Altered microRNA Research Articles

MicroRNA and Alternative mRNA Splicing Events in Cancer Drug Response/Resistance: Potent Therapeutic Targets.

Cancer is a multifaceted disease that involves several molecular mechanisms including changes in gene expression. Two important processes altered in cancer that lead to changes in gene expression include altered microRNA (miRNA) expression and aberrant splicing events. MiRNAs are short non-coding RNAs that play a central role in regulating RNA silencing and gene expression. Alternative splicing increases the diversity of the proteome by producing several different spliced mRNAs from a single gene for translation. MiRNA expression and alternative splicing events are rigorously regulated processes. Dysregulation of miRNA and splicing events promote carcinogenesis and drug resistance in cancers including breast, cervical, prostate, colorectal, ovarian and leukemia. Alternative splicing may change the target mRNA 3′UTR binding site. This alteration can affect the produced protein and may ultimately affect the drug affinity of target proteins, eventually leading to drug resistance. Drug resistance can be caused by intrinsic and extrinsic factors. The interplay between miRNA and alternative splicing is largely due to splicing resulting in altered 3′UTR targeted binding of miRNAs. This can result in the altered targeting of these isoforms and altered drug targets and drug resistance. Furthermore, the increasing prevalence of cancer drug resistance poses a substantial challenge in the management of the disease. Henceforth, molecular alterations have become highly attractive drug targets to reverse the aberrant effects of miRNAs and splicing events that promote malignancy and drug resistance. While the miRNA–mRNA splicing interplay in cancer drug resistance remains largely to be elucidated, this review focuses on miRNA and alternative mRNA splicing (AS) events in breast, cervical, prostate, colorectal and ovarian cancer, as well as leukemia, and the role these events play in drug resistance. MiRNA induced cancer drug resistance; alternative mRNA splicing (AS) in cancer drug resistance; the interplay between AS and miRNA in chemoresistance will be discussed. Despite this great potential, the interplay between aberrant splicing events and miRNA is understudied but holds great potential in deciphering miRNA-mediated drug resistance.

Identification and integrative analysis of microRNAs in myelodysplastic syndromes based on microRNAs expression profile

AbstractMyelodysplastic syndromes (MDS) are a group of malignant hematological disorders characterized by the abnormal development of hematopoietic stem cells and increased risk of acute myelogenous leukemia. Although the pathogenesis of MDS has not been fully understood, various alterations of microRNAs (miRNAs) have been reported in MDS. This study aimed to explore the molecular mechanisms of MDS by integrative bioinformatics analysis of miRNAs expression profile. The GSE81372 expression profile dataset was downloaded from Gene Expression Omnibus database. The differentially expressed miRNAs (DEMs) between MDS and normal controls were identified and targets of miRNAs were predicted. Subsequently, gene ontology (GO) functional and pathway enrichment analyses of target genes were performed. Finally, pathway relation network and miRNA–GO regulatory network were constructed and analyzed. A total of six upregulated and 35 downregulated DEMs were identified. The results showed that target genes of DEMs mainly participated in the process of signal transduction, blood coagulation, apoptotic process, cell proliferation, transmembrane transport, and angiogenesis. The significantly enriched pathways included MAPK signaling pathway, PI3K‐Akt signaling pathway, TGF‐beta signaling pathway, Hippo signaling pathway, and P53 signaling pathway. Moreover, miR‐195‐5p, miR‐4505, miR‐22‐3p, and miR‐148a‐3p were selected as hub miRNAs in miRNA–GO regulatory network and their aberrant expression might be closely associated with MDS pathogenesis. Our discovery provides a registry of miRNAs and pathways that are disrupted in MDS, which has the potential to be used in clinic for diagnosis and target therapy of MDS in future.

Vascular calcium signalling and ageing.

Changes in cellular Ca2+ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca2+ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing per se on vascular Ca2+ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca2+ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca2+ channels, Ca2+ -activated K+ channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca2+ handling was suppressed in ageing mainly via reduced expression of intracellular Ca2+ release channels, and Ca2+ reuptake or efflux pumps. An increase in mitochondrial Ca2+ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca2+ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca2+ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world's ageing population, future studies should be directed at solving specific ageing-induced Ca2+ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases.

