Changes In microRNA Expression Research Articles

Lentivirus-mediated Reprogramming of Somatic Cells in the Absence of Transgenic Transcription Factors

Retroviral vectors remain the most efficient and widely applied system for induction of pluripotency. However, mutagenic effects have been documented in both laboratory and clinical gene therapy studies, principally as a result of dysregulated host gene expression in the proximity of defined integration sites. Here, we report that cells with characteristics of pluripotent stem cells can be produced from normal human fibroblasts in the absence of reprogramming transcription factors (TFs) during lentiviral (LV) vector-mediated gene transfer. This occurred via induced alterations in host gene and microRNA (miRNA) expression and detrimental changes in karyotype. These findings demonstrate that vector-induced genotoxicity may alone play a role in somatic cell reprogramming derivation and urges caution when using integrating vectors in this setting. Clearer understanding of this process may additionally reveal novel insights into reprogramming pathways.

Integrating miRNA and mRNA expression profiles in response to heat stress-induced injury in rat small intestine

The molecular mechanisms underlying the pathophysiology of heat stress in the small intestine remain undefined. Furthermore, little information is available concerning changes in microRNA (miRNA) expression following heat stress. The present study sought to evaluate miRNA and mRNA expression profiles in the rat small intestine in response to heat stress. Male Sprague-Dawley rats were subjected to 2h of heat stress daily for ten consecutive days. Rats were sacrificed at specific time points immediately following heat treatment, and morphological changes in the small intestine were determined. The miRNA and mRNA expression profiles from sample of small intestine were evaluated by microarray analysis. Heat stress caused pronounced morphological damage in the rat small intestine, most severe within the jejunum after 3days of heat treatment. A mRNA microarray analysis found 270 genes to be up-regulated and 122 genes down-regulated (P ≤ 0.01, ≥2.0-fold change) in the jejunum after heat treatment. A miRNA microarray analysis found 18 miRNAs to be up-regulated and 11 down-regulated in the jejunum after heat treatment (P ≤ 0.05). Subsequent bioinformatic analyses of the differentially expressed mRNAs and miRNAs were carried out to integrate miRNA and mRNA expression and revealed that alterations in mRNA following heat stress were negatively correlated with miRNA expression. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of heat stress-induced injury in the small intestine, specifically with regard to miRNAs.

Expression profiling of cytogenetically normal acute myeloid leukemia identifies MicroRNAs that target genes involved in monocytic differentiation

MicroRNAs are short ribonucleic acids (RNAs) that play an important role in many aspects of cellular biology such as differentiation and apoptosis, due to their role in the regulation of gene expression. Using microRNA microarrays, we characterized the microRNA gene expression of 27 patients with acute myeloid leukemia (AML) with normal cytogenetics, focusing on the microRNAs differentially expressed between the M1 and M5 French-American-British (FAB) subtypes. An accurate delineation of these two AML entities was observed based on the expression of 12 microRNAs. We hypothesized that these microRNAs may potentially be involved in the differentiation block of M1 blasts and consequently monocytic differentiation. Using publically available mRNA data and microRNA target prediction software, we identified several key myeloid factors that may be targeted by our candidate microRNAs. The expression changes of the candidate microRNAs during monocytic differentiation of AML cell lines treated with Vitamin D and phorbol 12-myristate 13-acetate were examined. All six candidate microRNAs were significantly down-regulated over the time course by quantitative reverse transcriptase polymerase chain reaction suggesting a link between these microRNAs and monocytic differentiation. To further characterize these microRNAs, we confirmed by luciferase assays that these microRNA target several key myeloid factors such as MAFB, IRF8, and KLF4 identifying a possible mechanism for the control of differentiation by these microRNAs.

MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS), a kind of widely distributed environmentally organic compound, has been found to cause developmental toxicity. Although microRNAs (miRNAs) play an important role in many metabolic tasks, whether and how they are involved in the process of PFOS-induced toxicity is largely unknown. To address this problem, PFOS-induced changes in miRNAs and target gene expression in zebrafish embryos, and the potential mechanism of PFOS-induced toxic action were studied in this research. Zebrafish embryos were exposed to 1 µg ml(-1) PFOS or DMSO control from 6 h post-fertilization (hpf) to 24 or 120 hpf. Subsequently, RNA was isolated from the embryo pool and the expression profiles of 219 known zebrafish miRNAs were analyzed using microarray. Finally, quantitative real-time polymerase chain reaction was used to validate several miRNAs expression of microarray data. The analysis revealed that PFOS exposure induced significant changes in miRNA expression profiles. A total of 39 and 81 miRNAs showed significantly altered expression patterns after PFOS exposure 24 and 120 hpf. Of the changed miRNAs, 20 were significantly up-regulated and 19 were significantly down-regulated (p < 0.01) at 24 hpf, whereas 41 were significantly up-regulated and 40 were significantly down-regulated (p < 0.01) at 120 hpf. These miRNAs were involved in development, apoptosis and cell signal pathway, cell cycle progression and proliferation, oncogenesis, adipose metabolism and hormone secretion, whereas there is still little functional information available for 32 miRNAs. Our results demonstrate that PFOS exposure alters the expression of a suite of miRNAs and may induce developmental toxicity.

MiRNA Profile Associated with Replicative Senescence, Extended Cell Culture, and Ectopic Telomerase Expression in Human Foreskin Fibroblasts

Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

Early changes in micro RNA (miRNA) expression in the eutopic endometrium (EUE) in a baboon model of induced endometriosis

OBJECTIVE: To identify miRNAs in the EUE of baboons (Papio anubis) at 3 months following the induction of endometriosis.DESIGN: In the cycle prior to the induction of endometriosis, control EUE samples were obtained at laparotomy on day 10 post ovulation (PO) (n=4). Endometriosis was then induced in the same four animals by intraperitoneal inoculation of autologous menstrual tissue. Following laparoscopic confirmation of endometriosis, EUE samples were obtained at 3 months of disease.MATERIALS AND METHODS: Total RNA from the EUE was extracted and purified. A 8x15K miRNA microarray (Agilent) was applied. The signal data were preprocessed by AgiMiRna, a Limma package for R environment. Empirical Bayes model was used to estimate the P-values. The potential mRNA targets of differentially expressed miRNAs were predicted and compared against Affymetrix Human Genome Plus 2.0 expression data.Tabled 1Differentially Expressed miRNAs with Fold Change (FC) and P-values in baboons with endometriosis compared to controlsmiRNADisease/Control FCP-Valuehsa-miR-451-14.940.000036hsa-miR-181a-1.460.068hsa-miR-200a1.880.037hsa-miR-19b2.190.073hsa-miR-4242.430.027hsa-miR-212.490.014hsa-miR-29c4.460.009hsa-miR-1415.190.032 Open table in a new tab CONCLUSION: Induction of endometriosis causes rapid changes in the expression of several miRNAs in the baboon endometrium. miRNAs 200a, 424, 181a, 21, 29c, and 141, previously reported to be altered in women with endometriosis, were also altered in the endometrium of baboons with endometriosis, further validating this animal model. In addition, miR451, a previously unreported miRNA associated with the apoptotic pathway and cell survival, was significantly down regulated. OBJECTIVE: To identify miRNAs in the EUE of baboons (Papio anubis) at 3 months following the induction of endometriosis. DESIGN: In the cycle prior to the induction of endometriosis, control EUE samples were obtained at laparotomy on day 10 post ovulation (PO) (n=4). Endometriosis was then induced in the same four animals by intraperitoneal inoculation of autologous menstrual tissue. Following laparoscopic confirmation of endometriosis, EUE samples were obtained at 3 months of disease. MATERIALS AND METHODS: Total RNA from the EUE was extracted and purified. A 8x15K miRNA microarray (Agilent) was applied. The signal data were preprocessed by AgiMiRna, a Limma package for R environment. Empirical Bayes model was used to estimate the P-values. The potential mRNA targets of differentially expressed miRNAs were predicted and compared against Affymetrix Human Genome Plus 2.0 expression data. CONCLUSION: Induction of endometriosis causes rapid changes in the expression of several miRNAs in the baboon endometrium. miRNAs 200a, 424, 181a, 21, 29c, and 141, previously reported to be altered in women with endometriosis, were also altered in the endometrium of baboons with endometriosis, further validating this animal model. In addition, miR451, a previously unreported miRNA associated with the apoptotic pathway and cell survival, was significantly down regulated.

