miRNA Expression Analysis Research Articles

TMIC-05. AGING-SPECIFIC MICRORNA EXPRESSION SIGNATURE IN ELDERLY GLIOBLASTOMA

Abstract PURPOSE The rise in aging population worldwide is increasing death from cancer, including glioblastoma. MicroRNAs (miRNAs/miRs) are small non-coding RNAs that mediate the posttranscriptional silencing of specific target mRNAs and that are currently recognized as important regulators of tumorigenesis and development. Here, we explore the impact of brain aging and aging-specific miRNAs on elderly glioblastomas. METHODS We comprehensively analyzed miRNAs expression using the TCGA dataset and identified miRNAs that are specifically expressed in elderly glioblastomas. To create the Ehime Glioma Genome Atlas (EGGA), RNA sequencing was performed using tumor tissues from 8 glioblastoma patients (4 elderly and 4 non-elderly). First, we evaluated the impact of the identified miRNAs on prognosis using TCGA and CGGA datasets and searched for target genes of specific miRNAs using TargetScan. The expression patterns of the identified target genes were verified using EGGA, TCGA, and CGGA datasets. RESULTS We divided the TCGA dataset into elderly and non-elderly groups based on three ages (75 years-old, 70 years-old, and 65 years-old) and analyzed the expression of miRNAs in tumor tissues. There were 3 miRNAs that showed abnormalities (1 increased miRNA, 2 decreased miRNAs), and among these, miR-210 (increased expression) and miR-507 (increased expression) were identified as miRNAs related to prognosis. Next, we analyzed the expression of EGGA and identified 410 mRNAs (368 increased mRNAs, 42 decreased mRNAs) that showed significantly abnormal expression in the elderly group. The target genes searched using TargetScan were 4,048 genes for miR-210 and 4,704 genes for miR-507. When compared with the 410 genes identified using EGGA, PLA2G3, a target gene to miR-507, was identified. The expression of PLA2G3 was also specifically increased in elderly glioblastoma in the TCGA dataset, and it had a strong influence on prognosis. CONCLUSION Our results found an aging-specific miRNA “miR-507” and its target gene “PLA2G3” that suggested as potential new therapeutic target molecules for improving the prognosis of elderly glioblastomas.

Benchmarking miRNA reference genes in B-cell precursor acute lymphoblastic leukemia

MicroRNAs (miRNAs) play dual roles in acute lymphoblastic leukemia (ALL) as both tumor suppressors and oncogenes, and miRNA expression profiles can be used for patient risk stratification. Precise assessment of miRNA levels is crucial for understanding their role and function in gene regulation. Quantitative real-time polymerase chain reaction (qPCR) is a reliable, rapid, and cost-effective method for analyzing miRNA expression, assuming that appropriate normalization to stable references is performed to ensure valid data. In this study, we evaluated the stability of six commonly used miRNA references (5sRNA, SNORD44, RNU6, RNU1A1, miR-103a-3p, and miR-532-5p) across nine B-cell precursor (BCP) ALL cell lines, 22 patient-derived xenograft (PDX) BCP ALL samples from different organ compartments of leukemia bearing mice, and peripheral blood mononuclear cells (PBMCs) from six healthy donors. We used four different algorithms (Normfinder, ∆CT, geNorm, and BestKeeper) to assess the most stably expressed reference across all samples. Moreover, we validated our data in an additional set of 13 PDX ALL samples and six healthy controls, identifying miR-103a-3p and miR-532-5p as the most stable references for miRNA normalization in BCP ALL studies. Additionally, we demonstrated the critical importance of using a stable reference to accurately interpret miRNA data.

MicroRNA Analysis in Meningiomas with Different Degrees of Tissue Stiffness: A Potential Tool for Effective Preoperative Planning.

