Analysis Of microRNA Expression Research Articles

Genome-wide analysis of primary microRNA expression using H3K36me3 ChIP-seq data

MicroRNAs are key players in gene regulatory networks controlling cell homeostasis. Their altered expression has been previously linked to disease outcomes and microRNAs thus serve as biomarkers for disease diagnostics. However, their synthesis and its transcriptional regulation have been challenging to investigate. In this study, we validated the use of H3K36me3 histone modification for the quantification of microRNA transcription levels using data from the ENCODE Consortium and then applied this approach to provide new insight into the cell-type-specific regulationin tissues, cell line models and cardiac disease. In cardiomyocytes derived from patients suffering from septal defects, carrying a G296S mutation in the transcription factor GATA4, we show that microRNA gene transcription is altered in cardiomyocytes carrying this mutation and coincides with novel super-enhancers formed within regulatory domains defined using chromatin interaction profiles. The most prominently elevated primary transcript encodes for let-7a and miR-100 that may target genes in the Hippo signaling pathway. Collectively, our work presents a methodology to quantify microRNA gene expression using histone marker data and paves the way for functional studies of cell-type-specific transcriptional regulation occurring in disease pathology.

Identification of hypoxia-specific therapeutic targets in multiple myeloma

The bone marrow microenvironment is low in oxygen, promoting a hypoxic response which causes myeloma cells to acquire stem cell properties and enhanced therapy resistance. We performed comprehensive gene and microRNA expression analyses of samples from myeloma patients and cell lines cultured under hypoxia. Through this, we identified the histone demethylase KDM3A, the glycolytic enzyme HK2, and microRNA-210 as factors playing important roles in the behavior of cells under hypoxic conditions. These genes were regulated by the hypoxia-inducible factor HIF. However, we also found that the expression of IRF4 and MYC, factors required for maintenance of differentiation and proliferation was suppressed by hypoxia. This suggests that the regulatory factors that induce drug resistance and the anti-apoptotic capacity of myeloma cells fluctuate with the partial pressure of oxygen in their environment. Based on this premise, a dual treatment strategy in which a dominant clone and a dormant clone adapted to the hypoxic microenvironment are treated simultaneously with orthogonal drugs is a potentially viable strategy to achieve a cure for multiple myeloma.

Expression analysis of microRNAs and their target mRNAs of testes with high and low sperm motility in domestic pigeons (Columba livia)

Sperm motility is one of the most important indicators to evaluate poultry fertility. In order to explore key molecular regulation roles related to sperm motility, we employed testicular RNA sequencing of pigeon. A total of 705 known and 385 novel microRNAs were identified. Compared with the low sperm motility group, four upregulated and two downregulated miRNAs in the high sperm motility group were identified. A total of 3567 target mRNAs were predicted and four target mRNAs were selected to validate by qPCR. The miRNA-mRNA interaction network analysis, indicated that mmu-miR-183-5p /FOXO1 and PC-3p-244994_31/CHDH pairs might affect sperm motility. GO and KEGG annotation analysis showed that target genes of differentially expressed miRNAs were related to serine/threonine kinase activity, ATP binding, Wnt and MAPK signaling pathway. The study provided a global miRNAs transcriptome of pigeon and a novel insight into the expression of the miRNAs in testes that associated with sperm motility.

The role of the serum exosomal and endometrial microRNAs in recurrent implantation failure

Objective It has been identified that endometrium specific microRNAs have different expression levels in endometrial tissues and maternal serum during endometrial cycle. The aim of this study was to analyze microRNA expression levels in recurrent implantation failure patients and healthy controls endometrial samples for enlightening the aetiopathogenesis of the disease. The second aim was to search for a potential noninvasive molecular biomarker in early diagnosis and treatment of Recurrent Implantation Failure (RIF) patients. Methods Endometrium and serum samples in two different phases (PP; proliferative phase and SP; secretory phase) from the same cases (RIF; n = 12 and Control; n = 8) were obtained. The expression levels of the microRNA by RT-qPCR method were measured. The expression levels of the healthy controls and study group were compared. Lastly performed target genes analysis of significantly dysregulated miRNA by target analyze databases for obtained related biological pathways. Results This study showed that has-miR-145, has-miR-23b, has-miR-31 and has-miR-30b were significantly up-regulated in PP and down-regulated in SP endometrium samples. In serum samples, has-miR-145 and hsa-miR-23b were significantly down-regulated in both of PP and SP. Target gene and pathway analysis for dysregulated miRNAs identified important, validated and predicted genes for the implantation process. Conclusions This study is the first study to obtain endometrium and serum samples in two different phases from the same cases and measure the candidate miRNAs expression. Our finding suggests that expression level of four candidate miRNAs may be involved in RIF development in women. Furthermore, these miRNAs can be potential biomarker for early diagnosis of RIF patients.