Lack of miRNA-17 family mediates high glucose-induced PAR-1 upregulation in glomerular mesangial cells.

Upregulation of thrombin receptor protease-activated receptor 1 (PAR-1) is verified to contribute to chronic kidney diseases, including diabetic nephropathy; however, the mechanisms are still unclear. In this study, we investigated the effect of PAR-1 on high glucose-induced proliferation of human glomerular mesangial cells (HMCs), and explored the mechanism of PAR-1 upregulation from alteration of microRNAs. We found that high glucose stimulated proliferation of the mesangial cells whereas PAR-1 inhibition with vorapaxar attenuated the cell proliferation. Moreover, high glucose upregulated PAR-1 in mRNA level and protein expression while did not affect the enzymatic activity of thrombin in HMCs after 48h culture. Then high glucose induced PAR-1 elevation was likely due to the alteration of the transcription or post-transcriptional processing. It was found that miR-17 family members including miR-17-5p, -20a-5p, and -93-5p were significantly decreased among the eight detected microRNAs only in high glucose-cultured HMCs, but miR-129-5p, miR-181a-5p, and miR-181b-5p were markedly downregulated in both high glucose-cultured HMCs and equivalent osmotic press control compared with normal glucose culture. So miR-20a was selected to confirm the role of miR-17 family on PAR-1 upregulation, finding that miR-20a-5p overexpression reversed the upregulation of PAR-1 in mRNA and protein levels induced by high glucose in HMCs. In summary, our finding indicated that PAR-1 upregulation mediated proliferation of glomerular mesangial cells induced by high glucose, and deficiency of miR-17 family resulted in PAR-1 upregulation.

Sex-Specific Alterations in Inflammatory MicroRNAs in Mouse Brain and Bone Marrow CD11b+ Cells Following Traumatic Brain Injury.

Sex is a key biological variable in traumatic brain injury (TBI) and plays a significant role in neuroinflammatory responses. However, the molecular mechanisms contributing to this sexually dimorphic neuroinflammatory response remain elusive. Here we describe a significant and previously unreported tissue enrichment and sex-specific alteration of a set of inflammatory microRNAs (miRNAs) in CD11b+ cells of brain and bone marrow isolated from naïve mice as well as mice subjected to TBI. Our data from naïve mice demonstrated that expression levels of miR-146a-5p and miR-150-5p were relatively higher in brain CD11b+ cells, and that miR-155-5p and miR-223-3p were highly enriched in bone marrow CD11b+ cells. Furthermore, while miR-150-5p and miR-155-5p levels were higher in male brain CD11b+ cells, no significant sexual difference was observed for miR-146a-5p and miR-223-3p. However, TBI resulted in sex-specific differential responses of these miRNAs in brain CD11b+ cells. Specifically, miR-223-3p levels in brain CD11b+ cells were markedly elevated in both sexes in response to TBI at 3 and 24h, with levels in females being significantly higher than males at 24h. We then focused on analyzing several miR-223-3p targets and inflammation-related marker genes following injury. Corresponding to the greater elevation of miR-223-3p in females, the miR-223-3p targets, TRAF6 and FBXW7 were significantly reduced in females compared to males. Interestingly, anti-inflammatory genes ARG1 and IL4 were higher in females after TBI than in males. These observations suggest miR-223-3p and other inflammatory responsive miRNAs may play a key role in sex-specific neuroinflammatory response following TBI.

Pathophysiological mechanisms leading to muscle loss in chronic kidney disease.