Meningioma, the most common primary intracranial tumor, presents challenges in surgical treatment because of varying tissue stiffness. This study explores the molecular background of meningioma stiffness, a critical factor in surgical planning and prognosis, focusing on the utility of microRNAs (miRNAs) as diagnostic biomarkers of tissue stiffness. Patients with meningiomas treated surgically at the University Hospital Brno were included in this study. Total RNA, isolated from tumor tissue samples, underwent quality control and small RNA sequencing to analyze miRNA expression. Differentially expressed miRNAs were identified, and their association with tumor stiffness was assessed. This study identified specific miRNAs differentially expressed in meningiomas with different stiffness levels. Key miRNAs, such as miR-31-5p and miR-34b-5p, showed significant upregulation in stiffer meningiomas. These findings were validated using reverse transcription-quantitative polymerase chain reaction, revealing a potential link between miRNA expression and tumor consistency. The expression of miR-31-5p was most notably associated with the stiffness of the tumor tissue (sensitivity = 71% and specificity = 83%). This research highlights the potential of miRNAs as biomarkers for determining meningioma tissue stiffness. Identifying specific miRNAs associated with tumor consistency could improve preoperative planning and patient prognosis. These findings pave the way for further exploration of miRNAs in the clinical assessment of meningiomas.

Plasma exosomes may mediate the development of lupus nephritis in patients with systemic lupus erythematosus.

Lupus nephritis (LN) is the most serious complication of systemic lupus erythematosus (SLE), and plasma exosomes may serve as a bridge. MicroRNAs (miRNAs) are abundant in exosomes, so this study aimed to explore the role of exosome-derived miRNA in the development of LN. The publicly available data containing plasma exosomal miRNAs in SLE patients and healthy controls were researched, and differential expression and functional enrichment analysis of exosomal miRNA was conducted. Then, plasma exosomes from SLE patients were extracted, and the accuracy of differential expression and functional enrichment analysis was preliminarily verified. PKH26 dye was used to label exosomes to detect whether exosomes can enter HK2 cells. Evaluation of plasma exosomes impact on cell viability was done by utilizing CCK-8 assay. Flow cytometry was used to measure cell apoptosis. Plasma exosomes were successfully extracted and identified. Through differential expression analysis of the pulbilic data and subsequent qPCR validation, we observed that miR-20b-5p is overexpressed in plasma exosomes of SLE patients, whereas miR-181a-2-3p is downregulated. Then functional enrichment analysis revealed that these differential miRNAs primarily regulate processes such as apoptosis, autophagy, and inflammation. Then, flow cytometry analysis conducted after co-incubation of plasma exosomes and peripheral blood mononuclear cells confirmed that exosomes can indeed regulate apoptosis. And plasma exosomes can successfully enter HK2 cells without affecting cell activity. In addition, plasma exosomes promote HK2 cell apoptosis and autophagy. Overexpression of miR-181a-2-3p could inhibit HK2 cells apoptosis and upregulate the expression of bcl2, and beclin1. At the same time, a trend towards increased apoptosis rates was observed in HK2 overexpressed miR-20b-5p, although the difference did not reach statistical significance. And miR-20b-5p can enhance the expression of caspase3 and becin1 while suppressing the expression of bcl2 and LC3β. Our research indicates that the abundant presence of miR-20b-5p and the depletion of miR-181a-2-3p in plasma exosomes of SLE patients may mediate the promotion of apoptosis and autophagy in HK2 cells, thereby causing kidney damage and the development of LN.

Unveiling the microRNA landscape in pancreatic ductal adenocarcinoma patients and cancer cell models

BackgroundPancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to late-stage diagnoses resulting from nonspecific early symptoms and the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, and their abnormal expression is observed in various diseases, including cancer. Cancer stem cells (CSCs) are thought to act as a driving force in PDAC spread and recurrence. In pursuing the goal of unravelling the complexities of PDAC and its underlying molecular mechanisms, our study aimed to identify PDAC-associated miRNAs and relate them to disease progression, focusing on their involvement in various PDAC stages in patients and in reliable in vitro models, including pancreatic CSC (PaCSC) models.MethodsThe miRNA profiling datasets of serum and solid biopsies of PDAC patients deposited in GEO DataSets were analyzed by REML-based meta-analysis. The panel was then investigated by Real Time PCR in serum and solid biopsies of 37 PDAC patients enrolled in the study, as well as on BxPC-3 and AsPC-1 PDAC cell lines. We extended our focus towards a possible role of PDAC-associated miRNAs in the CSC phenotype, by inducing CSC-enriched pancreatospheres from BxPC-3 and AsPC-1 PDAC cell lines and performed differential miRNA expression analysis between PaCSCs and monolayer-grown PDAC cell lines.ResultsMeta-analysis showed differentially expressed miRNAs in blood samples and cancerous tissues of PDAC patients, allowing the identification of a panel of 9 PDAC-associated miRNAs. The results emerging from our patients fully confirmed the meta-analysis for the majority of miRNAs under investigation. In vitro tasks confirmed the aberrant expression of the panel of PDAC-associated miRNAs, with a dramatic dysregulation in PaCSC models. Notably, PaCSCs have shown significant overexpression of miR-4486, miR-216a-5p, and miR-216b-5p compared to PDAC cell lines, suggesting the recruitment of such miRNAs in stemness-related molecular mechanisms. Globally, our results showed a dual behaviour of miR-216a-5p and miR-216b-5p in PDAC while miR-4486, miR-361-3p, miR-125a-5p, miR-320d expression changes during the disease suggest they could promote PDAC initiation and progression.ConclusionsThis study contributed to an enhanced comprehension of the role of miRNAs in the development and progression of PDAC, shedding new light on the miRNA landscape in PDAC and its intricate interplay with CSCs, and providing specific insights useful in the development of miRNA-based diagnostic biomarkers and therapeutic targets.

LINCRNA01094 Promotes Renal Interstitial Fibrosis via the Mir-513b-5p/MELK/Smad3 Axis.

Chronic Kidney Disease (CKD) is a common chronic disease that is a threat to human health. Accumulating evidence showed that long noncoding RNAs (lncRNAs) are associated with various diseases and can function as competing endogenous RNAs (ceRNAs). However, the roles and functions of the lncRNA‒miRNA-mRNA network in CKD are still unclear. In this study, we performed differential expression analysis of lncRNAs, miRNAs, and mRNAs in CKD using the datasets GSE66494 and GSE80247 from the Gene Expression Omnibus. A total of 33 lncRNAs, 20 miRNAs, and 240 mRNAs were differentially expressed between CKD patients and healthy controls. Two ceRNA interaction modules composed of 11 hub nodes, namely, 2 lncRNAs (LINC01086, LINC01094), 2 miRNAs (hsa-miR-197-3p, hsamiR- 513b-5p) and 7 mRNAs (CENPF, TOP2A, ARHGAP11A, CEP55, MELK, DTL, and ANLN) were constructed. In vitro knockdown of LINC01094 expression in renal tubular epithelial HK2 cells significantly attenuated the phenotype of TGFβ1-induced cell fibrosis. The results of RNA immunoprecipitation (RIP) experiments and dual-luciferase reporter experiments based on constructed mutants confirmed that LINC01094 could mediate MELK expression by sponging miR-513b-5p. Our observations indicated that lowering the expression of LINC01094 can significantly attenuate the TGFβ1-induced fibrosis phenotype in HK2 cells and renal inflammation through the miR-513b-5p/MELK/Smad3 signalling axis.

Plasma miR-122-5p and miR-142-5p and their role in chemoresistance of colon cancer patients.