Aux/IAA14 Regulates microRNA-Mediated Cold Stress Response in Arabidopsis Roots.

The phytohormone auxin and microRNA-mediated regulation of gene expressions are key regulators of plant growth and development at both optimal and under low-temperature stress conditions. However, the mechanistic link between microRNA and auxin in regulating plant cold stress response remains elusive. To better understand the role of microRNA (miR) in the crosstalk between auxin and cold stress responses, we took advantage of the mutants of Arabidopsis thaliana with altered response to auxin transport and signal. Screening of the mutants for root growth recovery after cold stress at 4 °C revealed that the auxin signaling mutant, solitary root 1 (slr1; mutation in Aux/IAA14), shows a hypersensitive response to cold stress. Genome-wide expression analysis of miRs in the wild-type and slr1 mutant roots using next-generation sequencing revealed 180 known and 71 novel cold-responsive microRNAs. Cold stress also increased the abundance of 26–31 nt small RNA population in slr1 compared with wild type. Comparative analysis of microRNA expression shows significant differential expression of 13 known and 7 novel miRs in slr1 at 4 °C compared with wild type. Target gene expression analysis of the members from one potential candidate miR, miR169, revealed the possible involvement of miR169/NF-YA module in the Aux/IAA14-mediated cold stress response. Taken together, these results indicate that SLR/IAA14, a transcriptional repressor of auxin signaling, plays a crucial role in integrating miRs in auxin and cold responses.

Expression Analyses of MicroRNAs in Hamster Lung Tissues Infected by SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an infectious disease with multiple severe symptoms, such as fever over 37.5°C, cough, dyspnea, and pneumonia. In our research, microRNAs (miRNAs) binding to the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory-related coronavirus (MERS-CoV), and SARS-CoV-2 were identified by bioinformatic tools. Five miRNAs (hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-16-5p, and hsa-miR-196a-1-3p) were found to commonly bind to SARS-CoV, MERS-CoV, and SARS-CoV-2. We also identified miRNAs that bind to receptor proteins, such as ACE2, ADAM17, and TMPRSS2, which are important for understanding the infection mechanism of SARS-CoV-2. The expression patterns of those miRNAs were examined in hamster lung samples infected by SARS-CoV-2. Five miRNAs (hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-221-3p, hsa-miR-140-3p, and hsa-miR-422a) showed differential expression patterns in lung tissues before and after infection. Especially, hsa-miR-15b-5p and hsa-miR-195-5p showed a large difference in expression, indicating that they may potentially be diagnostic biomarkers for SARS-CoV-2 infection.

Analysis of microRNA regulating cell cycle-related tumor suppressor genes in endometrial cancer patients

Endometrial cancer remains the most common malignancy of the female genital system in developed countries. Tumor suppressor genes are responsible for controlling the cells fate in the cell cycle and preventing cancerogenesis. Gene expression affects cancer progression and is modulated by microRNAs defined as both tumor suppressors and oncogenes. These molecules indirectly regulate multiple processes like cell proliferation, differentiation and apoptosis. The aim of this study was to analyze miRNAs expression that can regulate the activity of tumor suppressor genes related to the cell cycle in patients with endometrioid endometrial cancer. The study group consisted of 12 samples that met the inclusion criteria from a total of 48 obtained. The 12 samples were used to analyze microRNA expression. Complementary miRNAs were identified using TargetScan Database and statistical analysis. MicroRNAs were determined for the tumor suppressor genes: CYR61, WT1, TSPYL5, HNRNPA0, BCL2L1 and BAK1. All the miRNAs were complementary to the described target genes based on TargetScan Database. There were five miRNAs differentially expressed that can regulate tumor suppressor genes related to the cell cycle. The distinguished miRNAs: mir-340-3p, mir-1236-5p, mir-874-3p, mir-873-5p.2 and mir-548-5p were differentially expressed in endometrial cancer in comparison to the control. Among the distinguished miRNAs, the most promising is mir-874-3p, which may have an important role in endometrial adenocarcinoma proliferation.