Loss of muscle proteins is a deleterious consequence of chronic kidney disease (CKD) that causes a decrease in muscle strength and function, and can lead to a reduction in quality of life and increased risk of morbidity and mortality. The effectiveness of current treatment strategies in preventing or reversing muscle protein losses is limited. The limitations largely stem from the systemic nature of diseases such as CKD, which stimulate skeletal muscle protein degradation pathways while simultaneously activating mechanisms that impair muscle protein synthesis and repair. Stimuli that initiate muscle protein loss include metabolic acidosis, insulin and IGF1 resistance, changes in hormones, cytokines, inflammatory processes and decreased appetite. A growing body of evidence suggests that signalling molecules secreted from muscle can enter the circulation and subsequently interact with recipient organs, including the kidneys, while conversely, pathological events in the kidney can adversely influence protein metabolism in skeletal muscle, demonstrating the existence of crosstalk between kidney and muscle. Together, these signals, whether direct or indirect, induce changes in the levels of regulatory and effector proteins via alterations in mRNAs, microRNAs and chromatin epigenetic responses. Advances in our understanding of the signals and processes that mediate muscle loss in CKD and other muscle wasting conditions will support the future development of therapeutic strategies to reduce muscle loss.

A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma.

Simple SummaryNeuroblastoma is a tumor of the sympathetic nervous system that substantially contributes to childhood cancer mortality. Neuroblastoma originates from the neural crest cells that are subjected to genetic alterations during embryonic development. These impairments hit key genes, whose expression is activated/repressed by transcription factors and inhibited by negative regulators, named microRNAs, thereby promoting tumorigenesis. Here, we have focused on the interactions between the transcription factors MYCN and PHOX2B with their target genes ALK and LIN28B and the miRNAs let-7, miR-34 and miR-204, which should act as negative regulators of their expression. In neuroblastoma, the physiologic regulatory networks among these genes and microRNAs are disrupted, leading to a complex and aberrant oncogene expression that underlies the development of the tumor. We also looked into the role of these genetic determinants of neuroblastoma starting from their physiological role in neural crest development and ending with their pathogenic dysregulation that leads to neuroblastoma oncogenesis.Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

Molecular mechanisms of pulmonary carcinogenesis by polycyclic aromatic hydrocarbons (PAHs): Implications for human lung cancer.

Lung cancer has the second highest incidence and highest mortality compared to all other cancers. Polycyclic aromatic hydrocarbon (PAH) molecules belong to a class of compounds that are present in tobacco smoke, diesel exhausts, smoked foods, as well as particulate matter (PM). PAH-derived reactive metabolites are significant contributors to lung cancer development. The formation of these reactive metabolites entails metabolism of the parent PAHs by cytochrome P4501A1/1B1 (CYP1A1/1B1) and epoxide hydrolase enzymes. These reactive metabolites then react with DNA to form DNA adducts, which contribute to key gene mutations, such as the tumor suppressor gene, p53 and are linked to pulmonary carcinogenesis. PAH exposure also leads to upregulation of CYP1A1 transcription by binding to the aryl hydrocarbon receptor (AHR) and eliciting transcription of the CYP1A1 promoter, which comprises specific xenobiotic-responsive element (XREs). While hepatic and pulmonary CYP1A1/1B1 metabolize PAHs to DNA-reactive metabolites, the hepatic CYP1A2, however, may protect against lung tumor development by suppressing both liver and lung CYP1A1 enzymes. Further analysis of these enzymes has shown that PAH-exposure also induces sustained transcription of CYP1A1, which is independent of the persistence of the parent PAH. CYP1A2 enzyme plays an important role in the sustained induction of hepatic CYP1A1. PAH exposure may further contribute to pulmonary carcinogenesis by producing epigenetic alterations. DNA methylation, histone modification, long interspersed nuclear element (LINE-1) activation, and non-coding RNA, specifically microRNA (miRNA) alterations may all be induced by PAH exposure. The relationship between PAH-induced enzymatic reactive metabolite formation and epigenetic alterations is a key area of research that warrants further exploration. Investigation into the potential interplay between these two mechanisms may lead to further understanding of the mechanisms of PAH carcinogenesis. These mechanisms will be crucial for the development of effective targeted therapies and early diagnostic tools.

