miRNA Expression Analysis Research Articles

MicroRNA expression profiling in the lungs of genetically different Ri chicken lines against the highly pathogenic avian influenza H5N1 virus.

The highly pathogenic avian influenza (HPAI) virus triggers infectious diseases, resulting in pulmonary damage and high mortality in domestic poultry worldwide. This study aimed to analyze miRNA expression profiles after infection with the HPAI H5N1 virus in resistant and susceptible lines of Ri chickens.For this purpose, resistant and susceptible lines of Vietnamese Ri chicken were used based on the A/G allele of Mx and BF2 genes. These genes are responsible for innate antiviral activity and were selected to determine differentially expressed (DE) miRNAs in HPAI-infected chicken lines using small RNA sequencing. A total of 44 miRNAs were DE after 3 days of infection with the H5N1 virus. Computational program analysis indicated the candidate target genes for DE miRNAs to possess significant functions related to cytokines, chemokines, MAPK signaling pathway, ErBb signaling pathway, and Wnt signaling pathway. Several DE miRNA-mRNA matches were suggested to play crucial roles in mediating immune functions against viral evasion. These results revealed the potential regulatory roles of miRNAs in the immune response of the two Ri chicken lines against HPAI H5N1 virus infection in the lungs.

Relationship between cluster miR-143/145 micro-RNAs with oncogenesis: tissue and cellular context

The purpose of the study was to present up-to-date data on the regulation of expression, function in normal tissues and multidirectional activity in the oncogenesis of miR-143/145 microRNAs cluster, as well as to evaluate the possibilities and limitations of the therapeutic use of microRNAs of this cluster in malignant neoplasms. Material and methods. The search for available domestic and foreign literary sources published in PubMed and RSCI databases over the past 10 years has been carried out. 427 articles were found, of which 41 were included in this review. Results. The conservative cluster miR-143/145 is one of the most intensively studied in tumors. Based on the results of the analysis of differential miRNA expression, in vitro experiments in cancer cell lines and in vivo in mouse tumor models, a decrease in miR-143 and miR-145 levels was shown in malignant neoplasms of epithelial origin. Until recently, these miRNAs were considered classical oncosuppressors. The data presented in the review demonstrate that the results of a number of studies taking into account the cellular aspects of microRNA expression contradict this concept. miR-143 microRNA, for example, is known to participate in the metabolic restructuring of the tumor and the activation of neoangiogenesis. It has been shown that the oncosuppressive or pro-oncogenic activity of miR-143 and miR-145 depend on the tissue and cellular context and can be explained by the presence of several regulated targets that have opposite effects on oncogenesis. Taken together, the data obtained suggest the need to exercise caution when choosing the microRNAs of the described cluster for exogenous therapeutic delivery. Conclusion. Further detailed decoding of the mechanisms of miR-143 and miR-145 functioning in various types of tissues and cells, as well as identification of new MRNA targets are necessary for a better understanding of the involvement of these molecules in oncogenesis.

P-673 Women exposed to higher levels of DEHP display altered microRNA expression profile in the pre-ovulatory follicular fluid