Chemoresistance represents a major issue affecting cancer therapy efficacy. Because microRNAs (miRNAs) regulate gene expression on multiple levels, their role in chemoresistance development is reasonably certain. In our previous study, miR-122-5p and miR-142-5p were identified as diagnostic, prognostic, and predictive biomarkers for primary and metastatic rectal cancer. The aim of the present study was to investigate whether these miRNAs can also reflect the disease course of colon cancer (CC) patients. Further, we focused on a deeper understanding of their involvement in 5-fluorouracil (5-FU) chemoresistance development. The expression analysis of both miRNAs was analysed in repeated whole plasma samplings (n=3, approximately every 6 months) of CC patients (n=49) by RT-qPCR. Expression levels of both miRNAs were determined in the 5-FU sensitive and resistant CC cell lines. From RNA-seq profiles of both sensitive and 5-FU resistant DLD-1 cell lines, the expression levels of miR-122-5p and miR-142-5p validated target genes were detected and compared. Significant differences in the expression levels of both miRNAs between T0 and T1 or T2 samplings were observed. Further, an association between the occurrence of relapse and miR-122-5p expression levels was noticed. Patients who did not relapse had higher expression of miR-122-5p at T1 (p=0.01; 3.16-fold change) and T2 (p=0.04; 2.79-fold change) samplings in comparison with T0 sampling. Out of all miR-122-5p validated targets (n=102), 25 genes were significantly differentially expressed between sensitive and 5-FU-resistant cell lines. Our data suggest that miR-122-5p may represent a predictive marker of tumour relapse in CC patients. In vitro data suggests that this aspect may be linked to the potential therapeutic targets of miR-122-5p related to 5-FU-based chemoresistance. However, deeper mechanistic studies are still needed for progress toward personalized medicine.

Abstract P350: Circulating Endothelial microRNAs in Patients with INOCA and Diabetes Mellitus

Ischemia with no obstructive coronary artery (INOCA) is a common cause of hospitalizations, resulting in poor outcomes and diminished quality of life. A key element in the pathophysiology of INOCA is endothelial dysfunction, which leads to myocardial ischemia despite the absence of significant coronary artery obstruction. Effective management of endothelial dysfunction is essential for alleviating symptoms and preventing cardiovascular events in INOCA patients. Recent studies have highlighted diabetes mellitus (DM) as a significant contributor to INOCA complications by exacerbating endothelial impairment and coronary microvascular dysfunction. MicroRNAs (miRNAs), which are known to regulate gene expression, have emerged as potential biomarkers and therapeutic targets in various biological processes, including endothelial dysfunction and cardiovascular diseases. However, there is a lack of research evaluating miRNA biomarkers specifically in INOCA patients. In our study, we examined a panel of circulating miRNAs associated with the regulation of endothelial function in INOCA patients with and without DM. Using RT-qPCR, we analyzed miRNA expression in a cohort of consecutive patients undergoing percutaneous coronary intervention (PCI) who were confirmed to have INOCA. We measured the expression levels of a previously validated panel of miRNAs in INOCA patients with DM compared to those without DM. Specifically, we observed an increased expression of several miRNAs previously associated with endothelial dysfunction and decreased expression of miRNAs known to protect endothelial function. Further analyses revealed that among these miRNAs, miR-363-5p was significantly downregulated (P<0.001) and miR-92a-3p was significantly upregulated (P<0.001) in INOCA patients with DM compared to those without DM, as illustrated in the volcano plot in Figure 1 . Taken together, our findings demonstrated significant dysregulation of miR-363-5p and miR-92a-3p in INOCA patients with DM compared to those without DM, indicating their potential role as biomarkers for endothelial dysfunction in INOCA.

MiRNA expression signatures induced by pasteurella multocida infection in goats lung

MicroRNAs (miRNAs) are important regulators of gene expression and are involved in bacterial pathogenesis and host–pathogen interactions. In this study, we investigated the function of miRNAs in the regulation of host responses to Pasteurella multocida infection. Using next-generation sequencing, we analyzed miRNA expression pattern and identified differentially expressed miRNAs in Pasteurella multocida-infected goat lungs. In addition, we investigated the function of differentially expressed miRNAs andtheir targeted signaling pathways in bacterial infection processes. The results showed that Pasteurella multocida infection led to 69 significantly differentially expressed miRNAs, including 28 known annotated miRNAs with miR-497-3p showing the most significant difference. Gene target prediction and functional enrichment analyses showed that the target genes were mainly involved in cell proliferation, regulation of the cellular metabolic process, positive regulation of cellular process, cellular senescence, PI3K-Akt signaling pathway, FoxO signaling pathway and infection-related pathways. In conclusion, these data provide a new perspective on the roles of miRNAs in Pasteurella multocida infection.