Amyloid peptide exerts a rapid induction of Dicer1 protein in neuron via reducing phosphorylation

A growing number of evidence suggests that altered microRNA network in the brain contributes to the risk of Alzheimer's disease(AD). Dicer1 is a type III riboendonuclease which cleaves pre-microRNA into functional microRNA. Reduction of Dicer1 or Dicer1 mutation has been involved in cancer, aging or age-related macular degeneration. Recently, we found a possible link between Dicer1 and AD. In particular, Dicer1 protein and Dicer1 mRNA is reduced in the hippocampus and the cortex of an animal model of AD and exposure to Aβ42 oligomer(AβO) longer than 6 h reduces the transcription of Dicer1 gene in neuron, via depletion of NF-E2-related factor-2. In this study, exposure to AβO at shorter time increased Dicer1 protein in neuron in a dose-dependent mode; but the mRNA level remained unaltered. Under this treatment regime,AβO reduced phosphorylation level of Dicer1 and of its binding partner, transactivation response element RNA-binding protein(TRBP). Addition of a JNK inhibitor,SP600125, or an ERK inhibitor,U0126, further increased Dicer1 protein compared to Aβo treatment alone, with simultaneaous reduction of phospho-Dicer1, but with different effects on phospho-TRBP. Finally, an inhibitor of calcineurin,FK506, further increased Dicer1 protein compared to Aβo treatment alone. Thus, phosphorylation of Dicer1 and TRBP was determined by mitogen activated protein kinases JNK,ERK, and protein phosphatase 2B(calcineurin) which together determined Dicer1 stability. In summary, reduced phosphorylation of Dicer1 accounted for the rapid induction of Dicer1 by AβO. This study highlights a novel way by which AβO regulates Dicer1.

Tissue microRNA expression in aortic aneurysm dissection

Abstract Background Dissection and other complications of ascending aortic aneurysms are potentially life-threatening. Several factors may be implicated in aneurysm progression and dissection. The role of tissue microRNAs may be of interest. Purpose To examine how serum biomarkers and tissue expression of microRNAs are associated with thoracic aortic aneurysms and dissection. Methods We compared three groups of patients; 21 patients with aneurysm of the aortic root, ascending aorta or aortic arch undergoing scheduled repair, 11 patients with acute Stanford type A aortic dissection who underwent emergency surgery and 18 patients with normal aortic diameter undergoing other cardiac surgery (control group). Prior to surgery, peripheral blood samples were obtained from patients, to assess osteoprotegerin and adiponectin levels with the ELISA method. Tissue samples from ascending aortic wall were obtained from patients during surgery. Following appropriate storage and homogenization, tissue Matrix Metalloproteinases (MMPs) 2 and 9 were measured with the ELISA method, while tissue microRNAs 29 and 195 were measured using qrtPCR, after RNA extraction. Results There was no significant difference among control, aneurysm and dissection groups in terms of age (62±10 years vs 66±12 years vs 59±12 years, p=0.052), gender distribution (77.8% male vs 81% male vs 90% male, p=0.28) or BMI (28.51±2.92 kg/m2 vs 25.72±3.09 kg/m2 vs 27.02±3.2 kg/m2, p=0.76). There was also no difference among control, aneurysm and dissection groups regarding hypertension (72% vs 62% vs 73%, p=0.73), diabetes mellitus (22% vs 19% vs 36%, p=0.54), smoking (44% vs 29% vs 46%, p=0.09) or dyslipidemia (78% vs 43% vs 55%, p=0.08). The groups of control subjects, aneurysms and dissections did not differ in osteoprotegerin [44 (28, 52) pmol/l vs 31 (28, 37) pmol/l vs 45 (24, 71) pmol/l, p=0.17], adiponectin [6,65 (2,39, 9,79) μg/ml vs 5,28 (2,34, 6,98) μg/ml vs 4,13 (2,49, 7,52) μg/ml, p=0.43], tissue MMP2 [0.97 (0.42, 27.66) ng/ml vs 9.12 (1.72, 61.49) ng/ml vs 2.51 (0.22, 235.72) ng/ml, p=0.34] and tissue MMP9 levels [0.96 (0.29, 8.56) ng/ml vs 10.31 (1.18, 25.58) ng/ml vs 2.76 (0.63, 54.83) ng/ml, p=0.09] (Figure 1). Importantly, tissue expression of mir29 was 2.11-fold higher in the dissection group (p=0.001) and 2.99-fold higher in the aneurysm group (p<0.001) compared to the control group. Tissue expression of mir195 was 2.72-fold higher in the dissection group (p<0.001) and 2.00-fold lower in the aneurysm group (p=0.08) compared to the control group (Figure 2). Conclusions These findings highlight the role of epigenetic modifications through altered microRNA tissue expression in aortic wall synthesis, extracellular matrix degradation and progress of aneurysm formation and dissection. The exact role of microRNA expression in aortic dilatation and dissection, as well as their role as potential biomarkers, merit further validation. Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