Abstract Study question Can higher concentrations of di-2-ethylhexyl phthalate (DEHP) in follicular fluid (FF) lead to an altered microRNA expression profile? Summary answer The present study indicates the upregulation of miR-203a-3p, miR-150-5p and miR-28-3p in the follicular fluid of women with higher DEHP concentrations (FDR<0.05). What is known already Women are exposed daily to endocrine-disrupting chemicals (EDCs) such as phthalates which can be found in hundreds of personal care products and plastic items. In studies of rodents exposed to certain phthalates, high doses have been shown to change hormone levels, disrupt folliculogenesis and cause birth defects. Recently, we reported a strong inverse association between DEHP metabolites and ovarian sensitivity index (OSI) in females undergoing ART treatment. Several mechanisms have been proposed to explain the association between DEHP and fertility in rodents, however not much is known about the mechanisms in humans. Study design, size, duration FF was collected from 96 women undergoing IVF treatment after controlled ovarian stimulation during ovarian puncture. 48 participants from Estonia were recruited at Nova Vita Clinic AS in Tallinn, Estonia in 2019 and 48 participants from Sweden were recruited at Carl von Linnekliniken in Uppsala, Sweden in 2016. In both cohorts, patients signed a written informed consent form. Both cohorts were matched for age, BMI, parity and the number of previous IVF treatments. Participants/materials, setting, methods DEHP metabolite concentrations in the FF samples were measured in our earlier study by LC-MS/MS. Cell free RNA was extracted from FF samples with Qiagen miRNeasy Micro kit, sequencing libraries were prepared with QIAseq miRNA library kit and sequenced on Illumina NextSeq 550 instrument. Differential miRNA expression analysis was performed with the DESeq2 package. Pathway enrichment analysis for differentially expressed miRNA targets was conducted with the miEAA tool integrated with WikiPathways. Main results and the role of chance In total, 465 miRNAs were observed at least twice in ≥ 24 samples. Differential expression analysis was conducted between DEHP-high and DEHP-low groups in combined cohorts using the cohort as a covariate. 16 miRNAs were found to be differentially expressed between DEHP groups with FDR<0.1. Three miRNAs: miR-203a-3p, miR-150-5p and miR-28-3p, were differentially expressed with FDR<0.05 and were more abundant in women with high DEHP-levels in both cohorts. Of these, miR-203a-3p was negatively correlated with OSI (Pearson R = -0.27, p = 0.0089). Six miRNAs (miR-10b-5p, miR-112-5p, miR-132-5p, miR-203-3p, miR-205-5p and miR-27-3p) were found to have predicted targets in the androgen receptor signalling pathway. Limitations, reasons for caution The miRNAs found in this study need to be validated in a larger set of patients as a cohort-dependent difference in the results was observed. There is a need for further functional studies to investigate miRNAs as possible mediators of the association between DEHP and ovarian sensitivity index. Wider implications of the findings The current study presents evidence that DEHP exposure alters the expression of FF miRNAs which are associated with androgen receptor signalling. These results provide novel evidence of the potential influence of endocrine-disrupting chemicals on female reproduction. Trial registration number NA

MiRNA Expression Analysis of the Hippocampus in a Vervet Monkey Model of Fetal Alcohol Spectrum Disorder Reveals a Potential Role in Global mRNA Downregulation.

MicroRNAs (miRNAs) are short-length non-protein-coding RNA sequences that post-transcriptionally regulate gene expression in a broad range of cellular processes including neuro- development and have previously been implicated in fetal alcohol spectrum disorders (FASD). In this study, we use our vervet monkey model of FASD to follow up on a prior multivariate (developmental age × ethanol exposure) mRNA analysis (GSE173516) to explore the possibility that the global mRNA downregulation we observed in that study could be related to miRNA expression and function. We report here a predominance of upregulated and differentially expressed miRNAs. Further, the 24 most upregulated miRNAs were significantly correlated with their predicted targets (Target Scan 7.2). We then explored the relationship between these 24 miRNAs and the fold changes observed in their paired mRNA targets using two prediction platforms (Target Scan 7.2 and miRwalk 3.0). Compared to a list of non-differentially expressed miRNAs from our dataset, the 24 upregulated and differentially expressed miRNAs had a greater impact on the fold changes of their corresponding mRNA targets across both platforms. Taken together, this evidence raises the possibility that ethanol-induced upregulation of specific miRNAs might contribute functionally to the general downregulation of mRNAs observed by multiple investigators in response to prenatal alcohol exposure.

Mitochondrial miR-23b-5p is a new biomarker of warm ischemic injury in donor livers and a candidate for graft evaluation: experimental studies.

