miRNA Regulation Research Articles

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy.

Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

Implications of GCLC in prognosis and immunity of lung adenocarcinoma and multi-omics regulation mechanisms

BackgroundFerroptosis is an iron-dependent type of regulated cell death, and has been implicated in lung adenocarcinoma (LUAD). Evidence has proved the key role of glutamate-cysteine ligase catalytic subunit (GCLC) in ferroptosis, but its role in LUAD remains unclear. Herein, we explored the implications of GCLC and relevant genes in LUAD prognosis and immunity as well as underlying molecular mechanisms.MethodsThis work gathered mRNA, miRNA, DNA methylation, somatic mutation and copy-number variation data from TCGA-LUAD. WGCNA was utilized for selecting GCLC-relevant genes, and a GCLC-relevant prognostic signature was built by uni- and multivariate-cox regression analyses. Immune compositions were estimated via CIBERSORT, and two immunotherapy cohorts of solid tumors were analyzed. Multi-omics regulatory mechanisms were finally assessed.ResultsOur results showed that GCLC was overexpressed in LUAD, and potentially resulted in undesirable survival. A prognostic model was generated, which owned accurate and independent performance in prognostication. GCLC, and relevant genes were notably connected with immune compositions and immune checkpoints. High GCLC expression was linked with better responses to anti-PD-L1 and anti-CTLA-4 treatment. Their possible DNA methylation sites were inferred, e.g., hypomethylation in cg19740353 might contribute to GCLC up-regulation. Frequent genetic mutations also affected their expression. Upstream transcription factors (E2F1/3/4, etc.), post-transcriptional regulation of miRNAs (hsa-mir-30c-1, etc.), lncRNAs (C8orf34-AS1, etc.), and IGF2BP1-mediated m6A modification were identified. It was also found NOP58-mediated SUMOylation post-translational modification.ConclusionsTogether, we show that GCLC and relevant genes exert crucial roles in LUAD prognosis and immunity, and their expression can be controlled by complex multi-omics mechanisms.

GSK-3: An "Ace" Among Kinases.

Background: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase known to participate in the regulation of β-catenin signaling (Wnt signaling). This aids in the establishment of a multicomponent destruction complex that stimulates phosphorylation, leading to the destruction of β-catenin. Evidence about the role of increasingly active β-catenin signaling is involved in many forms of human cancer. The understanding of GSK-3 remains elusive as recent research aims to focus on developing potent GSK-3 inhibitors to target this kinase. Objective: This short review aims to highlight the regulation of GSK-3 with emphasis on Wnt signaling while highlighting its interaction with miRNAs corresponding to pluripotency and epithelial mesenchymal transition substantiating this kinase as an "Ace" among kinases in regulation of cellular processes. Result: Significant findings of miRNA regulation by GSK-3 exemplify the underpinnings of kinase-mediated transcriptional regulation in cancers. Conclusion: The review provides evidence on the role of GSK-3 as a possible master regulator of proteins and noncoding RNA, thereby implicating the fate of a cell.

MEG3 in hematologic malignancies: from the role of disease biomarker to therapeutic target.

Maternally expressed gene 3 ( MEG3 ) is a noncoding RNA that is known as a tumor suppressor in solid cancers. Recently, a line of studies has emphasized its potential role in hematological malignancies in terms of tumorigenesis, metastasis, and drug resistance. Similar to solid cancers, MEG3 can regulate various cancer hallmarks via sponging miRNA, transcriptional, or posttranslational regulation mechanisms, but may regulate different key elements. In contrast with solid cancers, in some subtypes of leukemia, MEG3 has been found to be upregulated and oncogenic. In this review, we systematically describe the role and underlying mechanisms of MEG3 in multiple types of hematological malignancies. Particularly, we highlight the role of MEG3 in drug resistance and as a novel therapeutic target.

