Dysregulation Of miRNAs Research Articles

BIOM-53. SPECIFIC MICRORNA EXPRESSION PATTERN PREDICTS RECURRENCE IN ATYPICAL MENINGIOMA

Abstract Meningiomas are the most common primary tumors of the central nervous system (CNS). Atypical meningioma (AM) recurs in 40% of patients despite total resection and radiotherapy (RT). No consensus on optimal adjuvant management was found, and it is difficult to identify patients insensitive to RT in real-life clinical practice. A promising group of biomarkers represent microRNAs (miRNAs), short non-coding RNAs that regulate most biological processes, including cell proliferation, differentiation, and apoptosis. The study aims to identify tissue miRNAs capable of predicting patients with AM who could benefit from the indicated adjuvant RT. The study includes 80 patients with AM in the exploratory phase and 400 patients with meningioma in the validation phase. Total RNA enriched with miRNAs was isolated from FFPE tissue using the mirVana miRNA Isolation Kit (TF Scientific). Subsequently, RNA quantity and quality controls were measured using NanoDrop 2000 (TF Scientific) and Qubit 2.0 (TF Scientific) instruments. A global miRNA expression profile was generated using the TaqMan Array Human MicroRNA Cards (TF Scientific), which allow the detection of up to 754 miRNAs simultaneously. Obtained data were processed and integrated through bioinformatics algorithms with clinicopathological data of patients with AM.The study identified significantly dysregulated miRNAs among AM patients with and without recurrence (p < 0.05). Results also suggest dysregulated miRNA expression profile in AM patients with indicated RT who did/did not develop a recurrence (p < 0.05). Lists of individual miRNAs and detailed graphical analyses will be included in the conference presentation.The results will help predict the prognosis of surgically intervened patients more accurately and can help determine which patients will benefit from the adjuvant RT. This research is supported by the AZV grant from the Ministry of Health of the Czech Republic (reg. No. NV19-03-00559) and Conceptual development of research organization (FNBr, 65269705). All rights reserved.

PATH-02. ANALYSIS OF MICRORNA EXPRESSION IN BRAIN METASTASES USING NEXT-GENERATION SEQUENCING

Abstract BACKGROUND Brain metastases (BMs) are intracranial tumors that frequently occur in adult cancer patients. Because the prognosis of patients with BM individually varies, it would be useful to improve prognostic scoring tools by including new high-performance biomarkers. MicroRNAs (miRNAs) appear to be promising in this regard as they are highly stable and, thus, suitable for both next-generation sequencing (RNA-Seq) and retrospective analyses in formalin-fixed and paraffin-embedded (FFPE) tissues.Material and METHODS Total RNA enriched for miRNAs was isolated from 71 freshly frozen histopathologically confirmed BMs with origin in 5 tumor types (ca lung - 37%, melanoma - 23%, ca breast - 18%, RCC - 15%, CRC - 7%) using mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Sequencing libraries were prepared from RNA using the QIAseq miRNA Library Kit (Qiagen) and sequenced using the NextSeq 500 platform (Illumina). The miRNA molecules were subsequently transcribed from total RNA samples isolated from a retrospective set of 119 FFPE tissues using the TaqMan Advanced miRNA cDNA Synthesis Kit, and the expression of selected miRNAs was validated in a pilot experiment by qPCR using TaqMan Fast Advanced Master Mix and appropriate TaqMan MicroRNA Assays (all from Thermo Fisher Scientific). RESULTS The differential analysis identified 373 miRNAs with significantly different expression among the 5 BMs groups (p< 0.001). A molecular classifier based on the expression of 32 miRNAs was able to classify all samples correctly. Out of these, seven, including miR-122-5p, miR-141-3p, miR-146a-5p, miR-194-5p, miR-200c-3p, miR-211-3p, and miR-215-5p, were chosen for subsequent validation and their significantly different expression in 5 BMs groups was validated. CONCLUSIONS Presented results confirm the importance of studying dysregulated miRNA expression in BM and the diagnostic potential of validated miRNAs. The study was prepared with the grant support of the Ministry of Health of the Czech Republic - grant No. NV18-03-00398.