Warm ischemic injury (WII) stems from incorrect energy metabolism and is the main cause of graft dysfunction. Mitochondria, as the center of cellular metabolic activities, may be the key in identifying accurate indicators for evaluating the quality of grafts. Our research focuses on the screening, clinical application, and mechanism of the optimal WII mitochondrion biomarker. Using a 100% hepatic warm ischemia mouse model, without reperfusion, transmission electron microscopy demonstrated evident morphological changes of hepatic mitochondria at 15 min of ischemia. However, all 13 mt-mRNAs could not display continuously up-regulated consistency at 0-15-30-60 min during WII. High throughput analysis of miRNA expression in both purified mitochondria and liver tissues suggested miR-23b-5p was a potential mitochondrial microRNA (mitomiR) biomarker with high sensitivity and 0-15-30-60 min change consistency. Fluorescence in-situ hybridization and RT-qPCR further confirmed the results. Through overexpression and inhibition, the functionality of this mitomiR during WII was identified as a protective regulator in vitro and then verified in Dicer1fl/flAlbCre mice by down-regulation of other miRNAs and supplementation of mature mitomiR-23b-5p. Dual-luciferase reporter assay and the Seahorse XF analyzer determined that mitomiR-23b-5p reduced mitochondrial respiratory function by silencing mtRNR2 (16S). Clinically, mitomiR-23b-5p was positively correlated with serum ALT levels 3 days after the operation (P=0.032), and the C-statistic for 90-day graft survival rate was 0.698. MitomiR-23b-5p plays a protective regulatory role and implements a special mitochondrial regulation mechanism not yet reported in WII. Our clinical results further support the experimental result that the expression of MitomiR-23b-5p is closely related to the prognosis of clinical liver transplantation patients. This is a promising new biomarker for WII evaluation of donor livers.

Strategies for data normalization and missing data imputation and consequences for potential diagnostic microRNA biomarkers in epithelial ovarian cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression with diagnostic potential in different diseases, including epithelial ovarian carcinomas (EOC). As only a few studies have been published on the identification of stable endogenous miRNA in EOC, there is no consensus which miRNAs should be used aiming standardization. Currently, U6-snRNA is widely adopted as a normalization control in RT-qPCR when investigating miRNAs in EOC; despite its variable expression across cancers being reported. Therefore, our goal was to compare different missing data and normalization approaches to investigate their impact on the choice of stable endogenous controls and subsequent survival analysis while performing expression analysis of miRNAs by RT-qPCR in most frequent subtype of EOC: high-grade serous carcinoma (HGSC). 40 miRNAs were included based on their potential as stable endogenous controls or as biomarkers in EOC. Following RNA extraction from formalin-fixed paraffin embedded tissues from 63 HGSC patients, RT-qPCR was performed with a custom panel covering 40 target miRNAs and 8 controls. The raw data was analyzed by applying various strategies regarding choosing stable endogenous controls (geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder), missing data (single/multiple imputation), and normalization (endogenous miRNA controls, U6-snRNA or global mean). Based on our study, we propose hsa-miR-23a-3p and hsa-miR-193a-5p, but not U6-snRNA as endogenous controls in HGSC patients. Our findings are validated in two external cohorts retrieved from the NCBI Gene Expression Omnibus database. We present that the outcome of stability analysis depends on the histological composition of the cohort, and it might suggest unique pattern of miRNA stability profiles for each subtype of EOC. Moreover, our data demonstrates the challenge of miRNA data analysis by presenting various outcomes from normalization and missing data imputation strategies on survival analysis.

Analysis of miRNA Expression from the DLK1-DIO3 Locus in CD4+ and CD14+ Cells in Relapsing-Remitting Multiple Sclerosis

Analysis of miRNA Expression from the DLK1-DIO3 Locus in CD4+ and CD14+ Cells in Relapsing-Remitting Multiple Sclerosis

Identification of Functionally Significant Polymorphic Variants in miRNA Genes in Carotid Atherosclerosis