Dynamic changes in extracellular vesicle-associated miRNAs elicited by ultrasound in inflammatory bowel disease patients

Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to molecular shifts of EV contents, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the inflammation-specific molecular shifts in EVs like altered EV-miRNA expression, which in turn can be detected in the peripheral blood. 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 min after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing. Candidate inducible EV-miRNAs were functionally assessed in vitro by transfection of miRNA mimics and qPCR of predicted target genes. Serum EV-miRNA concentration at baseline correlated with disease severity, as determined by clinical activity scores and sonographic findings. Three miRNAs (miR-942-5p, mir-5588, mir-3195) were significantly induced by sonography. Among the significantly regulated EV-miRNAs, miR-942-5p was strongly induced in higher grade intestinal inflammation and correlated with clinical activity in Crohn’s disease. Prediction of target regulation and transfection of miRNA mimics inferred a role of this EV-miRNA in regulating barrier function in inflammation. Induction of mir-5588 and mir-3195 did not correlate with inflammation grade. This proof-of-concept trial highlights the principle of induced molecular shifts in EVs from inflamed tissue through transabdominal ultrasound. These inducible EVs and their molecular cargo like miRNA could become novel biomarkers for intestinal inflammation in IBD.

Impact of PEDV infection on the biological characteristics of porcine intestinal exosomes.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between these miRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets.

Construction of ferroptosis-related prediction model for pathogenesis, diagnosis and treatment of ruptured abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a dangerous cardiovascular disease, which often brings great psychological burden and economic pressure to patients. If AAA rupture occurs, it is a serious threat to patients' lives. Therefore, it is of clinical value to actively explore the pathogenesis of ruptured AAA and prevent its occurrence. Ferroptosis is a new type of cell death dependent on lipid peroxidation, which plays an important role in many cardiovascular diseases. In this study, we used online data and analysis of ferroptosis-related genes to uncover the formation of ruptured AAA and potential therapeutic targets. We obtained ferroptosis-related differentially expressed genes (Fe-DEGs) from GSE98278 dataset and 259 known ferroptosis-related genes from FerrDb website. Enrichment analysis of differentially expressed genes (DEGs) was performed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). Receiver Operating characteristic (ROC) curve was employed to evaluate the diagnostic abilities of Fe-DEGs. Transcription factors and miRNAs of Fe-DEGs were identified through PASTAA and miRDB, miRWalk, TargetScan respectively. Single-sample gene set enrichment analysis (ssGSEA) was used to observe immune infiltration between the stable group and the rupture group. DGIdb database was performed to find potential targeted drugs of DEGs. GO and KEGG enrichment analysis found that DEGs mainly enriched in "cellular divalent inorganic cation homeostasis," "cellular zinc ion homeostasis," "divalent inorganic cation homeostasis," "Mineral absorption," "Cytokine - cytokine receptor interaction," "Coronavirus disease - COVID-19." Two up-regulated Fe-DEGs MT1G and DDIT4 were found to further analysis. Both single and combined applications of MT1G and DDIT4 showed good diagnostic efficacy (AUC = 0.8254, 0.8548, 0.8577, respectively). Transcription factors STAT1 and PU1 of MT1G and ARNT and MAX of DDIT4 were identified. Meanwhile, has_miR-548p-MT1G pairs, has_miR-53-3p/has_miR-181b-5p/ has_miR-664a-3p-DDIT4 pairs were found. B cells, NK cells, Th2 cells were high expression in the rupture group compared with the stable group, while DCs, Th1 cells were low expression in the rupture group. Targeted drugs against immunity, GEMCITABINE and INDOMETHACIN were discovered. We preliminarily explored the clinical significance of Fe-DEGs MT1G and DDIT4 in the diagnosis of ruptured AAA, and proposed possible upstream regulatory transcription factors and miRNAs. In addition, we also analyzed the immune infiltration of stable and rupture groups, and found possible targeted drugs for immunotherapy.

Genetic Screening Revealed the Negative Regulation of miR-310~313 Cluster Members on Imd Pathway during Gram-Negative Bacterial Infection in Drosophila.