Unboxing the molecular modalities of mutagens in cancer.

The etiology of the majority of human cancers is associated with a myriad of environmental causes, including physical, chemical, and biological factors. DNA damage induced by such mutagens is the initial step in the process of carcinogenesis resulting in the accumulation of mutations. Mutational events are considered the major triggers for introducing genetic and epigenetic insults such as DNA crosslinks, single- and double-strand DNA breaks, formation of DNA adducts, mismatched bases, modification in histones, DNA methylation, and microRNA alterations. However, DNA repair mechanisms are devoted to protect the DNA to ensure genetic stability, any aberrations in these calibrated mechanisms provoke cancer occurrence. Comprehensive knowledge of the type of mutagens and carcinogens and the influence of these agents in DNA damage and cancer induction is crucial to develop rational anticancer strategies. This review delineated the molecular mechanism of DNA damage and the repair pathways to provide a deep understanding of the molecular basis of mutagenicity and carcinogenicity. A relationship between DNA adduct formation and cancer incidence has also been summarized. The mechanistic basis of inflammatory response and oxidative damage triggered by mutagens in tumorigenesis has also been highlighted. We elucidated the interesting interplay between DNA damage response and immune system mechanisms. We addressed the current understanding of DNA repair targeted therapies and DNA damaging chemotherapeutic agents for cancer treatment and discussed how antiviral agents, anti-inflammatory drugs, and immunotherapeutic agents combined with traditional approaches lay the foundations for future cancer therapies.Graphical abstract

293 Dacarbazine affects the percentage of G0-positive melanoma cells

Cancer cell quiescence can be defined as reversible state of growth arrest in which cells enter to G0-phase of cell cycle. Dormant tumors characterized by the balance between apoptosis and cell proliferation rates resulting in the absence of the progression and clinical symptoms. Acquisition quiescence by cancer cells is flexible process which can be induced by various factors including chemotherapy. We aim to determine if chemotherapeutic agent dacarbazine (DTIC) may induce melanoma cell quiescence. Therefore the IC50 value of DTIC was calculated and the final concentration of DTIC was chosen which was defined as of low-to-moderate toxicity for cell lines tested. SK-MEL-2 cells showed two times increase of G0-positive cells whereas BRO cells exhibited decrease of G0-positive cells after DTIC treatment. Next, we performed a transcriptomic study in SK-MEL-2 cells and focused our attention on signaling pathways where the differentially expressed genes were also components. We observed the top downregulated genes to be components of the vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor R2 (VEGFR2), PI3K/AKT, cell cycle, p53 transcriptional gene network, DNA damage response, Focal Adhesion-PI3K-Akt-mTOR-signaling pathways. Besides, several signal pathways associated with altered microRNAs expression and functioning were found to be upregulated. We performed microRNA profiling and dysregulated microRNAs were identified. One of such microRNA, miR-204-5p was studied where gain-and-loss of function experiments showed it’s role in mitosis regulation under DTIC treatment. We suppose that DTIC may be used for melanoma cell quiescence modeling. The role of microRNAs as epigenetic regulators of tumor dormancy should be further evaluated.