miRNAs are vital molecules of gene expression. They are involved in the pathogenesis of various common diseases, including atherosclerosis, its risk factors and complications. A detailed characterization of the spectrum of functionally significant polymorphisms of miRNA genes of patients with advanced carotid atherosclerosis is an actual research task. We analyzed miRNA expression and exome sequencing data of carotid atherosclerotic plaques of the same male patients (n = 8, 66–71 years of age, 67‒90% degree of carotid artery stenosis). For further study and analysis of the association between rs2910164 polymorphism of the MIR146A gene and advanced carotid atherosclerosis, we recruited 112 patients and 72 relatively healthy Slavic residents of Western Siberia. 321 and 97 single nucleotide variants (SNVs) were detected in the nucleotide sequences of pre- and mature miRNAs in carotid atherosclerotic plaques. These variants were located in 206 and 76 miRNA genes, respectively. Integration the data of exome sequencing and miRNA expression revealed 24 SNVs of 18 miRNA genes which were processed to mature form in carotid atherosclerotic plaques. SNVs with the greatest potential functional significance for miRNA expression predicted in silico were rs2910164:CG (MIR146A), rs2682818:AC (MIR618), rs3746444:AG (MIR499A), rs776722712:CT (MIR186), rs199822597:GA (MIR363). The expression of miR-618 was lower in carotid atherosclerotic plaques of patients with the AC rs2682818 genotype of the MIR618 gene compared with the CC genotype (log2FC = 4.8; p = 0.012). We also found the association of rs2910164:C (MIR146A) with the risk of advanced carotid atherosclerosis (OR = 2.35; 95% CI: 1.43–3.85; p = 0.001). Integrative analysis of polymorphism in miRNA genes and miRNA expression is informative for identifying functionally significant polymorphisms in miRNA genes. The rs2682818:AC (MIR618) is a candidate for regulating miRNA expression in carotid atherosclerotic plaques. The rs2910164:C (MIR146A) is associated with the risk of advanced carotid atherosclerosis.

Moyamoya disease-specific extracellular vesicle-derived microRNAs in the cerebrospinal fluid revealed by comprehensive expression analysis through microRNA sequencing.

To examine the specific changes that occur in the expression levels of extracellular vesicle-derived microRNAs (miRNAs) in intracranial cerebrospinal fluid (CSF) in moyamoya disease. Patients with arteriosclerotic cerebral ischemia were used as controls to eliminate the effects of cerebral ischemia. Intracranial CSF was collected from moyamoya disease and control patients during bypass surgery. Extracellular vesicles (EVs) were extracted from the CSF. Comprehensive expression analysis of miRNAs extracted from EVs by next-generation sequencing (NGS) and validation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed. Experiments were conducted on eight cases of moyamoya disease and four control cases. In the comprehensive miRNA expression analysis, 153 miRNAs were upregulated, and 98 miRNAs were downregulated in moyamoya disease compared to the control cases (q-value < 0.05 and |log2 fold change|> 1). qRT-PCR performed on the four most variable miRNAs (hsa-miR-421, hsa-miR-361-5p, hsa-miR-320a, and hsa-miR-29b-3p) associated with vascular lesions among the differentially expressed miRNAs gave the same results as miRNA sequencing. On gene ontology (GO) analysis for the target genes, cytoplasmic stress granule was the most significant GO term. This study is the first comprehensive expression analysis of EV-derived miRNAs in the CSF of moyamoya disease patients using NGS. The miRNAs identified here may be related to the etiology and pathophysiology of moyamoya disease.

Sequencing and bioinformatics analysis of miRNA from rat endplate chondrogenic exosomes.

Exosomes have a key role in various diseases, such as arthritis, heart disease and respiratory disease. Exosomes from various sources have also been indicated to improve intervertebral disc degeneration. However, the role of endplate chondrogenic exosomes in intervertebral disc degeneration has remained largely elusive. The aim of the present study was to compare exosomal microRNA (miRNA) expression patterns in endplate chondrocytes before and after degeneration, and their potential roles in the pathogenesis of intervertebral disc degeneration (IVDD). Endplate chondrocytes were extracted from rats and cultured to obtain pre- and post-degeneration chondrocytes. Exosomes were obtained from the chondrocytes by centrifugation. The two groups of exosomes were subjected to small RNA sequencing, miRNA identification, novel miRNA prediction, quantitative analysis of miRNA expression and differentially expressed (DE) miRNA screening, in addition to miRNA target gene (TG) prediction and TG functional annotation and enrichment analysis. The percentage of miRNAs isolated from the exosomes before and after degeneration was found to differ. A total of 58 DE miRNAs were analyzed, the expression levels of which were significantly different post-degeneration compared with pre-degeneration. Cell experiments were also performed, in which the exosomes were co-cultured with nucleus pulposus (NP) cells. The results indicated that the chondrocyte-derived exosomes were taken up by the NP cells and influenced the expression of aggrecan and collagen 1A and 2A, suggesting that they may inhibit IVDD via their action on NP cells. The specific miRNAs present in exosomes during IVDD may be used to develop new targets for the treatment and diagnosis of this condition. DE exosomal miRNAs derived from endplate cartilage pre- and post-degeneration may be associated with the risk of IVDD and could help to distinguish patients with IVDD. Furthermore, the expression of certain miRNAs may be associated with disease progression, which may contribute to understanding the pathophysiology of IVDD from an epigenetic perspective.