Innate immune response is the first line of host defense against pathogenic microorganisms, and its excessive or insufficient activation is detrimental to the organism. Many individual microRNAs (miRNAs) have emerged as crucial post-transcriptional regulators of immune homeostasis in Drosophila melanogaster. However, the synergistical regulation of miRNAs located within a cluster on the Imd-immune pathway remains obscured. In our study, a genetic screening with 52 transgenic UAS-miRNAs was performed to identify ten miRNAs or miRNA clusters, including the miR310~313 cluster, which may function on Imd-dependent immune responses. The miRNA RT-qPCR analysis showed that the expression of miR-310~313 cluster members exhibited an increase at 6-12 h post E. coli infection. Furthermore, the overexpression of the miR-310~313 cluster impaired the Drosophila survival. And the overexpression of miR-310/311/312 reduced Dpt expression, an indication of Imd pathway induced by Gram-negative bacteria. Conversely, the knockdown of miR-310/311/312 led to increases in Dpt expression. The Luciferase reporter expression assays and RT-qPCR analysis confirmed that miR-310~313 cluster members directly co-targeted and inhibited Imd transcription. These findings reveal that the members of the miR-310~313 cluster synergistically inhibit Imd-dependent immune responses by co-targeting the Imd gene in Drosophila.

A role for miRNAs in the regulation of brown adipose tissue whitening in goats (Capra Hircus).

A study of the mechanism of and metabolic regulation of brown adipose tissue (BAT) production is important for improving the survival rate of young animals. In the present study, we observed that perirenal adipose tissue in goats undergoes a rapid BAT whitening after birth. However, the underlying regulatory mechanism remains unknown. To address this further, we investigated the role of miRNAs in regulating the whitening process of BAT in goats. First, we identified the dynamic expression profiles of miRNAs during the whitening of BAT in Dazu black goat using RNA-seq. We identified a total of 1374 miRNAs, including 408 existing miRNAs, 693 known miRNAs, and 273 novel miRNAs. By analysis of the differentially expressed miRNAs (DE miRNAs), we found that 102 highly expressed miRNAs, including chi-miR-144-3p, chi-miR-144-5p, chi-miR-378-5p, chi-miR-136-3p, chi-miR-381, chi-miR-323b, chi-miR-1197-3p, chi-miR-411b-3p, and chi-miR-487a-3p, were enriched in BAT. In addition, 60 highly expressed miRNAs, including chi-miR-184, chi-miR-193a, chi-miR-193b-3p, chi-let-7c-5p, and chi-let-7e-5p, were enriched in white fat-like tissue. An analysis of miRNAs that were linearly downregulated (profile 0) or linearly upregulated (profile 19) over the D0-D28 period found that these DE miRNAs were mainly enriched in the Hippo signaling pathway, Cytokine-cytokine receptor interactions, and the TGF-beta signaling pathway. Furthermore, we confirmed that chi-let-7e-5p promotes the proliferation and differentiation of brown adipocytes. These results should facilitate a better understanding of the molecular regulation of miRNAs involved in BAT whitening in goats.

Resveratrol in the Treatment of Gynecological Cancer: Mechanisms and Therapeutic Potential.

Gynecological cancers, encompassing endometrial, ovarian, and cervical cancer, pose significant challenges in clinical practice, often marked by high mortality rates and treatment resistance. Despite advances in standard therapies, including chemoradiation and surgery, tumor recurrence and metastasis remain formidable obstacles. In this context, there is a pressing need to explore novel therapeutic strategies that offer improved efficacy and reduced side effects. Herbal medicine, particularly compounds like resveratrol, has garnered attention for its diverse biological properties, including anticancer effects. Resveratrol, a multipotential nutraceutical, holds promise in gynecological cancer therapy through its modulation of key cellular and molecular processes. This review aims to provide an overview of the current status, challenges, and opportunities in utilizing resveratrol for gynecological cancer treatment. We discuss its role in miRNA regulation, clinical trial findings, and the development of effective formulations. By elucidating the underlying mechanisms of resveratrol's anticancer effects and exploring innovative delivery systems, we aim to shed light on the potential avenues for optimizing its therapeutic benefits in gynecological cancers.