The hsa-miR-516a-5p and hsa-miR-516b-5p microRNAs reduce the migration and invasion on T98G glioblastoma cell line

microRNAs (miRNAs) are involved in numerous functions and processes in the brain and other organs through the regulation of gene and protein expression. miRNA dysregulation is associated with the development of several diseases, including the brain and Central Nervous System cancer (CNS). The hsa-miR-516a-5p and hsa-miR-516b-5p are involved in proliferation, migration, and invasion in different tumor models, but their antitumor effect has not been evaluated in cancer of CNS. Therefore, we aimed to assess the effect of the miRNAs hsa-miR-516a-5p and miRNA hsa-miR-516b-5p on the Glioblastoma cell line (T98G). We used synthetic miRNA mimics to induce the overexpression of both miRNAs in the cell line, which was corroborated by RT-qPCR. Next, we evaluated the effect on proliferation, migration, and invasion using the CyQuant direct kit, ThinCert ™ inserts and invasion BioCoat ™ Matrigel® Invasion Chambers. We found upregulation of these miRNAs induced significant changes on the migration and invasion processes of T98G cells, but not affected the proliferation rate. These results suggest that both microRNAs could be playing an important role in the control of tumor progression towards metastasis. The bioinformatics analysis showed that target genes for these miRNAs are involved in different biological processes such as in cell adhesion molecule binding and cell junction disassembly, which are important for cancer progression. Further studies and experimental validation are needed to identify the genes regulated by microRNAs.

Differential Expression of lncRNA-miRNA-mRNA and Their Related Functional Networks in New-Onset Type 2 Diabetes Mellitus among Chinese Rural Adults.

Increasing evidence suggested that the expression and inter-regulation of long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) were related to the development of diabetes. Based on bioinformatics analysis, this study aimed to comprehensively analyze the dysregulated RNA molecules related to new-onset type 2 diabetes mellitus (T2DM). Twenty-four patients with new-onset T2DM were included as cases, and sex- and age-matched participants were included as controls. The differentially expressed lncRNAs, miRNAs, and mRNAs between the two groups were screened by RNA sequencing. LncRNA-miRNA-mRNA network and enrichment analysis were used to reveal the RNA molecules that were potentially associated with T2DM and their early changes. A total of 123 lncRNAs, 49 miRNAs, and 312 mRNAs were differentially expressed in the new-onset T2DM (fold change ≥ 1.5 and p value < 0.05). Functional analysis revealed that differentially expressed RNAs were likely to play essential roles in diabetes-related pathways. In addition, the protein-protein interaction (PPI) network screened multiple hub mRNAs, and lncRNA-miRNA-mRNA networks showed that a single miRNA could be related to multiple lncRNAs, and then they coregulated more mRNAs. SLC25A4, PLCB1, AGTR2, PRKN, and SCD5 were shown to be important mRNAs in T2DM, and miR-199b-5p, miR-202-5p, miR-548o-3p as well as miR-1255b-5p could be involved in their regulation. In conclusion, several new and previously identified dysregulated lncRNAs, miRNAs, and mRNAs were found to be vital biomarkers in T2DM. Their alterations and interactions could modulate the pathophysiology of T2DM. Those findings may provide new insights into the molecular mechanisms underlying the development of T2DM.

A Comprehensive Overview of Recent Advances in Epigenetics in Pediatric Acute Lymphoblastic Leukemia.

Recent years have brought a novel insight into our understanding of childhood acute lymphoblastic leukemia (ALL), along with several breakthrough treatment methods. However, multiple aspects of mechanisms behind this disease remain to be elucidated. Evidence suggests that leukemogenesis in ALL is widely influenced by epigenetic modifications. These changes include: DNA hypermethylation, histone modification and miRNA alteration. DNA hypermethylation in promoter regions, which leads to silencing of tumor suppressor genes, is a common epigenetic alteration in ALL. Histone modifications are mainly caused by an increased expression of histone deacetylases. A dysregulation of miRNA results in changes in the expression of their target genes. To date, several hundred genes were identified as suppressed by epigenetic mechanisms in ALL. What is promising is that epigenetic alterations in ALL may be used as potential biomarkers for classification of subtypes, predicting relapse and disease progression and assessing minimal residual disease. Furthermore, since epigenetic lesions are potentially reversible, an activation of epigenetically silenced genes with the use of hypomethylating agents or histone deacetylase inhibitors may be utilized as a therapeutic strategy for ALL. The following review summarizes our current knowledge about epigenetic modifications in ALL and describes potential uses of epigenetics in the clinical management of this disease.

The role of altered microRNAexpression in premalignant and malignant head and neck lesions with epithelial origin.