Unique Interplay Between Molecular miR-181b/d Biomarkers and Health Related Quality of Life Score in the Predictive Glioma Models.

In the last decade, an increasing amount of research has been conducted analyzing microRNA expression changes in glioma tissue and its expressed exosomes, but there is still sparse information on microRNAs or other biomarkers and their association with patients’ functional/psychological outcomes. In this study, we performed a combinational analysis measuring miR-181b and miR-181d expression levels by quantitative polymerase chain reaction (qPCR), evaluating isocitrate dehydrogenase 1 (IDH1) single nucleotide polymorphism (SNP), and O-6-methylguanine methyltransferase (MGMT) promoter methylation status in 92 post-surgical glioma samples and 64 serum exosomes, including patients’ quality of life evaluation applying European Organization for Research and Treatment of Cancer (EORTC) questionnaire for cancer patients (QLQ-30), EORTC the Brain Cancer-Specific Quality of Life Questionnaire (QLQ-BN20), and the Karnofsky performance status (KPS). The tumoral expression of miR-181b was lower in grade III and glioblastoma, compared to grade II glioma patients (p < 0.05). Additionally, for the first time, we demonstrated the association between miR-181 expression levels and patients’ quality of life. A positive correlation was observed between tumoral miR-181d levels and glioma patients’ functional parameters (p < 0.05), whereas increased exosomal miR-181b levels indicated a worse functional outcome (p < 0.05). Moreover, elevated miR-181b exosomal expression can indicate a significantly shorter post-surgical survival time for glioblastoma multiforme (GBM) patients. In addition, both tumoral and exosomal miR-181 expression levels were related to patients’ functioning and tumor-related symptoms. Our study adds to previous findings by demonstrating the unique interplay between molecular miR-181b/d biomarkers and health related quality of life (HRQOL) score as both variables remained significant in the predictive glioma models.

Computational Approaches towards Micro-RNA Expression Analysis for Identification of Biomarkers in Inflammatory Bowel Disease Subtypes

Background and Aim: The potential biomarkers of inflammatory bowel diseases (IBDs) were analyzed from GSE53867 dataset. The current study aimed to identify miRNAs as potential diagnostic and prognostic biomarkers to delineate the IBD subtypes. Methods: Differentially expressed microRNAs (DEMs)-genes and protein-protein interaction networks were constructed and hub genes were selected using Cytoscape. Differentially expressed genes were analyzed for GO (Gene Ontology) and Reactome-pathway. Results: Seven DEMs were upregulated in Crohn’s disease (CD), 4 downregulated in ulcerative colitis (UC), 8 upregulated and 2 downregulated in IBD. A 620, 2377 and 1821 target-genes were in CD, UC and IBD, respectively. SOCS3, upregulated by miR-650, was hub gene in CD, induced by cytokines, through NFKB-signalling pathway to mediate ubiquitin-proteasomal degradation. CIRH1A, downregulated by miR-16, was hub gene of UC, acted by impairing ribosomebiogenesis. SKP2 and ASB1, up- and downregulated, by miR-142 and miR-665, respectively, were hub genes of IBD, induced cytokines through activation of TLR- and TNF-signalling pathways to mediate ubiquitin-proteasomal degradation. Conclusion: The SOCS3, CIRH1A, SKP2 and ASB1 genes might serve as valuable biomarkers to differentiate CD, UC and IBD.

Analytical and clinical validation of pairwise microRNA expression analysis to identify medullary thyroid cancer in thyroid fine-needle aspiration samples.