Altered microRNA and mRNA profiles during heart failure in the human sinoatrial node

Heart failure (HF) is frequently accompanied with the sinoatrial node (SAN) dysfunction, which causes tachy-brady arrhythmias and increased mortality. MicroRNA (miR) alterations are associated with HF progression. However, the transcriptome of HF human SAN, and its role in HF-associated remodeling of ion channels, transporters, and receptors responsible for SAN automaticity and conduction impairments is unknown. We conducted comprehensive high-throughput transcriptomic analysis of pure human SAN primary pacemaker tissue and neighboring right atrial tissue from human transplanted HF hearts (n = 10) and non-failing (nHF) donor hearts (n = 9), using next-generation sequencing. Overall, 47 miRs and 832 mRNAs related to multiple signaling pathways, including cardiac diseases, tachy-brady arrhythmias and fibrosis, were significantly altered in HF SAN. Of the altered miRs, 27 are predicted to regulate mRNAs of major ion channels and neurotransmitter receptors which are involved in SAN automaticity (e.g. HCN1, HCN4, SLC8A1) and intranodal conduction (e.g. SCN5A, SCN8A) or both (e.g. KCNJ3, KCNJ5). Luciferase reporter assays were used to validate interactions of miRs with predicted mRNA targets. In conclusion, our study provides a profile of altered miRs in HF human SAN, and a novel transcriptome blueprint to identify molecular targets for SAN dysfunction and arrhythmia treatments in HF.

Bile reflux and hypopharyngeal cancer (Review).

Laryngopharyngeal reflux, a variant of gastroesophageal reflux disease, has been considered a risk factor in the development of hypopharyngeal cancer. Bile acids are frequently present in the gastroesophageal refluxate and their effect has been associated with inflammatory and neoplastic changes in the upper aerodigestive tract. Recent in vitro and in vivo studies have provided direct evidence of the role of acidic bile refluxate in hypopharyngeal carcinogenesis and documented the crucial role of NF-κB as a key mediator of early oncogenic molecular events in this process and also suggested a contribution of STAT3. Acidic bile can cause premalignant changes and invasive squamous cell cancer in the affected hypopharynx accompanied by DNA damage, elevated p53 expression and oncogenic mRNA and microRNA alterations, previously linked to head and neck cancer. Weakly acidic bile can also increase the risk for hypopharyngeal carcinogenesis by inducing DNA damage, exerting anti-apoptotic effects and causing precancerous lesions. The most important findings that strongly support bile reflux as an independent risk factor for hypopharyngeal cancer are presented in the current review and the underlying mechanisms are provided.

Altered expression of microRNAs in the rat diaphragm in a model of ventilator-induced diaphragm dysfunction after controlled mechanical ventilation

BackgroundVentilator-induced diaphragm dysfunction (VIDD) is a common complication of life support by mechanical ventilation observed in critical patients in clinical practice and may predispose patients to severe complications such as ventilator-associated pneumonia or ventilator discontinuation failure. To date, the alterations in microRNA (miRNA) expression in the rat diaphragm in a VIDD model have not been elucidated. This study was designed to identify these alterations in expression.ResultsAdult male Wistar rats received conventional controlled mechanical ventilation (CMV) or breathed spontaneously for 12 h. Then, their diaphragm tissues were collected for RNA extraction. The miRNA expression alterations in diaphragm tissue were investigated by high-throughput microRNA-sequencing (miRNA-seq). For targeted mRNA functional analysis, gene ontology (GO) analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were subsequently conducted. qRT-PCR validation and luciferase reporter assays were performed. We successfully constructed a model of ventilator-induced diaphragm dysfunction and identified 38 significantly differentially expressed (DE) miRNAs, among which 22 miRNAs were upregulated and 16 were downregulated. GO analyses identified functional genes, and KEGG pathway analyses revealed the signaling pathways that were most highly correlated, which were the MAPK pathway, FoxO pathway and Autophagy–animal. Luciferase reporter assays showed that STAT3 was a direct target of both miR-92a-1-5p and miR-874-3p and that Trim63 was a direct target of miR-3571.ConclusionsThe current research supplied novel perspectives on miRNAs in the diaphragm, which may not only be implicated in diaphragm dysfunction pathogenesis but could also be considered as therapeutic targets in diaphragm dysfunction.