The premalignant lesions of the oral cavity carry a risk of transformation to malignancy. Hence, early diagnosis followed by timely intervention remarkably affects the prognosis of patients. During tumorigenesis, particular microRNAs (miRNAs) show altered expressions and because of their post transcriptionally regulatory role could provide favorable diagnostic, therapeutic, or prognostic values in head and neck cancers. In this review, we have demonstrated diagnostic, prognostic, and potential therapeutic roles of some miRNAs associated with oral premalignant and malignant lesions based on previous validate studies. It is previously documented that dysregulation of miRNAs contributes to cancer development and progression. MiRNAscould be tumor suppressors that normally suppress cell proliferation, differentiation, and apoptosis or play as oncogenes that improved tumorigenesis process. Altered expression of miRNAs has also been reported in premalignant oral epithelial lesions such as leukoplakia, oral submucous fibrosis, oral lichen planus and some malignant carcinoma like oral squamous cell, verrucous, spindle cell, Merkel cell carcinoma and basal cell. Some of miRNAs could be new therapeutic candidates in miRNA-based target gene therapy. Although more investigations are required to identify the most favorable miRNA candidate, altered expression of some miRNAs could be used as biomarkers in premalignant lesions and oral cancers with high sensitivity and specificity.

Increased Serum Levels of miR-125b and miR-132 in Fragile X Syndrome: A Preliminary Study.

Background and ObjectivesFragile X syndrome (FXS) is a neurodevelopmental disorder, identified as the most common cause of hereditary intellectual disability and monogenic cause of autism spectrum disorders (ASDs), caused by the loss of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein, a regulator of translation that plays an important role in neurodevelopment, and its loss causes cognitive and behavioral deficits. MicroRNAs (miRNAs) are small molecules that regulate gene expression in diverse biological processes. Previous studies found that the interaction of FMRP with miR-125b and miR-132 regulates the maturation and synaptic plasticity in animal models and miRNA dysregulation plays a role in the pathophysiology of FXS. The present study aimed to analyze the expression of miR-125b-5p and miR-132-3p in the serum of patients with FXS.MethodsThe expressions of circulating miRNAs were studied in the serum of 10 patients with FXS and 20 controls using the real-time quantitative retrotranscribed method analyzed by relative quantification. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated to assess the diagnostic values of the miRNAs.ResultsWe found that both miR-125b and miR-132 were increased in the serum of patients with FXS compared with controls and likely involved with FMRP loss. The AUC (95% confidence interval) of miR-125b and miR-132 was 0.94 (0.86–1.0) and 0.89 (0.77–1.0), respectively. Databases allowed for the identification of possible target genes for miR-125b and miR-132, whose products play an important role in the homeostasis of the nervous system.DiscussionOur results indicate that serum miR-125b and miR-132 may serve as potential biomarkers for FXS. The increased expression of circulating miR-125b and miR-132 seems to be associated with the genotype of FXS. Predicted gene targets of the differentially regulated miRNAs are involved in cognitive performance and ASD phenotype.Classification of EvidenceThis study provides Class III evidence that miR-125b and miR-132 distinguish men with FXS from normal controls.

MiRNAs in anti-cancer drug resistance of non-small cell lung cancer: Recent advances and future potential.

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors worldwide. Clinical success is suboptimal owing to late diagnosis, limited treatment options, high recurrence rates, and the development of drug resistance. MicroRNAs (miRNAs), a range of small endogenous non-coding RNAs that are 22 nucleotides in length, have emerged as one of the most important players in cancer initiation and progression in recent decades. Current evidence has revealed the pivotal roles of miRNAs in regulating cell proliferation, migration, invasion, and metastasis in NSCLC. Recently, several studies have demonstrated that miRNAs are strongly associated with resistance to anti-cancer drugs, ranging from traditional chemotherapeutic and immunotherapy drugs to anti-vascular drugs, and even during radiotherapy. In this review, we briefly introduce the mechanism of miRNA dysregulation and resistance to anti-tumor therapy in NSCLC, and summarize the role of miRNAs in the malignant process of NSCLC. We then discuss studies of resistance-related miRNAs in chemotherapy, radiotherapy, targeted therapy, immunotherapy, and anti-vascular therapy in NSCLC. Finally, we will explore the application prospects of miRNA, an emerging small molecule, for future anti-tumor therapy. This review is the first to summarize the latest research progress on miRNAs in anti-cancer drug resistance based on drug classification, and to discuss their potential clinical applications.