BackgroundMedullary thyroid carcinoma (MTC) is an aggressive malignancy originating from the parafollicular C cells. Preoperatively, thyroid nodule fine‐needle aspiration cytology (FNAC) and pathogenic gene mutations are definitive in approximately one‐half of cases. MicroRNAs (miRNAs) are endogenous, noncoding, single‐stranded RNAs that regulate gene expression, a characteristic that confers the potential for identifying malignancy. In the current study, the authors hypothesized that differential pairwise (diff‐pair) analysis of miRNA expression levels would reliably identify MTC in FNA samples.MethodsThe relative abundance of 10 different miRNAs in total nucleic acids was obtained from ThyraMIR test results. Diff‐pair analysis was performed by subtracting the critical threshold value of one miRNA from the critical threshold values of other miRNAs. Next‐generation sequencing with the ThyGeNEXT panel identified oncogenic gene alterations. The discovery cohort consisted of 30 formalin‐fixed, paraffin‐embedded benign and malignant thyroid neoplasms, including 4 cases of MTC. After analytical validation, clinical validation was performed using 3 distinct cohorts (total of 7557 specimens).ResultsIn the discovery cohort, 9 diff‐pairs were identified as having significant power using the Kruskal‐Wallis test (P < .0001) to distinguish MTC samples from non‐MTC samples. The assay correctly classified all MTC and non‐MTC samples in the analytical validation study and in the 3 clinical validation cohorts. The overall test accuracy was 100% (95% confidence interval, 99%‐100%). In indeterminate FNAC samples, the sensitivity of the diff‐pair analysis was greater than that of the MTC‐specific mutation analysis (100% vs 25%; P = .03).ConclusionsPairwise miRNA expression analysis of ThyraMIR results were found to accurately predict MTC in thyroid FNA samples, including those with indeterminate FNAC findings.

Body temperature-dependent microRNA expression analysis in rats: rno-miR-374-5p regulates apoptosis in skeletal muscle cells via Mex3B under hypothermia

Forensic diagnosis of fatal hypothermia is considered difficult because there are no specific findings. Accordingly, exploration of novel fatal hypothermia-specific findings is important. To elucidate the molecular mechanism of homeostasis in hypothermia and identify novel molecular markers to inform the diagnosis of fatal hypothermia, we focused on microRNA expression in skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We generated rat models of mild, moderate, and severe hypothermia, and performed body temperature-dependent microRNA expression analysis of the iliopsoas muscle using microarray and quantitative real-time PCR (qRT-PCR). The results show that rno-miR-374-5p expression was significantly induced only by severe hypothermia. Luciferase reporter assay and qRT-PCR results indicated that Mex3B expression was regulated by rno-miR-374-5p and decreased with decreasing body temperature. Gene ontology analysis indicated the involvement of Mex3B in positive regulation of GTPase activity. siRNA analysis showed that Mex3B directly or indirectly regulated Kras expression in vitro, and significantly changed the expression of apoptosis-related genes and proteins. Collectively, these results indicate that rno-miR-374-5p was activated by a decrease in body temperature, whereby it contributed to cell survival by suppressing Mex3B and activating or inactivating Kras. Thus, rno-miR-374-5p is a potential supporting marker for the diagnosis of fatal hypothermia.

Genome-wide profiling and analysis of microRNA expression in buffalo milk exosomes

Milk microRNA are being studied as a bioactive component of milk. A major fraction of milk microRNA are encapsulated in the lipid bilayered nano-vesicles called exosomes which mediate cell-cell communication. Therefore, the present study was aimed to identify and characterize microRNA in buffalo milk exosomes. Milk exosomes were characterized and used for total RNA isolation. Small RNA sequencing identified 351 microRNA and 17 previously unannotated microRNA in the buffalo milk exosomes. Bta-miR-148a, bta-miR-30a-5p, bta-miR-21-5p, bta-miR-99a-5p, bta-miR-27b, bta-miR-200a, bta-miR-26a, bta-miR-26c, bta-let-7g and bta-let-7i were the 10 most abundant microRNA in buffalo milk exosomes. Out of 5 validated microRNA, bta-miR-148a and −30a-5p were differentially expressed whereas bta-miR-21-5p, bta-miR-200a and bta-let-7g were consistently expressed across lactation stages. Gene targets of the 10 most abundant microRNA were identified using TargetScan taking Homo sapiens as the reference genome. All the targets having context scores < −0.4 with a total of 1539 were taken for network and pathway analysis. In silico analyses predicted 9 significant gene clusters in buffalo milk exosomal microRNA, which were found to be involved in important cellular processes. Analyses of hub genes interactions, string and cytoscape network analysis indicated that ubiquitin proteasomal degradation is the most regulated cellular process by buffalo milk exosomal microRNA. In summary, the repertoire of microRNA identified along with in silico analysis has provided insights into the physiological functionality of bovine milk microRNA in humans.