A Review of the Clinical and Therapeutic Implications of Neuropathic Pain

Understanding neuropathic pain presents several challenges, given the various mechanisms underlying its pathophysiological classification and the lack of suitable tools to assess its diagnosis. Furthermore, the response of this pathology to available drugs is still often unpredictable, leaving the treatment of neuropathic pain still questionable. In addition, the rise of personalized treatments further extends the ramified classification of neuropathic pain. While a few authors have focused on neuropathic pain clustering, by analyzing, for example, the presence of specific TRP channels, others have evaluated the presence of alterations in microRNAs to find tailored therapies. Thus, this review aims to synthesize the available evidence on the topic from a clinical perspective and provide a list of current demonstrations on the treatment of this disease.

Mesenchymal Stem Cells Alter MicroRNA Expression and Attenuate Thoracic Aortic Aneurysm Formation

Thoracic aortic aneurysms (TAA) are a progressive disease characterized by inflammation, smooth muscle cell activation and matrix degradation. We hypothesized that mesenchymal stem cells (MSCs) can immunomodulate vascular inflammation and remodeling via altered microRNA (miRNAs) expression profile to attenuate TAA formation. C57BL/6 mice underwent topical elastase application to form descending TAAs. Mice were also treated with MSCs on days 1 and 5 and aortas were analyzed on day 14 for aortic diameter. Cytokine array was performed in aortic tissue and total RNA was tagged and hybridized for miRNAs microarray analysis. Immunohistochemistry was performed for elastin degradation and leukocyte infiltration. Treatment with MSCs significantly attenuated aortic diameter and TAA formation compared to untreated mice. MSC administration also attenuated T-cell, neutrophil and macrophage infiltration and prevented elastic degradation to mitigate vascular remodeling. MSC treatment also attenuated aortic inflammation by decreasing proinflammatory cytokines (CXCL13, IL-27, CXCL12 and RANTES) and upregulating anti-inflammatory interleukin-10 expression in aortic tissue of elastase-treated mice. TAA formation demonstrated activation of specific miRNAs that are associated with aortic inflammation and vascular remodeling. Our results also demonstrated that MSCs modulate a different set of miRNAs that are associated with decrease leukocyte infiltration and vascular inflammation to attenuate the aortic diameter and TAA formation. These results indicate that MSCs immunomodulate specific miRNAs that are associated with modulating hallmarks of aortic inflammation and vascular remodeling of aortic aneurysms. Targeted therapies designed using MSCs and miRNAs have the potential to regulate the growth and development of TAAs.

MicroRNAs in blood act as biomarkers of colorectal cancer and indicate potential therapeutic targets.

Association studies have linked alterations of blood‐derived microRNAs (miRNAs) with colorectal cancer (CRC). Here, we performed a microarray‐based comparison of the profiles of 2549 miRNAs in 80 blood samples from healthy donors and patients with colorectal adenomas, colorectal diverticulitis and CRC at different stages. Confirmation by quantitative real‐time PCR (RT‐PCR) was complemented by validation of identified molecules in another 36 blood samples. No variations in miRNA levels were observed in samples from patients with colorectal adenomas and diverticulitis or from healthy donors. However, there were 179 CRC‐associated miRNAs of differential abundance compared to healthy controls. Only three – miR‐1225‐5p, miR‐1207‐5p and miR‐4459 – exhibited increased levels at all CRC stages. Most deregulated miRNAs (128/179, 71%) specifically predicted metastatic CRC. Pathway analysis found several cancer‐related pathways to which the miRNAs contribute in various ways. In conclusion, miRNA levels in blood vary throughout CRC progression and affect cellular functions relevant to haematogenous CRC progression and dissemination. The identified biomarker and therapeutic candidates require further confirmation of their clinical relevance.