Heavy Metal-Laden Particulate Dust Alters Transcriptional Regulation in Human Colonoids

Chronic heavy metals exposure in the southwestern United States co-occurs with greater incidences of metabolic and digestive diseases, particularly in the Indigenous and Hispanic populations. There is a need to understand how particulate heavy metals drive this dysbiotic state and propose a molecular explanation as a result of these exposures. Previously, our group demonstrated human colonoids to be a relevant model for investigating the physiological impact of environmental toxicants on the intestinal tract. The objective of this study is to elucidate the mechanism behind the transcriptional and cellular changes present in the intestinal epithelia upon acute exposure to non fissile uranium particulate dust. We hypothesize that non-fissile uranium exposure biases the differentiation of progenitor cells in the crypt to secretory roles through miRNA regulation. Colonoids were exposed (18 hours) to non-fissile uranium dust obtained from the Jackpile uranium mine, one of the largest open pit uranium mines, located on the Laguna Pueblo in New Mexico. Control and dust-exposed colonoids (n=3 unique donors) were digested into single cells and processed for droplet-based single cell sequencing (scRNAseq). Upon exposure, enteroendocrine cells (EEC) expanded >4 fold, along with increases in serotonin and PYY, as well as goblet cells (>3 fold) which indicates changes to the secretory lineage. Downstream pathway analysis found microRNAs miR-625-3p, miR-320a, miR-29b-3p to be differentially expressed. These results suggest that acute uranium dust exposure induces changes in differentiation pathways which are modulated by regulatory miRNAs. We would like to expand these findings by performing miRNA sequencing and comparing miRNA expression profiles between UBD exposed and control samples. Our results also provide an improved understanding of uranium dust’s impact on the gastrointestinal system, which may inform other physiological studies on the health of individuals exposed to heavy metals. This study was funded by K01DK106323, R56ES034400, P42ES025589, P20GM121176, P20GM130422, and AGA SURF. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of lncRNA-related studies of ivermectin-sensitive and -resistant strains of Haemonchus contortus.

In this study, 858 novel long non-coding RNAs (lncRNAs) were predicted as sensitive and resistant strains of Haemonchus contortus to ivermectin. These lncRNAs underwent bioinformatic analysis. In total, 205 lncRNAs significantly differed using log2 (difference multiplicity) > 1 or log2 (difference multiplicity) < - 1 and FDR < 0.05 as the threshold for significant difference analysis. We selected five lncRNAs based on significant differences in expression, cis-regulation, and their association with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. These expressions of lncRNAs, namely MSTRG.12610.1, MSTRG.8169.1, MSTRG.6355.1, MSTRG.980.1, and MSTRG.9045.1, were significantly downregulated. These findings were consistent with the results of transcriptomic sequencing. We further investigated the relative expression of target gene mRNAs and the regulation of mRNA and miRNA, starting with lncRNA cis-regulation of mRNA, and constructed a lncRNA-mRNA-miRNA network regulation. After a series of statistical analyses, we finally screened out UGT8, Unc-116, Fer-related kinase-1, GGPP synthase 1, and sart3, which may be involved in developing drug resistance under the regulation of their corresponding lncRNAs. The findings of this study provide a novel direction for future studies on drug resistance targets.

Upregulation of miR-361-3p Is Associated With Impaired AT2 Receptor Expression in Cardiometabolic Diseases