Genome-wide microRNA expression analysis in human placenta reveals sex-specific patterns: an ENVIRONAGE birth cohort study

ABSTRACT There is an increasing interest in microRNAs (miRNAs) as they are of utmost importance in gene regulation at the posttranscriptional level. Sex-related susceptibility for non-communicable diseases later in life could originate in early life. Until now, no data on sex-specific miRNA expression are available for the placenta. Therefore, we investigated the difference by sex of newborn’s miRNA expression in human placental tissue. Within the ENVIRONAGE birth cohort, miRNA and mRNA expression profiling was performed in 60 placentae (50% boys) using Agilent (8 × 60 K) microarrays. The distribution of chromosome locations was studied and pathway analysis of the identified sex-specific miRNAs in the placenta was carried out. Of the total 2558 miRNAs on the array, 597 miRNAs were expressed in over 70% of the samples and were included for further analyses. A total of 142 miRNAs were significantly (FDR<0.05) associated with the newborn’s sex. In newborn girls, 76 miRNAs had higher expression (hsa-miR-361-5p as most significant) and 66 miRNAs had lower expression (hsa-miR-4646-5p as most significant) than in newborn boys. In the same study population, placental differentially expressed genes by sex were also identified using a whole genome approach. The placental gene expression revealed 27 differentially expressed genes by comparing girls to boys. Ultimately, we studied the miRNA-RNA interactome and identified 14 miRNA–mRNA interactions as sex-specific. Sex differences in placental m(i)RNA expression may reveal sex-specific patterns already present during pregnancy, which may influence physiological conditions in early or later life. These molecular processes might play a role in sex-specific disease susceptibility in later life.

Abstract 267: microRNA expression analysis in hrHPV positive FFPE tissues for the detection of cervical disease

Abstract Cervical cancer ranks first in cancer mortality among women of low-middle income countries where 80% of the 570,000 cases and 311,000 worldwide deaths estimated for 2018 occurred. Persistent infections with high-risk HPV (hrHPV) genotypes can lead to cervical high-grade lesions (Cervical Intraepithelial Neoplasia grade 2 or more severe disease, CIN-II+), that if left untreated progress to cancer. hrHPV test has high sensitivity but because many women infected with hrHPV genotypes will clear the infection spontaneously, it has low specificity to detect CIN-II+. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and show differential profiles in cervical intraepithelial lesions. Our aim was to identify miRNAs differentially expressed in CIN-II+ and to evaluate their potential use as biomarkers to distinguish low from high-grade lesions in hrHPV positive women. Methods We compared the miRNAs expression pattern between Formalin-Fixed Paraffin-Embedded (FFPE) tissues from hrHPV positive women with low-grade lesions (n=10) and women with high-grade lesions (n=10) of Medellin, Colombia, using the QIAseq miRNA Library Kit (Qiagen) and sequencing reagents and protocols from Illumina. The miRNAs with low coefficient of variation and high fold change were further validated by RT-PCR (miR-133a-3p, 143-3p, 143-5p, 29a-3p and 30b-5p) using miRCURY LNA miRNA PCR kit (Qiagen) and SNORD44 as housekeeping gene. Receiver Operating Characteristic (ROC) analyses with 95% confidence interval of the area under the curve were used to evaluate the diagnostic accuracy of the 5 miRNA and their combinations and the maxim Youden index to determine the optimal specificity and sensitivity. Results The differential miRNAs expression analysis identified 25 miRNAs overexpressed in high- versus low-grade lesions. The relative expression by RT-PCR of 5 of those miRNAs was higher in high-grade when compared to low-grade, and the levels of miR-143-3p, 143-5p and 30b-5p increased with disease progression (from healthy, CIN-I, CIN-II, CIN-III+). ROC analyses showed that miR-143-5p and the combination with miR-133a-3p exhibits the highest AUC 79% (95% CI: 65-93). However, the highest sensitivity is given by miR-29a-3p, 87.50% (95% CI: 67.64 - 97.34), and the highest specificity is given by miR-30b-5p, 95.45% (95% CI: 77.16-99-88), with cut-offs of &amp;gt;1,157 and &amp;gt;3,345 respectively. In conclusion, we identified 25 candidate miRNAs biomarkers and validated 5 of them with good diagnostic performance to distinguish high-grade from low-grade cervical lesions. Further validation on a larger cohort of samples is needed to confirm the potential role of these microRNAs to triage HPV positive women. Citation Format: Martha I. Gonzalez, Jone Garai, Jovanny Zabaleta, Gloria I. Sanchez. microRNA expression analysis in hrHPV positive FFPE tissues for the detection of cervical disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 267.