Serum extracellular vesicle microRNA dysregulation and childhood trauma in adolescents with major depressive disorder.

Major depressive disorder (MDD) seriously endangers adolescent mental and physical health. Extracellular vesicles (EVs) are mediators of cellular communication and are involved in many physiological brain processes. Although EV miRNAshave been implicated in adults with major psychiatric disorders, investigation into their effects in adolescent MDDremains scarce. In discovery set, we conducted a genome-wide miRNA sequencing of serum EVs from 9 untreated adolescents with MDD and 8 matched healthy controls (HCs), identifying 32 differentially expressed miRNAs (18 upregulated and 14 downregulated). In the validation set, 8 differentially expressed and highly enriched miRNAs were verified in independent samples using RT-PCR, with 4 (miR-450a-2-3p, miR-3691-5p, miR-556-3p, and miR-2115-3p) of the 8 miRNAs found to be significantly elevated in 34 untreated adolescents with MDD compared with 38 HCs and consistent with the sequencing results. After the Bonferroni correction, we found that three miRNAs (miR-450a-2-3p, miR-556-3p, and miR-2115-3p) were still significantly different. Among them, miR-450a-2-3p showed the most markeddifferential expression and was able to diagnose disease with 67.6% sensitivity and 84.2% specificity. Furthermore, miR-450a-2-3p partially mediated the associations between total childhood trauma, emotional abuse, and physical neglect and adolescent MDD. We also found that the combination of miR-450a-2-3p and emotional abuse could effectively diagnose MDD in adolescents with 82.4% sensitivity and 81.6% specificity. Our data demonstrate the association of serum EV miRNA dysregulation with MDD pathophysiology and, furthermore, show that miRNAs may mediate the relationship between early stress and MDD susceptibility. We also provide a valid integrated model for the diagnosis of adolescent MDD.

Current Insights into miRNA and lncRNA Dysregulation in Diabetes: Signal Transduction, Clinical Trials and Biomarker Discovery.

Diabetes is one of the most frequently occurring metabolic disorders, affecting almost one tenth of the global population. Despite advances in antihyperglycemic therapeutics, the management of diabetes is limited due to its complexity and associated comorbidities, including diabetic neuropathy, diabetic nephropathy and diabetic retinopathy. Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are involved in the regulation of gene expression as well as various disease pathways in humans. Several ncRNAs are dysregulated in diabetes and are responsible for modulating the expression of various genes that contribute to the ‘symptom complex’ in diabetes. We review various miRNAs and lncRNAs implicated in diabetes and delineate ncRNA biological networks as well as key ncRNA targets in diabetes. Further, we discuss the spatial regulation of ncRNAs and their role(s) as prognostic markers in diabetes. We also shed light on the molecular mechanisms of signal transduction with diabetes-associated ncRNAs and ncRNA-mediated epigenetic events. Lastly, we summarize clinical trials on diabetes-associated ncRNAs and discuss the functional relevance of the dysregulated ncRNA interactome in diabetes. This knowledge will facilitate the identification of putative biomarkers for the therapeutic management of diabetes and its comorbidities. Taken together, the elucidation of the architecture of signature ncRNA regulatory networks in diabetes may enable the identification of novel biomarkers in the discovery pipeline for diabetes, which may lead to better management of this metabolic disorder.

Cardiovascular Diseases and their Novel Therapeutic Interventions: A Literature Review