Cardiometabolic homeostasis has been shown to be regulated by the brain Renin-Angiotensin System (RAS), including via epigenetic mechanisms. MicroRNA (miRNA) regulation is one of the most common mechanisms controlling the expression of RAS genes and has previously been implicated in hypertension, cardiac ischemia, and aging. Recently, we observed that hypercaloric diet (HCD) programming resulted in differentially expressed miRNA in the hypothalamus of only the male progeny, including miR-361-3p which is thought to target angiotensin-II type 2 receptors (AT2R). Here, we aimed to investigate whether miR-361-3p regulate AT2R expression in a mouse model of cardiometabolic disease (CMD) exhibiting hypertension, diabetes and obesity. C57BI/6J mice (3-week-old, both sexes, n=10) were fed a HCD (D12492. 60 kcal% Fat, Research Diets, USA) while weight and fasting blood glucose levels were measured weekly to ensure the acquisition of metabolic syndrome. At 12 weeks of age, the mice were infused subcutaneously (osmotic pump model 1004, Alzet) with Ang-II (600 ng/kg/min/4 weeks). BP, food intake and glycemia were recorded weekly while intraperitoneal glucose (GTT), insulin (ITT) tolerance tests and body composition (EchoMRI) were assessed at the end of the study. Gene expression of miR-361-3p (QRT-PCR) was increased in the hypothalamus of CMD male mice (2.0 ±0.5 vs.1.2 ±0.2, p&lt;0.05) but not in females (2.7 ±0.8 vs.2.5 ±0.4, P&gt;0.05). However, both sexes showed an increase in the heart (Males: 2.0 ±0.5 vs.1.0 ±0.3; Females: 1.6 ±0.4 vs. 0.8 ±0.2, p&lt;0.05) which was associated with decreased AT2R cardiac expression (Capillary Western) in CMD male (0.9 ±0.0 vs.1.0 ±0.0, p&lt;0.05) and females (0.1 ±0.0 vs.0.6 ±0.1, p&lt;0.001). Together, our data suggest that in CMD, upregulation of miR-361-3p is associated with the decrease of AT2R in the heart, possibly contributing to impaired cardiac function in this model. This positions inhibitors of miR-361-3p as a possible new therapeutic strategy to control RAS overactivity in CMD. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential expression of microRNAs in diapause and non-diapause gonads of Aspongopus chinensis Dallas (Hemiptera: Dinidoridae): implications for reproductive control.

Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae), an edible and medicinal insect, usually found in China and Southeast Asia, offers substantial potential for various applications. The reproductive cycle of this particular insect occurs annually because of reproductive diapause, leading to inadequate utilization of available natural resources. Despite its considerable ecological importance, the precise mechanisms underlying diapause in A. chinensis are not yet well understood. In this study, we conducted an analysis of comparing the microRNA (miRNA) regulation in the diapause and non-diapause gonads of A. chinensis and identified 303 differentially expressed miRNAs, among which, compared with the diapause group, 76 miRNAs were upregulated and 227 miRNAs downregulated. The results, regarding the Enrichment analysis of miRNA-targeted genes, showed their involvement in several essential biological processes, such as lipid anabolism, energy metabolism, and gonadal growth. Interestingly, we observed that the ATP-binding cassette pathway is the only enriched pathway, demonstrating the capability of these targeted miRNAs to regulate the reproductive diapause of A. chinensis through the above essential pathway. The current study provided the role of gonadal miRNA expression in the control of reproductive diapause in A. chinensis, the specific regulatory mechanism behind this event remained unknown and needed more investigation.

Machine Learning Strategies in MicroRNA Research: Bridging Genome to Phenome.

MicroRNAs (miRNAs) have emerged as a prominent layer of regulation of gene expression. This article offers the salient and current aspects of machine learning (ML) tools and approaches from genome to phenome in miRNA research. First, we underline that the complexity in the analysis of miRNA function ranges from their modes of biogenesis to the target diversity in diverse biological conditions. Therefore, it is imperative to first ascertain the miRNA coding potential of genomes and understand the regulatory mechanisms of their expression. This knowledge enables the efficient classification of miRNA precursors and the identification of their mature forms and respective target genes. Second, and because one miRNA can target multiple mRNAs and vice versa, another challenge is the assessment of the miRNA-mRNA target interaction network. Furthermore, long-noncoding RNA (lncRNA)and circular RNAs (circRNAs) also contribute to this complexity. ML has been used to tackle these challenges at the high-dimensional data level. The present expert review covers more than 100 tools adopting various ML approaches pertaining to, for example, (1) miRNA promoter prediction, (2) precursor classification, (3) mature miRNA prediction, (4) miRNA target prediction, (5) miRNA- lncRNA and miRNA-circRNA interactions, (6) miRNA-mRNA expression profiling, (7) miRNA regulatory module detection, (8) miRNA-disease association, and (9) miRNA essentiality prediction. Taken together, we unpack, critically examine, and highlight the cutting-edge synergy of ML approaches and miRNA research so as to develop a dynamic and microlevel understanding of human health and diseases.