A novel orthotopic mouse model replicates human lung cancer cachexia.

Cancer cachexia, highly prevalent in lung cancer, is a debilitating syndrome characterized by involuntary loss of skeletal muscle mass and is associated with poor clinical outcome, decreased survival and negative impact on tumour therapy. Various lung tumour-bearing animal models have been used to explore underlying mechanisms of cancer cachexia. However, these models do not simulate anatomical and immunological features key to lung cancer and associated muscle wasting. Overcoming these shortcomings is essential to translate experimental findings into the clinic. We therefore evaluated whether a syngeneic, orthotopic lung cancer mouse model replicates systemic and muscle-specific alterations associated with human lung cancer cachexia. Immune competent, 11weeks old male 129S2/Sv mice, were randomly allocated to either (1) sham control group or (2) tumour-bearing group. Syngeneic lung epithelium-derived adenocarcinoma cells (K-rasG12D ; p53R172HΔG ) were inoculated intrapulmonary into the left lung lobe of the mice. Body weight and food intake were measured daily. At baseline and weekly after surgery, grip strength was measured and tumour growth and muscle volume were assessed using micro cone beam CT imaging. After reaching predefined surrogate survival endpoint, animals were euthanized, and skeletal muscles of the lower hind limbs were collected for biochemical analysis. Two-third of the tumour-bearing mice developed cachexia based on predefined criteria. Final body weight (-13.7±5.7%; P<0.01), muscle mass (-13.8±8.1%; P<0.01) and muscle strength (-25.5±10.5%; P<0.001) were reduced in cachectic mice compared with sham controls and median survival time post-surgery was 33.5days until humane endpoint. Markers for proteolysis, both ubiquitin proteasome system (Fbxo32 and Trim63) and autophagy-lysosomal pathway (Gabarapl1 and Bnip3), were significantly upregulated, whereas markers for protein synthesis (relative phosphorylation of Akt, S6 and 4E-BP1) were significantly decreased in the skeletal muscle of cachectic mice compared with control. The cachectic mice exhibited increased pentraxin-2 (P<0.001) and CXCL1/KC (P<0.01) expression levels in blood plasma and increased mRNA expression of IκBα (P<0.05) in skeletal muscle, indicative for the presence of systemic inflammation. Strikingly, RNA sequencing, pathway enrichment and miRNA expression analyses of mouse skeletal muscle strongly mirrored alterations observed in muscle biopsies of patients with lung cancer cachexia. We developed an orthotopic model of lung cancer cachexia in immune competent mice. Because this model simulates key aspects specific to cachexia in lung cancer patients, it is highly suitable to further investigate the underlying mechanisms of lung cancer cachexia and to test the efficacy of novel intervention strategies.

Abstract 6076: Compartment-specific multiomic characterisation of ovarian carcinosarcoma