Abstract 5448: Integrative analysis of microRNA expression identifies two biologically distinct and clinically relevant subtypes of head and neck squamous cell carcinoma

Abstract Purpose: The objective of this study is to investigate the potential role of microRNAs (miRNA) as molecular markers of tumor heterogeneity in head and neck squamous cell carcinoma (HNSCC). Experimental Design: Here, we analyzed 562 HNSCC samples, 88 from a novel cohort and 474 from The Cancer Genome Atlas (TCGA), using miRNA-microarray and miRNA-seq, respectively. Using an integrative correlations method followed by miRNA expression-based hierarchical clustering, we validated miRNA clusters across cohorts, and evaluation of clusters by logistic regression and gene ontology approaches revealed subtype-based clinical and biological characteristics. Results: We identified two statistically significant tumor subtypes and named them ‘epithelial' and ‘stromal' based on correlations with functional target gene ontology in relation to differing stages of epithelial cell differentiation. Each subtype demonstrated significant differences in terms of mRNA signature (p &amp;lt; 0.001), primary tumor sites (p &amp;lt; 0.001) and HPV status (p &amp;lt; 0.001), and multivariate analysis associated the stromal subtype with a worse prognosis (HR = 1.5646, p = 0.006). We also observed several dysregulated miRNA families and clusters that function as regulators of subtype-specific gene expression networks in HNSCC. Conclusion: Our findings suggest miRNAs determine HNSCC subclassifications through coordinating growth and differentiation programs in epithelial cells. These results delineate developmental miRNA signatures characterizing the phenotypic diversity between HNSCC subtypes, providing an expanded framework for the pathogenesis and personalized treatment of HNSCC. Citation Format: Jeremiah R. Holt, Heejoon Jo, Vonn Walter, Xiaobei Zhao, David Neil Hayes, Yoon Ho Ko. Integrative analysis of microRNA expression identifies two biologically distinct and clinically relevant subtypes of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5448.

MicroRNA-27a-3p directly targets FosB to regulate cell proliferation, apoptosis, and inflammation responses in immunoglobulin a nephropathy

Immunoglobulin A nephropathy (IgAN) constitutes the most common primary glomerulonephritis worldwide; however, the exact pathogenesis of IgAN is unknown. Previous genome-wide analysis of microRNA (miRNA) expression in the kidney has confirmed that miRNAs are closely related to the pathological changes of IgAN. Accordingly, in this study we found that miR-27a-3p is upregulated in IgAN kidney tissues in addition to human podocytes and tubule epithelial HK2 but not mesangial cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), flow cytometry, real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays were used to verify the regulatory effects of miR-27a-3p and its inhibition on cell proliferation, apoptosis, and release of inflammatory factors in podocytes and HK2 cells. The target genes of miR-27a-3p were predicted using bioinformatics software; the identity of FosB as a target gene of miR-27a-3p was confirmed by luciferase report assay and western blot. Overall, our findings demonstrated that miR-27a-3p regulates cell apoptosis, cell proliferation, and the release of inflammatory cytokines of human podocytes and HK2 cells by directly targeting FosB. Our results therefore suggested that miR-27a-3p might be associated with the pathophysiology of IgAN and may represent a potential target for further studies related to IgAN mechanism or therapeutics.