Sensitization of hepatocellular carcinoma cells to HDACi is regulated through hsa-miR-342-5p/CFL1

BackgroundIncreased prevalence of hepatocellular carcinoma (HCC) remains a global health challenge. HCC chemoresistance is a clinical obstacle for its management. Aberrant miRNA expression is a hallmark for both cancer progression and drug resistance. However, it is unclear which miRNAs are involved in HCC chemoresistance.MethodsMicroRNA microarray analysis revealed a differential expression profile of microRNAs between the hepatocellular carcinoma HA22T cell line and the HDACi-R cell line, which was validated by quantitative real-time PCR (qRT-PCR). To determine the biological function of miR-342-5p and the mechanism of the microRNA-342-5p/CFL1 axis in hepatocellular carcinoma HDACi resistance, loss- and gain-of-function studies were conducted in vitro.ResultsHere we demonstrated the molecular mechanism of histone deacetylase inhibitor (HDACi) resistance in HCC. Differential miRNA expression analysis showed significant down regulation of miR-342-5p in HDACi-R cells than in parental HA22T cells. Mimics of miR-342-5p enhanced apoptosis through upregulation of Bax, cyto-C, cleaved-caspase-3 expressions with concomitant decline in anti-apoptotic protein (Bcl-2) in HDACi-R cells. Although HDACi did not increase cell viability of HDACi-R, overexpression of miR-342-5p decreased cofilin-1 expression, upregulated reactive oxygen species (ROS) mediated apoptosis, and sensitized HDACi-R to HDACi in a dose-dependent manner.ConclusionOur findings demonstrated the critical role of miR-342-5p in HDACi resistance of HCC and that this mechanism might be attributed to miR-342-5p/cofilin-1 regulation.

Unique miRNA Expression Profile in MSI- and EMAST-Unstable Sporadic Colon Cancer.

MicroRNAs (miRNAs) are critical post-transcriptional gene regulators and their involvement in sporadic colon cancer (CRC) tumorigenesis has been confirmed. In this study we investigated differences in miRNA expression in microsatellite stable (MSS/EMAST-S), microsatellite unstable marked by high elevated microsatellite alterations at selected tetranucleotide repeats (MSS/EMAST-H), and high microsatellite unstable (MSI-H/EMAST-H) tumor subgroups as well as in tumors with different clinicopathologic characteristics. An RT-qPCR analysis of miRNA expression was carried out on 45 colon cancer and adjacent normal tissue samples (15 of each group). Overall, we found three differentially expressed miRNAs between the subgroups. miR-92a-3p and miR-224-5p were significantly downregulated in MSI-H/EMAST-H tumors in comparison to other subgroups. miR-518c-3p was significantly upregulated in MSS/EMAST-H tumors in comparison to stable and highly unstable tumors. Furthermore, we showed that miR-143-3p and miR-145-5p were downregulated in tumors in comparison to normal tissues in all subgroups. In addition, we showed overexpression of miR-125b-5p in well-differentiated tumors and miR-451a in less advanced tumors. This is the first report on differences in miRNA expression profiles between MSS/EMAST-S, MSS/EMAST-H, and MSI-H/EMAST-H colorectal cancers. Our findings indicate that the miRNA expression signatures differ in CRC subgroups based on their instability status.

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.

Bioinformatic Exploration of Circulating microRNAs Related to Functional Outcomes in Patients With Acute Ischemic Stroke: An Exploratory Prospective Study.