MiRNA Regulation of Cerebrovascular Inwardly Rectifying K+ Channels During Onset of Alzheimer Disease: Role of Cholesterol and Cannabidiol Treatment

Currently, more than 6 million Americans live with Alzheimer disease (AD). Recent studies demonstrate that impaired blood flow in the brain is integral to the development of AD. Vascular inwardly rectifying K+ channels, such as KIR2.x and KIR6.x, are important for regulating vasoconstriction and vasodilation to tune cerebral perfusion in response to metabolic demand. We’ve previously identified that disruption in membrane cholesterol during advanced AD pathology restores function of KIR2.1 channels to that of young, healthy conditions or better. Additionally, we’ve identified specific microRNAs (miRNAs) in cerebral vessels that can be used to track early development of AD. In this study, we seek to understand molecular pathways stemming from miRNAs that directly or indirectly modulate activity of KIR2.x ( Kcnj2, Kcnj12) and KIR6.x ( Kcnj8, Kcnj11) channels. These ion channels are abundant in cerebrovascular endothelium and smooth muscle cells while particularly sensitive to dyslipidemia. We hypothesize that cerebrovascular miRNAs marking AD pathology are involved in dysregulation of inwardly rectifying K+ channels via cholesterol signaling. With employing QIAGEN’s Ingenuity Pathway Analysis (IPA), we first identified miRNAs that generally regulate each of the target genes for select KIR channels. With applying acquired knowledge of cerebrovascular miRNAs implicated in AD, we found which of these miRNAs also regulate cellular membrane cholesterol levels. Finally, we pinpointed miRNAs sensitive to cannabidiol (CBD) treatment during the development of AD as a potential therapeutic alternative to statins and cyclodextrins. In total, there are 233 miRNAs that regulate Kcnj2, 43 for Kcnj8, 203 for Kcnj11, and 177 for Kcnj12. When factoring in miRNAs that are AD-related, remaining miRNAs include nine that regulate Kcnj2, and one each for Kcnj8, Kcnj11, and Kcnj12. Remaining miRNAs that are cholesterol-related include one for Kcnj2, 43 for Kcnj8, 191 for Kcnj11, and one for Kcnj12. In general, at AD onset, CBD upregulates KIR2.2 and downregulates KIR2.1, KIR6.1, and KIR6.2 channels. CBD reversed expression of miRNAs involved in KIR2.x channel regulation in endothelial and smooth muscle/pericytes (e.g., miR-133 and miR-145) during AD onset in 3xTg-AD animals relative to wild-type controls. CBD also reverses expression of miR-129 and miR-151 to effectively upregulate genes modulating KIR6.x channels from onset of AD relative to wild-type. Altogether, we evidence specific pathways underpinning regulation of KIR2.x and KIR6.x channels in the transition from a healthy to diseased brain. This work was supported by the National Institutes of Health (R01AG073230). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Dependent Exacerbation of Salt Sensitive Hypertension and Cardiac Dysfunction Is Associated With Upregulation of miRNA Targeting ADAM17 and ACE2 in the Brain