3072 – MICRORNA-127-3P CONTROLS MURINE HEMATOPOIETIC STEM CELL MAINTENANCE BY LIMITING DIFFERENTIATION

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p over-expression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological down-regulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

MicroRNA and protein-coding gene expression analysis in idiopathic pulmonary fibrosis yields novel biomarker signatures associated to survival

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology that poses significant challenges in early diagnosis and prediction of progression. Analyses of microRNA and gene expression in IPF have yielded potentially predictive information. However, the relationship between microRNA/gene expression and quantitative phenotypic value in IPF remains controversial, as is the added value of this approach to current molecular signatures in IPF. To identify biomarkers predictive of survival in IPF via a microRNA-driven strategy. We profiled microRNA and protein-coding gene expression in peripheral blood mononuclear cells from 70 IPF subjects in a discovery cohort. We linked the microRNA/gene expression level with the quantitative phenotypic variation in IPF, including diffusing capacity of the lung for carbon monoxide and the forced vital capacity percent predicted. In silico analyses of expression profiles and quantitative phenotypic data allowed the generation of 2 sets of IPF molecular signatures (unique for microRNAs and protein-coding genes) that predict IPF survival. Each signature performed well in a validation cohort comprised of IPF patients aggregated from distinct patient populations recruited from different sites. Resampling test suggests that the protein-coding gene based signature is comparable and potentially superior to published IPF prognostic gene signatures. In conclusion, these results highlight the utility of microRNA-driven peripheral blood molecular signatures as valuable and novel biomarkers associated to individuals at high survival risk and for potentially facilitating individualized therapies in this enigmatic disorder.

Optimization of reference genes for qRT-PCR analysis of microRNA expression under abiotic stress conditions in sweetpotato

Sweetpotato (Ipomoea batatas. L) is an important food crop, harvested for its nutrient-rich tuberous roots. Drought and salt stresses are two major factors limiting the sweetpotato production. Since microRNAs (miRNAs) are well known to play crucial roles in regulation of plant stress responses, quantitative profiling of miRNA expression under stress conditions will facilitate identification and genetic manipulation of novel miRNAs to improve stress tolerance. Real-time quantitative reverse transcription PCR (qRT-PCR) is a commonly used tool for this purpose, but not without challenges. Although stem-loop and poly(A)-tail modified qRT-PCR methods were developed for characterizing miRNA expression, accurate profiling of miRNAs is still difficult in many plant species because of a lack of reliable reference genes for normalizing miRNA transcripts. To identify reference genes that are suitable for normalizing miRNA expression in sweetpotato, the expression stability of eight candidate miRNAs and two commonly used reference genes were tested in 96 samples involving four tissues and two cultivars under drought and salt stress treatments. Data analysis using the geNorm, NormFinder and Bestkeeper algorithms demonstrated that miRn60, miR482, and their combination were reliable references. We further validated the reference genes by expression analysis of the well-characterized miR319 and miR156 that regulate drought and salt stress responses, respectively. The reference genes identified in this study will facilitate future miRNA analysis under abiotic stress conditions in sweetpotato.

Comprehensive expression analysis of mRNA and microRNA for the investigation of compensatory mechanisms in the rat kidney after unilateral nephrectomy.

Compensation is a physiological response that occurs during chemical exposure to maintain homeostasis. Because compensatory responses are not usually considered adverse effects, it is important to understand compensatory mechanisms for chemical risk assessment. Although the kidney is a major target organ for toxicity, there is controversy over whether hyperplasia or hypertrophy contributes to the compensatory mechanism, and there is limited information to apply for chemical risk assessment. In the present study, compensatory mechanisms of the kidney were investigated in a unilateral nephrectomy (UNx) model using adult male and female F344 rats. In residual kidneys of male and female rats after UNx, 5-bromo-2'-deoxyuridine-labeling indices and mRNA expression of cell cycle-related genes were increased, although there were no fluctuations in mRNA expression of transforming growth factor-β1, which contributes to hypertrophy in renal tubules. Pathway analysis using mRNA expression data from a complementary DNA (cDNA) microarray revealed that canonical pathways related to cell proliferation were mainly activated and that forkhead box M1 (FOXM1) was an upstream regulator of compensatory cell proliferation in residual kidneys of male and female rats. cDNA microarray for microRNAs (miRNAs) demonstrated that nine miRNAs were downregulated in residual kidneys, and mRNA/miRNA integrated analysis indicated that miRNAs were associated with the expression of factors downstream of FOXM1. Overall, these results suggested that FOXM1-mediated hyperplasia rather than hypertrophy contributed to compensatory mechanisms in the kidney and that miRNAs regulated downstream FOXM1 signaling. These results will be beneficial for evaluating nephrotoxicity in chemical risk assessment and for developing new biomarkers to predict nephrotoxicity.