Background Although epigenetic modifications have been expected to play an important role in neuroplasticity for stroke recovery, the role of dynamic microRNA (miRNA) regulation related to functional outcomes after ischemic stroke remains unclear. Therefore, the current study performed a comprehensive miRNA expression analysis in serum to identify specifically altered circulating miRNAs associated with different grades of functional outcomes in patients with acute ischemic stroke (AIS). Methods Twelve patients with AIS in the middle cerebral artery region were included in this study. Peripheral blood samples were collected from patients one or two days after hospitalization. Total RNA, including small RNAs, was extracted from 400 µL of serum, and comprehensive miRNA expression analysis was performed to identify specifically altered circulating miRNAs associated with different grades of functional outcomes. Functional outcomes were evaluated three months after stroke onset using the modified Rankin Scale (mRS), classified as favorable (mRS score of 0 or 1) or unfavorable (mRS score of 2 to 5). Differentially expressed miRNAs were analyzed using the DESeq2 package. Target genes of the miRNAs were explored using miRTargetLink 2.0. Results Acute miRNA expression dynamics were characterized by differences in the patients' functional outcomes following ischemic stroke. The favorable outcome group exhibited significantly downregulated miRNAs, including hsa-miR-218-1, hsa-miR-218-2, hsa-miR-320e, hsa-miR-320d-1, hsa-miR-320d-2, hsa-miR-326, and hsa-miR-4429. In addition, 15 miRNAs, including hsa-miR-223, hsa-miR-18a, hsa-miR-411, and hsa-miR-128-1, were significantly upregulated in the favorable outcome group compared to the unfavorable outcome group. Interesting and strong validated networks between miRNAs and their target genes were identified. Conclusion This study identified specifically altered circulating miRNAs in serum associated with varying grades of functional outcomes in AIS patients and explored miRNA-target gene networks that might contribute to these outcomes. Although further studies are needed, this study highlights their potential role as biomarkers for predicting functional outcomes in patients with AIS.

Selection of references for quantitative real-time PCR analysis of microRNAs in Nile tilapia (Oreochromis niloticus) under osmotic stress

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices.

Endothelial microRNAs in INOCA patients with diabetes mellitus

Ischemia with non-obstructive coronary artery (INOCA) is a common cause of hospital admissions, leading to negative outcomes and reduced quality of life. Central to its pathophysiology is endothelial dysfunction, which contributes to myocardial ischemia despite the absence of significant coronary artery blockage. Addressing endothelial dysfunction is essential in managing INOCA to alleviate symptoms and prevent cardiovascular events. Recent studies have identified diabetes mellitus (DM) as a significant factor exacerbating INOCA complications by promoting endothelial impairment and coronary microvascular dysfunction. MicroRNAs (miRNAs) have emerged as potential biomarkers and therapeutic targets in various biological processes, including endothelial dysfunction and cardiovascular diseases. However, research on miRNA biomarkers in INOCA patients is sparse. In this study, we examined a panel of circulating miRNAs involved in the regulation of endothelial function in INOCA patients with and without DM. We analyzed miRNA expression using RT-qPCR in a cohort of consecutive INOCA patients undergoing percutaneous coronary intervention. We detected a significant dysregulation of miR-363-5p and miR-92a-3p in INOCA patients with DM compared to those without DM, indicating their role as biomarkers for predicting and monitoring endothelial dysfunction in INOCA patients with DM.

Analysis of Carcinogenic Involvement of MicroRNA Pattern in Peripheral Non-Cancerous Tissues and Chronic Viral Liver Injury.

Risk factors for hepatocarcinogenesis include chronic inflammation due to viral infection, liver fibrosis, and aging. In this study, we separated carcinogenic and non-carcinogenic cases due to hepatitis C virus (HCV) infection, aiming to comprehensively analyze miRNA expression in liver tissues by age, and identify factors that contribute to carcinogenesis. Total RNA was extracted from 360 chronic hepatitis C (CH), 43 HCV infected hepatocellular carcinoma (HCC), and surrounding non-tumor (SNT) tissues. MicroRNA (miRNA) expression patterns were analyzed using microarray. Using machine learning, we extracted characteristic miRNA expression patterns for each disease and age. There were no age-dependent changes in miRNA expression in the disease-specific comparisons; however, miRNA expression differed among the age groups of 50, 60, and 70 years of age between CH and SNT. The expression of miRNA was different between SNT and HCC only in patients in their 70s. Of the 55 miRNAs with significant differences in expression between CH and SNT, 34 miRNAs showed significant differences in expression even in the degree of liver fibrosis. The observation that miRNAs involved in hepatocarcinogenesis differ at different ages suggests that the mechanisms of carcinogenesis differ by age group as well. We also found that many miRNAs whose expression did not affect liver fibrosis were involved in carcinogenesis. These findings are expected to define biomarkers for detection of HCC at early stage, and develop novel therapeutic targets for HCC.