Abstract Background: Ovarian carcinosarcoma (OCS) is the most aggressive ovarian cancer type. Risk of relapse and death is high across all patient groups, including those diagnosed at early stage. OCS is biphasic, comprising both carcinomatous and sarcomatous populations, leading to its historic consideration alongside true sarcomas. However, we now recognize that OCS in fact represent metaplastic carcinomas, with the sarcomatous component having undergone complete epithelial to mesenchymal transition (EMT) from carcinoma. Detailed understanding of shared and compartment-specific OCS biology has the potential to identify therapeutic opportunities to improve patient survival. Methods: We recently curated the largest pathologically-confirmed OCS cohort to date. Here, we perform compartment-specific profiling of 12 cases by mRNA sequencing, whole exome sequencing, microRNA (miRNA) profiling and quantification of tumor-infiltrating lymphocytes. These data paint a detailed picture of shared and compartment-specific biology between matched carcinomatous and sarcomatous compartments. Results: The sarcomatous compartments harbored significantly fewer infiltrating CD8+ cells (P=0.006), with a significantly lower CD8+:CD3+ cell ratio compared to the carcinomatous components (P=0.002). In 11 of 12 cases, identical TP53 mutations were identified in both cell populations; the remaining case was TP53 wild-type in both compartments. Paired analysis of mRNA sequencing identified 1477 significantly differentially expressed transcripts at FDR&amp;lt;0.01. The sarcomatous compartments demonstrated significantly higher MAPK activity, as determined by the MPAS transcriptomic score (P=0.042). Principal component analysis and unsupervised hierarchical clustering of miRNA expression grouped samples by compartment (carcinomatous versus sarcomatous), rather than by patient, suggesting global differences in the miRNA landscape. Paired analysis identified 131 significantly differentially expressed miRNAs between the two populations (FDR&amp;lt;0.01). Significantly enriched miRNA gene targets were identified using The MiRNA Enrichment Analysis and Annotation Tool (miEAA), identifying key EMT-associated gene targets, including SIRT1, PRDM16, ZEB1, ZEB2 and TGFB signaling components, among other biological processes. Conclusions: Carcinomatous and sarcomatous compartments of OCS demonstrate marked differences in transcriptomic profiles, immune engagement and miRNA expression patterns. Identification of shared TP53 mutations between compartments supports the notion that OCS represent metaplastic carcinomas. Shared biological events represent opportunities for targeted interventions that may be efficacious against both cell populations, while compartment-specific biological events allude to mechanisms by which the carcinomatous population undergoes EMT to form the sarcomatous compartment. Citation Format: Robert L. Hollis, Ailsa Oswald, Lorna J. Stillie, Ian Croy, Michael Churchman, Charlie Gourley, C. Simon Herrington. Compartment-specific multiomic characterisation of ovarian carcinosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6076.

Abstract 3796: Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model

Abstract Introduction: Pancreatic cancer (PC) is believed to be a difficult disease because of its radio-resistance and metastases. Radiotherapy reported to modify tumor microenvironment (TME) and to affect PC progression. Exosomes containing such as microRNAs (miRNAs), messenger RNAs (mRNAs), and proteins of cells, play essential roles in cell-to-cell communications. However, characteristics and mechanisms for radiation responsive exosomes remain unclear. Here, we first report mechanisms of exosomes-mediated radiation response and regulation in PC metastasis with focusing on intra/intercellular communications via miRNA. Methods: MIAPaCa-2 cells, human pancreatic carcinoma, was used. To isolate exosomes, cell culture media was ultracentrifuged at 100,000 x g for 90 min at 4°C. Exosomes derived from non-irradiated culture media was named ‘0 Gy-Exo’ and those from irradiated with 5 Gy culture media is called ‘5 Gy-Exo’. Exosome sizes and features were evaluated by transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) levels induced by exosomes and by X-ray irradiation were evaluated by the C-H2DCF dye staining. To detect DNA damage, cells were fixed and stained by γ-H2AX antibody. The expression of superoxide dismutase 1 (SOD1) was evaluated by using immunoblots. Total RNA was extracted from 0 Gy-Exo and 5 Gy-Exo, and then comprehensive miRNA expression analysis was performed to analyze 2,565 human miRNA sequences. The Gene Expression Omnibus database (GSE163133) and the TargetScan were used for specific miRNAs identification. Living MIAPaCa-2 cells in the presence or absence of those exosomes were injected in the spleen of Balb/c nude mice to establish liver metastatic models. Eight weeks after the injection, mice were sacrificed, and then the spleen and the liver were harvested as a whole organ. H-E staining and immunohisitochemical analyses using S100A4 and SMAD4 antibodies were performed to detect liver metastases. Results: Isolated exosomes were shaped in the form of closed, round vesicles with a diameter of 10~100 nm. Intracellular ROS levels and the numbers of γ-H2AX foci in the cells with 5 Gy-Exo were significantly increased compared to those the control (without adding exosomes, p&amp;lt;0.01). Intracellular SOD1 expression in the cells with the 5 Gy-Exo was decreased compared with that in the control. The miR-6823-5p was identified to have a complementary base sequence to SOD1. The average occupied proportions with MIAPaCa-2 cells in the liver was modulated according to those exosomes. The SMAD4 expression was highly observed in the liver and the spleen suggesting that the SMAD4 could be a useful marker of liver metastases. Conclusion: Exosomes increased radiation sensitivity through increase of intracellular ROS levels via exosomal miRNAs and also could affect liver metastases in pancreatic cancer models. Citation Format: Ai Nakaoka, Makiko Nakahana, Sachiko Inubushi, Yasuyuki Shimizu, Kazuma Iwashita, Naritoshi Mukumoto, Kana Kobayashi, Takeaki Ishihara, Daisuke Miyawaki, Ryohei Sasaki. Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3796.

