, resulting in semi-quantitative or qualitative outcomes. DNA-peptide technology offers a promising alternative. It has successfully translated nucleic acid quantification into peptide quantification, particularly in the context of miRNA analysis. However, the intricate structure of long-chain lncRNAs presents a challenge not encountered with miRNAs, making the design and acquisition of specific DNA-peptide probes for direct quantification more difficult. In this study, a strategy for lncRNA quantification was developed using a DNA-peptide probe coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quasi-targeted proteomics. The inherent structural complexity of long-chain lncRNAs was overcome by designing DNA-peptide probes based on their unique sequence segments, enabling direct quantification without the need for amplification. Highly upregulated in liver cancer (HULC) was identified as a target lncRNA through bioinformatics analysis using GEO2R online software. Subsequently, the target lncRNA (i.e., HULC), which had been biotinylated and bound to streptavidin agarose beforehand, was hybridized with the probe. Following trypsin digestion, the reporter peptide was released and quantified using LC-MS/MS. Finally, this method was applied to analyze HULC levels in liver cancer cell lines and serum samples, enabling quantitative assessment and early diagnosis. The results demonstrated that liver cancer diagnostics could be enhanced through the integration of mass spectrometry and DNA-peptide probe technology for lncRNA quantification.

Micro RNA Dysregulation in Keratinocyte Carcinomas: Clinical Evidence, Functional Impact, and Future Directions.

The keratinocyte carcinomas, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC), are the most common cancers in humans. Recently, an increasing body of literature has investigated the role of miRNAs in keratinocyte carcinoma pathogenesis, progression and their use as therapeutic agents and targets, or biomarkers. However, there is very little consistency in the literature regarding the identity of and/or role of individual miRNAs in cSCC (and to a lesser extent BCC) biology. miRNA analyses that combine clinical evidence with experimental elucidation of targets and functional impact provide far more compelling evidence than studies purely based on clinical findings or bioinformatic analyses. In this study, we review the clinical evidence associated with miRNA dysregulation in KCs, assessing the quality of validation evidence provided, identify gaps, and provide recommendations for future studies based on relevant studies that investigated miRNA levels in human cSCC and BCC. Furthermore, we demonstrate how miRNAs contribute to the regulation of a diverse network of cellular functions, and that large-scale changes in tumor cell biology can be attributed to miRNA dysregulation. We highlight the need for further studies investigating the role of miRNAs as communicators between different cell types in the tumor microenvironment. Finally, we explore the clinical benefits of miRNAs as biomarkers of keratinocyte carcinoma prognosis and treatment.

Abstract We003: Identification of CircRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathways Related to Cardiomyocyte Proliferation in Neonatal Mice

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

Exosomal MicroRNAs modulate the cognitive function in fasudil treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice model of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by cognitive decline stemming from the accumulation of beta-amyloid (Aβ) plaques and the propagation of tau pathology through synapses. Exosomes, crucial mediators in neuronal development, maintenance, and intercellular communication, have gained attention in AD research. Yet, the molecular mechanisms involving exosomal miRNAs in AD remain elusive. In this study, we treated APPswe/PSEN1dE9 transgenic (APP/PS1) mice, a model for AD, with either vehicle (ADNS) or fasudil (ADF), while C57BL/6 (control) mice received vehicle (WT). Cognitive function was evaluated using the Y-maze test, and AD pathology was confirmed through immunostaining and western blot analysis of Aβ plaques and phosphorylated tau. Exosomal RNAs were extracted, sequenced, and analyzed from each mouse group. Our findings revealed that fasudil treatment improved cognitive function in AD mice, as evidenced by increased spontaneous alternation in the Y-maze test and reduced Aβ plaque load and phosphorylated tau protein expression in the hippocampus. Analysis of exosomal miRNAs identified three miRNAs (mmu-let-7i-5p, mmu-miR-19a-3p, mmu-miR-451a) common to both ADNS vs ADF and WT vs ADNS groups. Utilizing miRTarBase software, we predicted and analyzed target genes associated with these miRNAs. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of miRNA target genes indicated that mmu-miR-19a-3p and mmu-miR-451a are implicated in signal transduction, immune response, cellular communication, and nervous system pathways. Specifically, mmu-miR-19a-3p targeted genes involved in the sphingolipid signaling pathway, such as Pten and Tnf, while mmu-miR-451a targeted Nsmaf, Gnai3, and Akt3. Moreover, mmu-miR-451a targeted Myc in signaling pathways regulating the pluripotency of stem cells. In conclusion, fasudil treatment enhanced cognitive function by modulating exosomal MicroRNAs, particularly mmu-miR-451a and mmu-miR-19a-3p. These miRNAs hold promise as potential biomarkers and therapeutic targets for novel AD treatments.Graphical

Comparison of serum exosome miRNA from patients with Raynaud's phenomenon with positive and negative serum antinuclear antibodies.

To compare the microRNAs (miRNAs) contained within serum exosomes isolated from patients with Raynaud's phenomenon (RP) and negative antinuclear antibodies (ANA) to the miRNA contained in serum exosomes isolated from patients with RP and positive ANA. Serum exosomes were isolated employing a polymer precipitation procedure. Next Generation Sequencing (NGS) was used to identify the miRNAs contained in the exosomes isolated from the two clinical cohorts and to analyse the differences in their contents. The NGS results identified six miRNAs that displayed significant differences in their content between serum exosomes from patients with RP with negative serum ANA compared to miRNAs contained in serum exosomes from patients with ANA-positive RP. A comparative analysis of miRNAs contained within serum exosomes of patients with RP and negative ANA vs. samples from patients with RP and positive ANA identified several differentially expressed miRNAs that may represent non-invasive biomarkers to assist in the identification of patients with RP at risk of evolving into systemic sclerosis.

G2/M Checkpoint Modulation: Insights from miRNA Profiles in FAM and Breast Cancer.

The aim of this research is to understand the role of microRNA in cell cycle regulation especially on G2M Checkpoint from Luminal A samples Indonesian population. The profile results are used as biomarkers and therapeutic targets for breast cancer. For this reason, analysis was carried out on the comparison of miRNA expression between Luminal A and Fibroadenoma mamae (FAM) using Nanostring nCounter. In this study, 5 (Formalin-Fixed Paraffin-Embedded) FFPE Luminal A tissues and 4 FFPE FAM samples were used. RNA was isolated from cancer tissue samples. Differential expression analysis of miRNA was conducted using Nanostring nCounter technology, subsequently followed by the expression analysis between FAM and Luminal A using nSolver softwere. Elevated expression levels of miRNAs were subjected to pathway and gene regulation analysis using KEGG and GSEA MsigDB databases. Data visualization was performed utilizing Cytoscape, NetworkAnalyst, and SRplot tools. Based on 792 miRNAs detected on Nanostring nCounter, it was found that 60 miRNAs were upregulated and 6 miRNAs were downregulated. The 15 upregulated miRNAs analyzed show their role in the G2M Checkpoint through several pathways. The five miRNAs that significantly regulate the G2M Checkpoint are hsa-miR-196b-5p, hsa-miR-218-5p, hsa-miR-7-5p, hsa-miR-19a-5p, and hsa-miR-18a-5p Where each of these miRNAs regulates the CDKN1B gene. Significant differences in the expression of multiple miRNAs between Luminal A and FAM samples were observed. Furthermore, several of these miRNAs were found to modulate the G2M Checkpoint in Luminal A cancer by suppressing tumor suppressor genes.