Small RNA and Degradome Deep Sequencing Reveal Regulatory Roles of MicroRNAs in Response to Sugarcane Mosaic Virus Infection on Two Contrasting Sugarcane Cultivars.

MicroRNAs (miRNAs) play an essential regulatory role in plant-virus interaction. However, few studies have focused on the roles of miRNAs and their targets after sugarcane mosaic virus (SCMV) infection in sugarcane. To address this issue, we conducted small RNA (sRNA) and degradome sequencing on two contrasting sugarcanes (SCMV-resistant 'Fuoguo1' [FG1] and susceptible 'Badila') infected by SCMV at five time points. A total of 1,578 miRNAs were profiled from 30 sRNA libraries, comprising 660 known miRNAs and 380 novel miRNAs. Differential expression analysis of miRNAs revealed that most were highly expressed during the SCMV exponential phase in Badila at 18 h postinfection, with expression profiles positively correlated with virus replication dynamics as observed through clustering. Analysis of degradome data indicated a higher number of differential miRNA targets in Badila compared to FG1 at 18 h postinfection. Gene ontology (GO) enrichment analysis significantly enriched the stimulus-response pathway, suggesting negative regulatory roles to SCMV resistance. Specifically, miR160 upregulated expression patterns and validated in Badila through quantitative real-time PCR (qRT-PCR) in the early stages of SCMV multiplication. Our research provides new insights into the dynamic response of plant miRNA and virus replication and contributes valuable information on the intricate interplay between miRNAs and SCMV infection dynamics. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Early changes of microRNAs in blood one month after bariatric surgery

BackgroundChanges in microRNAs (miRNAs) are relevant to bariatric surgery and its comorbidities. The characteristics of changes in miRNAs of the early postoperative period following both bariatric procedures, sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB), as well as the factors that related to the effectiveness of early weight loss remain unclear.MethodsWe recruited 18 patients who performed SG and 15 patients who performed RYGB. Their preoperative and 1-month postoperative clinical data and fasting serum samples were collected, and the latter were analyzed by RNA-sequencing. Differential expression analysis of miRNAs was performed by the R-tool. Functional classification annotation and pathway enrichment analysis of targeted genes were analyzed by KOBAS software. The change profiles of miRNAs for both surgeries and their correlation with clinical characteristics and weight loss effectiveness were further analyzed.ResultsA total of 85 differentially expressed miRNAs were identified before and after SG, while a total of 76 were found before and after RYGB. The target genes of these miRNAs were similar in the Gene Ontology enrichment analysis in SG and RYGB, and the enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes was mainly related to metabolic pathways. Hsa-miR-493-5p, hsa-miR-184, and hsa-miR-3199 exhibited similar changes in SG and RYGB, and the former two were correlated with clinical characteristics. Hsa-miR-6729-5p, hsa-miR-4659b-5p, and hsa-miR-2277-5p were correlated with the weight loss effectiveness of SG, while hsa-miR-4662a-5p was correlated with the weight loss effectiveness of RYGB.ConclusionsShort-term metabolic improvement and weight loss occurring after SG and RYGB surgery might be related to changes in miRNAs, which act on multiple biological pathways by regulating genes. In addition, some clinical characteristics and miRNAs were related to the effectiveness of early weight loss after SG and RYGB surgery.Clinical Trial RegistrationChiCTR2200058333.

P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality

Abstract Study question Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance? Summary answer This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status. What is known already We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology. Study design, size, duration In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR. Participants/materials, setting, methods Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change>2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results. Main results and the role of chance We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis. Limitations, reasons for caution This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing. Wider implications of the findings The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions. Trial registration number not applicable

Vitrification of pig embryos dysregulates the microRNA transcriptome profile

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4–6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139–3p, miR-214 and miR-885–3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-β signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-β (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.