Cardiomyocyte-produced miR-339-5p mediates pathology in Duchenne muscular dystrophy cardiomyopathy.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by severe, progressive muscle wasting. Cardiomyopathy has emerged as a leading cause of death in patients with DMD. The mechanisms contributing to DMD cardiac disease remain under investigation and specific therapies available are lacking. Our prior work has shown that DMD-iPSC-derived cardiomyocytes (DMD-iCMs) are vulnerable to oxidative stress injury and chronic exposure to DMD-secreted exosomes impaired the cell's ability to protect against stress. In this study, we sought to examine a mechanism by which DMD cardiac exosomes impair cellular response through altering important stress-responsive genes in the recipient cells. Here, we report that DMD-iCMs secrete exosomes containing altered microRNA (miR) profiles in comparison to healthy controls. In particular, miR-339-5p was upregulated in DMD-iCMs, DMD exosomes and mdx mouse cardiac tissue. Restoring dystrophin in DMD-iCMs improved the cellular response to stress and was associated with downregulation of miR-339-5p, suggesting that it is disease-specific. Knockdown of miR-339-5p was associated with increased expression of MDM2, GSK3A and MAP2K3, which are genes involved in important stress-responsive signaling pathways. Finally, knockdown of miR-339-5p led to mitochondrial protection and a reduction in cell death in DMD-iCMs, indicating miR-339-5p is involved in direct modulation of stress-responsiveness. Together, these findings identify a potential mechanism by which exosomal miR-339-5p may be modulating cell signaling pathways that are important for robust stress responses. Additionally, these exosomal miRs may provide important disease-specific targets for future therapeutic advancements for the management and diagnosis of DMD cardiomyopathy.

P2. Unsupervised anomaly detection for diagnosing brain disorders from EEG recordings – Results from a rodent epilepsy model

We present a general machine learning (ML) framework for diagnosing brain disorders using EEG recordings. It could detect any systematic deviations from the baseline (BL) EEG recordings. We test the approach using a rodent model of an acquired mesial temporal lobe epilepsy (mTLE) via electrical perforant pathway stimulation (PPS) [1] followed by usually a latent period of several weeks of epileptogenesis (EPG) progression without any seizures. Identifying ongoing EPG during this latent period prior to the first spontaneous seizure might enable early intervention. Previous supervised approaches have proven that deep neural networks can distinguish intracranial EEG signals between the BL period prior to PPS and the EPG period after PPS [2]. Here, we formulate the problem as an unsupervised anomaly detection task, which allows learning the statistics of the input data without the need for any labels as in supervised approaches. Specifically, we first train an autoencoder (AE) to learn to encode EEG data from the BL period. We then apply the trained AE on data from the EPG period. We expect that activity patterns systematically deviating from those observed during BL should be characterized by higher reconstruction errors (REs). Indeed, our results show that REs averaged over one hour gradually increase as a function of time after the PPS (Fig. 1). We also find that the distribution of REs from the BL and the EPG phases are significantly different (Fig. 1B). EEG segments with particularly high REs exhibit spikes and high frequency oscillations (Fig. 1B, right inset). Interestingly, there are an increased large number of segments with unusually low REs (Fig. 1B, left inset), which exhibits EEG slowing and delta rhythm (1.5–3 Hz). In conclusion, we showed that unsupervised ML with AEs allows the automatic detection of systematic drifts in brain activity, which may facilitate rapid EEG recordings for early diagnosis of brain disorders and open the door for early interventions. References Costard, L.S., Neubert, V., Venø, M.T., Su, J., Kjems, J., Connolly, N.M., Prehn, J.H., Schratt, G., Henshall, D.C., Rosenow, F. and Bauer, S., 2019. Electrical stimulation of the ventral hippocampal commissure delays experimental epilepsy and is associated with altered microRNA expression. Brain Stimulation, 12(6), pp. 1390-1401. Lu, D., Bauer, S., Neubert, V., Costard, L.S., Rosenow, F. and Triesch, J., 2020, September. Towards Early Diagnosis of Epilepsy from EEG Data. In Machine Learning for Healthcare Conference (pp. 80-96). PMLR. Fig. 1. A. The reconstruction error evolution throughout the whole recording period of rat #1B. The distribution of the reconstruction errors from the BL and the EPG phases from the recordings of rat #1. We also show some typical EPG segments whose reconstruction errors are below the 10th percentile and over the 90th percentile, highlighted with a solid and a dashed black box, respectively. Other stimulated rats show a similar trend.