Introduction: Cardiovascular diseases (CVD) are responsible for millions of deaths worldwide every year and remain one of the main causes of death in low- middle-income countries. Current methods of treating CVD involve the use of lipid- lowering drugs although these patients continue to suffer from atherosclerotic disease risk. Novel RNA therapeutic strategies are being brought to light with the advancement in our understanding of cellular mechanisms and communication, however, these need to be evaluated critically before their clinical use. Methods: Electronic literature databases such as PubMed and Google Scholar were used to access review papers and research studies done in the past 25 years. Studies most relevant to RNA cardiovascular therapeutics were used to study therapeutic interventions and their limitations. Results: MicroRNAs (miRNAs), a subset of non-coding RNAs play an important function in cell-cell communication and microenvironment remodeling due to their role in cellular processes such as differentiation, proliferation, and apoptosis. Dysregulation of miRNA synthesis has been shown to drive disease pathology. Administration of the miRNAs downregulated during disease or silencing the activity of pathogenic miRNA can be used to establish the genetic composition of a healthy individual. Exosomes are cell-derived bilipid layer extracellular vesicles, 40-150 nm in size, which conduct paracrine signaling by carrying a cargo of mRNAs, non-coding RNAs, and proteins. They could be used as an efficient delivery method for miRNAs. Gene silencing therapies targeting the ApoCIII gene have emerged as novel therapeutic interventions to treating CVD with genome-wide association studies demonstrating enhanced cardioprotective function with ApoCIII deficiency. Gene silencing through miRNA delivery and antisense oligonucleotides reveals new avenues of CVD treatment. Discussion: Novel therapeutics addressing miRNA dysregulation and gene expression regulation come with caveats that need to be addressed before they are prescribed. This review describes the role of the gene silencing interventions and the implementation barriers that delay their approval for use in treating heart disease. Conclusion: The treatments and limitations addressed in this review suggest more studies are needed to determine the pharmacokinetic aspects of RNA drugs prior to establishing the use of RNA therapeutics along with conventional cholesterol-lowering drugs to ameliorate CVD risk.

Recent advances in the diagnostic and therapeutic roles of microRNAs in colorectal cancer progression and metastasis.

Colorectal cancer (CRC) is the third most common malignancy in the world and one of the leading causes of cancer death; its incidence is still increasing in most countries. The early diagnostic accuracy of CRC is low, and the metastasis rate is high, resulting in a low survival rate of advanced patients. MicroRNAs (miRNAs) are a small class of noncoding RNAs that can inhibit mRNA translation and trigger mRNA degradation, and can affect a variety of cellular and molecular targets. Numerous studies have shown that miRNAs are related to tumour progression, immune system activity, anticancer drug resistance, and the tumour microenvironment. Dysregulation of miRNAs occurs in a variety of malignancies, including CRC. In this review, we summarize the recent research progress of miRNAs, their roles in tumour progression and metastasis, and their clinical value as potential biomarkers or therapeutic targets for CRC. Furthermore, we combined the roles of miRNAs in tumorigenesis and development with the therapeutic strategies of CRC patients, which will provide new ideas for the diagnosis and treatment of CRC.

MicroRNAs Dysregulation as Potential Biomarkers for Early Diagnosis of Endometriosis.

Endometriosis is a benign chronic disease in women that is characterized by the presence of active foci of the endometrium or endometrial tissue occurring outside of the uterus. The disease causes disabling symptoms such as pelvic pain and infertility, which negatively affect a patient’s quality of life. In addition, endometriosis imposes an immense financial burden on the healthcare system. At present, laparoscopy is the gold standard for diagnosing the disease because other non-invasive diagnostic tests have less accuracy. In addition, other diagnostic tests have low accuracy. Therefore, there is an urgent need for the development of a highly sensitive, more specific, and non-invasive test for the early diagnosis of endometriosis. Numerous researchers have suggested miRNAs as potential biomarkers for endometriosis diagnosis due to their specificity and stability. However, the greatest prognostic force is the determination of several miRNAs, the expression of which varies in a given disease. Despite the identification of several miRNAs, the studies are investigatory in nature, and there is no consensus on them. In the present review, we first provide an introduction to the dysregulation of miRNAs in patients with endometriosis and the potential use of miRNAs as biomarkers in the detection of endometriosis. Then we will describe the role of the mir-200 family in endometriosis. Several studies have shown that the expression of the mir-200 family changes in endometriosis patients, suggesting that they could be used as a diagnostic biomarker and therapeutic target for endometriosis.

Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development.

MicroRNA Biology on Brain Development and Neuroimaging Approach.

Proper brain development requires the precise coordination and orchestration of various molecular and cellular processes and dysregulation of these processes can lead to neurological diseases. In the past decades, post-transcriptional regulation of gene expression has been shown to contribute to various aspects of brain development and function in the central nervous system. MicroRNAs (miRNAs), short non-coding RNAs, are emerging as crucial players in post-transcriptional gene regulation in a variety of tissues, such as the nervous system. In recent years, miRNAs have been implicated in multiple aspects of brain development, including neurogenesis, migration, axon and dendrite formation, and synaptogenesis. Moreover, altered expression and dysregulation of miRNAs have been linked to neurodevelopmental and psychiatric disorders. Magnetic resonance imaging (MRI) is a powerful imaging technology to obtain high-quality, detailed structural and functional information from the brains of human and animal models in a non-invasive manner. Because the spatial expression patterns of miRNAs in the brain, unlike those of DNA and RNA, remain largely unknown, a whole-brain imaging approach using MRI may be useful in revealing biological and pathological information about the brain affected by miRNAs. In this review, we highlight recent advancements in the research of miRNA-mediated modulation of neuronal processes that are important for brain development and their involvement in disease pathogenesis. Also, we overview each MRI technique, and its technological considerations, and discuss the applications of MRI techniques in miRNA research. This review aims to link miRNA biological study with MRI analytical technology and deepen our understanding of how miRNAs impact brain development and pathology of neurological diseases.