Investigation into miRNA profile in patient groups with and without ST elevation

Abstract Objectives Because acute myocardial infarction (AMI) may occur suddenly and cannot be predicted, it is critical to identify early diagnosis markers. In recent years, in addition to the developing diagnostic methods, other markers that may be related to the diagnosis of AMI are miRNA molecules. The specificity of miRNAs involved in the pathogenesis of atherosclerosis and regulation of cardiac functions has been shown. The present study aimed to investigate the changes in miRNA expression levels in MI patients with ST-elevation (STEMI) and without ST elevation (NSTEMI). Methods In this study, 25 patients with STEMI, 25 with NSTEMI, and 20 healthy participants were included. Expression analysis of miRNAs isolated from the plasma of individuals was performed using high-throughput real-time PCR. Results Compared with the control group, miR-221-3p and miR-30d-5p were downregulated, while miR-25-3p, miR-92a-3p, miR-34a-5p and miR-150 upregulated in the patient group with NSTEMI; miR-221-3p, miR-25-3p, miR-30d-5p and miR-92a-3p were downregulated while miR-208a-3p was upregulated in patients with STEMI (p&gt;0.05). Conclusions In conclusion, miRNAs may be an early biomarker for diagnosing AMI; however, further and larger studies are needed.

Identifying Key Regulators of Keratinization in Lung Squamous Cell Cancer Using Integrated TCGA Analysis

Keratinization is one of lung squamous cell cancer's (LUSC) hallmark histopathology features. Epithelial cells produce keratin to protect their integrity from external harmful substances. In addition to their roles as cell protectors, recent studies have shown that keratins have important roles in regulating either normal cell or tumor cell functions. The objective of this study is to identify the genes and microRNAs (miRNAs) that act as key regulators of the keratinization process in LUSC. To address this goal, we classified LUSC samples from GDC-TCGA databases based on their keratinization molecular signatures. Then, we performed differential analyses of genes, methylation, and miRNA expression between high keratinization and low keratinization samples. By reconstruction and analysis of the differentially expressed genes (DEGs) network, we found that TP63 and SOX2 were the hub genes that were highly connected to other genes and displayed significant correlations with several keratin genes. Methylation analysis showed that the P63, P73, and P53 DNA-binding motif sites were significantly enriched for differentially methylated probes. We identified SNAI2, GRHL3, TP63, ZNF750, and FOXE1 as the top transcription factors associated with these binding sites. Finally, we identified 12 miRNAs that influence the keratinization process by using miRNA-mRNA correlation analysis.

A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads

Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

Comparative analysis of miRNA expression in dedifferentiated and well-differentiated components of dedifferentiated chondrosarcoma

Dedifferentiated chondrosarcoma (DDCS) is a rare malignant cartilage tumor arising out of a low-grade chondrosarcoma, whereby the well-differentiated and the dedifferentiated components coexist in the same localization. DDCS has a massively increased metastatic potential in comparison to low-grade chondrosarcoma. So far, the underlying mechanisms of DDCS development and the increased malignancy are widely unknown. Targeted DNA sequencing revealed no genetic differences between both tissue components. Besides genetic events, alterations in epigenetic control may play a role in DDCS development. In this preliminary study, we have analyzed the differential miRNA expression in paired samples of both components of four primary DDCS cases and a rare lung metastasis with both components using the nCounter MAX analysis system from NanoString technologies. We identified 21 upregulated and two downregulated miRNAs in the dedifferentiated components of the primary cases. Moreover, three miRNAs were also significantly deregulated in the dedifferentiated component of the lung metastasis, supporting their possible role in DDCS development. Additionally, validated targets of the 23 deregulated miRNAs are involved in signaling pathways, like PI3K/Akt, Wnt/β-catenin, and TGF-β, as well as in cellular processes, like cell cycle regulation, apoptosis, and dedifferentiation. Further investigations are necessary to confirm and understand the role of the identified miRNAs in DDCS development.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Profiling of Targeted miRNAs (8-nt) for the Genes Involved in Type 2 Diabetes Mellitus and Cardiac Hypertrophy