Background: The DOCA-salt model has been widely used to study salt-sensitive hypertension and we previously reported that it is associated with ADAM17-mediated ACE2 shedding in the central nervous system. However, previously reported data are mainly from male mice, and it is unclear whether female mice exhibit the same phenotypes. Objective: We aimed to compare sex specific development of salt sensitive hypertension and cardiac dysfunction and investigate miRNA regulation associated with these changes. Methods: C57 mice (male and females, n = 8 -15) underwent uninephrectomy and telemetry implantation (DSI Inc.) for blood pressure (BP) conscious recording. After baseline recording, a DOCA pellet (50 mg, 21 days, Innovative Research of America) was implanted subcutaneously and drinking water was replaced by 1% NaCl solution to induce salt sensitive hypertension. Cerebral Spinal fluid (CSF) was collected at baseline and after 3 weeks of DOCA-salt treatment for exosome miRNA sequencing (Creative Biolab). In another set of mice, cardiac function was assessed by echocardiography (Visualsonics Vevo F2) and invasive left ventricular hemodynamics (Transonic). Mortality from each group was analyzed as well. Results: Baseline BP was not different between male and female mice. Following DOCA-salt treatment for 3 weeks, BP was increased in both sexes, with higher systolic BP observed in males compared to females (active phase: 173.0 ± 19.6 vs. 154.2 ± 19.1 mmHg, p&lt;0.05). Although ejection fraction was not changed in either male or female mice, mitral peak early diastolic filling velocity and tissue doppler showed that E/E’ was increased in both sexes compared to baselines (Male: 33.4 ± 4 vs. 26.6 ± 1.9; Female: 30.3 ± 5.0 vs. 26.4 ± 4), with a greater increase in males (25.3% vs. 14.4%). In addition, hemodynamics revealed increased left ventricular end-diastolic pressure (11.1 ± 1.9 vs. 4.5 ± 0.5 mmHg, p&lt;0.01) and relaxation time constant Tau (6.7 ± 1.0 vs. 4.9 ± 0.5 msec, p&lt;0.01) in DOCA-salt treated males compared to non-treated controls, with no changes shown in female mice. These data suggest worsened cardiac diastolic function in males compared to females following DOCA-salt treatment. Moreover, a 20% mortality was observed in males while no death occured in females, confirming a more severe phenotype in males. Furthermore, miRNAseq analysis of CSF exosomes showed several upregulated miRNA in males but not in females following DOCA-salt treatment. Notably, miR206 associated with increased ADAM17 activity was increased by 8-fold in males but only 3-fold n females. Conclusion: DOCA-salt treatment promotes exacerbated hypertension, impaired cardiac function and mortality in a sex dependent manner, associated with upregulation of key miRNA targeting the brain renin-angiotensin system. The changes of ADAM17 and ACE2 targeted exosome miRNAs in the central nervous system might contribute to the sex difference in the cardiovascular dysfunction phenotypes in this model. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MiR-770-5p-induced cellular switch to sensitize trastuzumab resistant breast cancer cells targeting HER2/EGFR/IGF1R bidirectional crosstalk.

Studies highlighted the bidirectional crosstalk between the HER family members in breast cancer as resistance mechanism to anti-HER agents. Cross-signaling between HER2/EGFR and ER/IGF1R could play role in the development of resistance to therapeutics hence stimulating cell growth. To overcome this resistance, combined therapies targeting both pathways simultaneously have been proposed as an effective strategy. The involvement of miRNAs in resistance of targeted therapies like trastuzumab was demonstrated in recent studies. Hence the regulation of miRNAs in resistance state could reverse the cell behaviour to drugs. Previously we found that overexpression of miR-770-5p downregulated AKT and ERK expression through HER2 signaling and potentiated the effect of trastuzumab. In this study we examined the impact of miR-770-5p on trastuzumab resistance. Cells were treated with tamoxifen or trastuzumab to examine their role in bidirectional crosstalk. The molecule mechanism of miR-770-5p on HER2/EGFR/IGF1R bidirectional crosstalk was explored by western blot. The expression of miR-770-5p in trastuzumab resistant cells was examined by q-PCR. To investigate the effect of miR-770-5p on cancer cell proliferation in trastuzumab resistance state, resistant cells were analyzed by iCELLigence real-time cell analyzer. miR-770-5p expression was significantly downregulated in trastuzumab-resistant BT-474 and SK-BR-3 cells. Overexpression of miR-770-5p sensitized the resistant cells to trastuzumab, as evidenced by reduced cell proliferation and increased cell viability. Additionally, in resistant cells, increased expression and activation of EGFR and IGF1R were observed. However, miR-770-5p overexpression resulted in decreased phosphorylation of AKT and ERK, indicating its suppressive role in EGFR/HER2 signaling. Furthermore, miR-770-5p downregulated the expression of IGF1R and mTOR, suggesting its involvement in regulating the escape signaling mediated by IGF1R in resistance. In conclusion, our findings demonstrate the critical role of miR-770-5p in regulating bidirectional crosstalk and overcoming trastuzumab resistance in breast cancer cells. These results highlight the potential of miR-770-5p as a therapeutic target to improve the efficacy of targeted therapies and address resistance mechanisms in breast cancer.