Crosstalk of miRNAs with signaling networks in bladder cancer progression: Therapeutic, diagnostic and prognostic functions

Urological cancers are considered as life-threatening diseases around the world. Bladder cancer is one of the most malignant urological tumors with high mortality and morbidity. Bladder cancer is a heterogenous disease and genetic alterations have shown to be key players in regulating its progression. Although conventional therapies are somewhat beneficial in improving prognosis and survival, bladder cancer patients suffer from recurrence. MicroRNAs (miRNAs) are endogenous short RNA molecules that do not encode proteins and show dysregulated expression in human cancers. miRNAs are regulators of vital biological processes in cells such as proliferation, migration, differentiation and apoptosis. Dysregulation of miRNAs is observed in bladder cancer and they are used as biomarkers for diagnosis and prognosis of patients. LncRNAs and circRNAs are modulators of bladder cancer progression via miRNA expression regulation. Overexpression of onco-suppressor miRNAs impairs bladder cancer progression, while oncogenic miRNAs drive tumor progression. Glycolysis and EMT mechanisms are two important factors for proliferation and migration of bladder cancer that are modulated by miRNAs. Furthermore, miRNAs can affect STAT3 and Wnt/β-catenin as instances of molecular factors in regulating bladder tumor progression. Bladder tumor response to drug therapy and radiotherapy is regulated by miRNAs. Hence, aim of current review is to provide function of miRNAs in bladder cancer based on their crosstalk with other molecular pathways and interaction with biological processes.

A miRNA derived from the 17/92 cluster is a novel player in programming pulmonary hypertension in response to gestational hypoxia via NADPH oxidases and the mTOR pathway

Abstract Background Gestational hypoxia can lead to intrauterine growth restriction (IUGR) and programming of cardiovascular diseases in adulthood via different, although not completely understood, epigenetic mechanisms. We have previously shown that reactive oxygen species (ROS) derived from NADPH oxidases contribute to hypoxia induced pulmonary hypertension (PH). However, their role in disease programming by gestational hypoxia is not resolved. Purpose We aimed to elucidate the role of NADPH oxidases and redox sensitive miRNAs in the cardiovascular response of adult offspring to transient gestational hypoxia. Methods Wildtype (WT) and nmf333 mice, lacking a functional p22phox dependent NADPH oxidase, were exposed to gestational hypoxia from E10.5 to E11.5. Embryos and adult offspring were analyzed by functional, immunohistochemical and molecular methods including mRNA and miRNA profiling. miRNA mimics or inhibitors were applied in vivo and in vitro using embryoid bodies (EBs). Heart samples from patients with right heart disease associated with congenital heart disease (CHD) were used to validate target miRNA expression. Results Transient gestational hypoxia was sufficient to induce IUGR in embryos and pulmonary hypertension (PH) characterized by right ventricular hypertrophy, increased right ventricular pressure and pulmonary vascular remodeling in adult offspring from WT mice while p22phox-deficient nmf333 mice were protected. Cardiac mRNA and miRNA profiling followed by qPCR validation identified several single miRNAs derived from the 17/92 miRNA cluster as downregulated by gestational hypoxia and NADPH oxidases in embryos and adult offspring hearts. In vitro analyses using EBs validated a single miRNA from this cluster to be sufficient to induce cardiac differentiation in response to hypoxia and p22phox, and target analyses identified the mTOR pathway as dysregulated. Treatment with an inhibitor targeting this miRNA was sufficient to induce PH in adult mice, while treating offspring derived from gestational hypoxia pregnancies with an miRNA mimic prevented the development of PH in the adult and dysregulation of the mTOR pathway. Importantly, expression of this single miRNA from the miRNA 17/92 cluster was negatively correlated to right ventricular pressure in CHD patients. Conclusions In this study, we demonstrate that gestational hypoxia programs PH in adulthood by upregulating NADPH oxidases and downregulating miRNAs derived from the 17/92 cluster subsequently affecting the mTOR pathway. Since dysregulation of a single miRNA from this cluster induced not only PH but was also associated with right ventricular pressure in CHD patients, these findings might open novel options for diagnosis, prevention and treatment of right heart disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): DZHK (German Centre for Cardiovascular Research)