Type 2 Diabetes Mellitus (T2DM) and cardiac hypertrophy (CH) are among the top ten leading cause of deaths, worldwide. T2DM and cardiac hypertrophy are the chronic diseases, have close association and direct life-threatening complications like stroke, myocardial infarction, retinopathy, nephropathy, and limb amputation. In addition to other medical approaches, miRNAs-based strategy is considered most efficient for early detection of chronic diseases and also has potential for the treatment of T2DM and cardiac hypertrophy like it is being used for cancer in clinical trials. MicroRNAs (miRNAs) are single stranded (non-coding) of 20 to 22 nucleotides sequences which bind to their target mRNA upon the complimentary basis, to silence the protein expression at post transcriptional level. Bioinformatic databases are used like online mendelian inheritance in man (OMIM), gene testing registry (GTR), TargetScan and ShinyGO for validation of disease linked genes and sorting the common miRNAs in both diseases, such as miR-30-5p/ 101-3p.2/ 190-5p/ 506-3p/ 9-5p/ 128-3p/ 137/ 96-5p/ 7-5p/ 107/ 101-3p.1/ 98-5p/ 124-3p.2/ 124-3p.1/ 16-5p/ 15-5p/ 497-5p/ 424-5p/ 195-5p/ 1271-5p, let-7-5p. Aforementioned databases were also used for the miRNAs which have more than one disease linked genes target in each pathological condition. Such miRNAs for cardiac hypertrophy are: miR-19-3p/ 183-5p.2/ 153-3p/ 372-3p/ 302-3p/ 520-3p/ 373-3p/ 129-5p/ 144-3p/ 139-5p and for T2DM are: miR-27-3p/ 206/ 1-3p/ 181-5p. This finding would be helpful for the appropriate selection of miRNAs and to design applicable research project in future. It will require more validation by using the miRNAs expression analysis, mimic, and anti-miRNA approach to check their potential against cardiac hypertrophy and T2DM.

Integrated mRNA and miRNA expression analyses for Cryptocaryon irritans resistance in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. However, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatened the healthy and sustainable development of L. crocea industry. Recently, a new C. irritans resistance strain of L. crocea (RS) has been bred using genomic selection technology in our laboratory work. However, the molecular mechanisms for C. irritans resistance of RS have not been fully understood. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that are post-transcriptional regulators, and they play vital roles in immune process of bony fish. Identification of anti-C.irritans relevant miRNA signatures could, therefore, be of tremendous translational value. In the present study, integrated mRNA and miRNA expression analysis was used to explore C. irritans resistance mechanisms of the L. crocea. RS as well as a control strain (CS) of L. crocea, were artificially infected with C. irritans for 100 h, and their gill was collected at 0 h (pre-infection), 24 h (initial infection), and 72 h (peak infection) time points. The total RNA from gill tissues was extracted and used for transcriptome sequencing and small RNA sequencing. After sequencing, 23,172 known mRNAs and 289 known miRNAs were identified. The differential expression was analyzed in these mRNAs and mRNAs and the interactions of miRNA-mRNA pairs were constructed. KEGG pathway enrichment analyses showed that these putative target mRNAs of differentially expressed miRNAs (DEMs) were enriched in different immune-related pathways after C. irritans infection in RS and CS. Among them, necroptosis was the immune-related pathway that was only significantly enriched at two infection stages of RS group (RS-24 h/RS-0h and RS-72 h/RS-0h). Further investigation indicates that necroptosis may be activated by DEMs such as miR-133a-3p, miR-142a-3p and miR-135c, this promotes inflammation responses and pathogen elimination. These DEMs were selected as miRNAs that could potentially regulate the C. irritans resistance of L. crocea. Though these inferences need to be further verified, these findings will be helpful for the research of the molecular mechanism of C. irritans resistance of L. crocea and miRNA-assisted molecular breeding of aquatic animals.