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors.

In early embryonic development, the cross-regulation of transcription factors and signaling pathways are critical in mediating developmental and physiological processes. Additionally, many studies have shown the importance of post-transcriptional regulation of signaling and network components mediated by microRNAs (miRNAs); however, how miRNAs are transcriptionally regulated is poorly understood. miRNAs are critical fine-tuners of many biological processes and their dysregulation leads to a variety of diseases and developmental defects. Previously, we have shown that miRNAs are dynamically expressed throughout sea urchin development, suggesting that miRNAs are likely to be under transcriptional regulation. Here, we used pharmacological inhibitors, genetic constructs, and loss-of-function reagents to assess the impact of key signaling pathways (Wnt, Nodal, MAPK, Sonic Hedgehog, Delta/Notch, VEGF, and BMP) and transcription factors (Alx1, Ets1/2, and Tbr) on the transcript levels of the evolutionarily conserved miR-1, miR-31, miR-92 and miR-124; the invertebrate-specific miR-71; and the echinoderm-specific miR-2002, miR-2007, and miR-2012. We also used computational methods to identify potential transcription factor binding sites of these miRNAs. Lists of binding motifs for transcription factors (TFs) were acquired from the MEME-Suite Motif Database and used as inputs for the algorithm FIMO (Find Individual Motif Occurrences), which detects short nucleotide motifs within larger sequences. Based on experimental data on miRNA expression in conjunction with bioinformatic predictions, we propose that the transcription factors Tbr, Alx1, and Ets1 regulate SpmiR-1, SpmiR-31, and SpmiR-71, respectively. We additionally observed significant effects on miRNA levels as a result of perturbations to Wnt, Nodal, MAPK, and Sonic Hedgehog signaling pathways, while no significant change on miRNA levels were observed with perturbations to Delta/Notch, VEGF, or BMP signaling pathways. Overall, this study provides insights into the transcriptional regulation of miRNAs by signaling pathways and transcription factors and contribute to our overall understanding of the genetic regulation of developmental processes.

Characterization and expression profiles of the ZmLBD gene family in Zea mays.

The Lateral Organ Boundaries Domain (LBD) gene family is a family of plant-specific transcription factors (TFs) that are widely involved in processes such as lateral organ formation, stress response, and nutrient metabolism. However, the function of LBD genes in maize remains poorly understood. In this study, a total of 49 ZmLBD genes were identified at the genome-wide level of maize, they were classified into nine branches based on phylogenetic relationships, and all of them were predicted to be nuclear localized. The 49 ZmLBD genes formed eight pairs of segmental duplicates, and members of the same branches' members had similar gene structure and conserved motif composition. The promoters of ZmLBD genes contain multiple types of cis-acting elements. In addition, by constructing the regulatory network of ZmLBD and other genes and miRNAs, 12 and 22 ZmLBDs were found to be involved in the gene regulatory network and miRNA regulatory network, respectively. The expression pattern analysis suggests that ZmLBD genes may be involved in different biological pathways, and drought stress induced the expressions of two inbred lines. The findings enhance our comprehension of the potential roles of the ZmLBD gene family in maize growth and development, which is pivotal for genetic enhancement and breeding efforts pertaining to this significant crop.