Molecular and haemodynamic interplay in bicuspid aortic valve aortopathy: segmental differences across the aortic circumference

Abstract Background Bicuspid aortic valve (BAV) patients develop ascending aortic (AAo) dilation. The pathogenesis of BAV-aortopathy (genetic vs. hemodynamic) remains unclear. Purpose To identify regional changes around the AAo wall in BAV patients with aortopathy integrating clinical imaging and molecular data. Methods BAV patients with aortopathy (n=20) were recruited prospectively to surgically collect aortic tissue and perform four-dimensional cardiac magnetic resonance (CMR) scans. Molecular markers were measured analysing the tissue biopsies (n=15) and wall shear stress (WSS) across the AAo circumference was calculated from the 4D CMR data (n=11 patients, and n=7 additional healthy volunteers for comparison). Dilated (anterior/right) vs. non-dilated (posterior/left) circumferential segments were profiled for whole genomic microRNAs (Next Generation RNA-Sequencing, miRCURY LNA PCR), proteins content (Tandem Mass Spectrometry) and elastin fragmentation and degeneration (histomorphometric analysis). Picture 1 summarises the study approach (including a map of the AAo divided in the following segments: A=anterior, AR=anterior-right, PR=posterior-right, P=posterior, PL=posterior-left, AL=anterior-left). Results Integrated bioinformatic analyses of RNA-sequencing and proteomic datasets identified 5 microRNAs (miR-128-3p, miR-210-3p, miR-150-5p, miR-199b-5p, and miR-21-5p) differentially expressed across the AAo circumference. Among them, three miRNAs (miR-128-3p, miR-150-5p, and miR-199b-5p) were predicted to have an effect on eight common target genes, whose expression was dysregulated according to proteomic analyses and involved in vascular endothelial growth factor signalling, hippo signaling and arachidonic acid pathway. Decreased elastic fibre levels and elastic layer thickness were observed in the dilated segments in the histomorphometric analysis. Increased WSS and flow-velocity, helical streamlines and asymmetrical flow were observed at the anterior/right wall in asymmetrically and symmetrically dilated patients (the latter exhibiting higher WSS), compared to healthy volunteers. Conclusion This study has newly revealed concomitant expressional dysregulation of miRNAs, proteins, and elastic fibres on the anterior/right wall in dilated BAV patients, corresponding to regions of elevated WSS, contributing to advance the understanding of the interplay of genetic and hemodynamic factors underpinning BAV aortopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation and Above &amp; Beyond

MiR‑92a represses the viability and migration of nerve cells in Hirschsprung's disease by regulating the KLF4/PI3K/AKT pathway.

Hirschsprung's disease (HSCR) is an intestinal disease caused by defects in neural crest cell migration, proliferation, differentiation, and survival. Many reports have proposed that miRNA dysregulation is related to the occurrence of HSCR. However, the roles and mechanisms of miRNAs have not been thoroughly studied. The levels of miR‑92a and KLF4 were examined using qRT‑PCR and immunohistochemistry, respectively. Cell viability, migration and apoptosis were evaluated by MTT, Transwell and flow cytometry assays, respectively. A dual‑luciferase reporter assay was employed to verify the binding relationship between miR‑92a and KLF4. Levels of PI3K/AKT signals were further determined by western blot assay. Herein, elevated expression of miR‑92a and reduced expression of KLF4 were found in HSCR tissues, and their expression patterns were negatively correlated. Overexpression of miR‑92a inhibited cell viability and migration but enhanced cell apoptosis. However, overexpression of KLF4 had the opposite effects. Mechanistically, KLF4 was a target of miR‑92a and it negatively affected biological functions by activating PI3K/AKT signaling. These results proved that miR‑92a inhibited the proliferation and metastasis of nerve cells by regulating the KLF4/PI3K/AKT axis.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes

BackgroundRheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PurposeThis study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. MethodsWe identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. ResultsOur results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. ConclusionOur research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.