Epigenetic alteration of microRNAs in DNMT3B-mutated patients of ICF syndrome

Immunodeficiency, Centromeric region instability, Facial anomalies (ICF; OMIM #242860) syndrome, due to mutations in the DNMT3B gene, is characterized by inheritance of aberrant patterns of DNA methylation and heterochromatin defects. Patients show variable agammaglobulinemia and a reduced number of T cells, making them prone to infections and death before adulthood. Other variable symptoms include facial dysmorphism, growth and mental retardation. Despite the recent advances in identifying the dysregulated genes, the molecular mechanisms, which underlie the altered gene expression causing ICF phenotype complexity, are not well understood. Held the recently-shown tight correlation between epigenetics and microRNAs (miRNAs), we searched for miRNAs regulated by DNMT3B activity, comparing cell lines from ICF patients with those from healthy individuals. We observe that eighty-nine miRNAs, some of which involved in immune function, development and neurogenesis, are dysregulated in ICF (LCLs) compared to wild-type cells. Significant DNA hypomethylation of miRNA CpG islands was not observed in cases of miRNA up-regulation in ICF cells, suggesting a more subtle effect of DNMT3B deficiency on their regulation; however, a modification of histone marks, especially H3K27 and H3K4 trimethylation, and H4 acetylation, was observed concomitantly with changes in microRNA expression. Functional correlation between miRNA and mRNA expression of their targets allow us to suppose a regulation either at mRNA level or at protein level. These results provide a better understanding of how DNA methylation and histone code interact to regulate the class of microRNA genes and enable us to predict molecular events possibly contributing to ICF condition.

MicroRNA-101 Regulates Amyloid Precursor Protein Expression in Hippocampal Neurons

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Abeta) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3'-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Abeta load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1beta (IL-lbeta). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Abeta, suggesting a possible role for miR-101 in neuropathological conditions.

Abstract 721: An anaplastic lymphoma kinase antibody targets pancreatic allografts, prevents the progression of pancreatic cancer, and induces changes in microRNA expression

Abstract Anaplastic lymphoma kinase (ALK) is a transmembrane tyrosine kinase receptor that has an increased expression in various tumors and areas of active angiogenesis. In pancreatic tissues of mice with endogenously expressed mutant KrasG12D under embryonic acinar promoter p48, we detected ALK expression in pre maligant pancreatic intra-epithelial neoplastic ducts (PanIN) as well as malignant adenocarcinoma (PDAC). We have developed an antagonistic mouse monoclonal IgG antibody to the ALK receptor (anti-ALK IgG). To determine the antibody's binding efficacy, we used a quantum dot labeled anti-ALK IgG for the fluorescent visualization of pancreatic allografts in nude mice. Intravenously injected Qdot labeled anti-ALK IgG concentrated in allograft areas more than a Qdot labeled control antibody. In a prospective treatment study, we treated KrasG12D mice to determine the antibody's efficacy at preventing the progression from early to late PanIN and ultimately PDAC. Mice of three different age cohorts were treated with the antibody inta-peritoneal for six weeks. Upon completion of antibody treatment, the pancreata of mice were histologically examined for the appearance of early PanIN, late PanIN, or PDAC. Anti-ALK IgG treatment was most effective at preventing the progression of late PanIN to PDAC, with 8.3% of antibody treated mice harboring PDAC compared to 33.3% of saline treated control mice. Metastasis was also prevented in mice treated with antibody. Anti-ALK IgG treatment did not prevent the progression of early or late PanINs better than control treatment. Preliminary experiments have determined a change in microRNA expression in the pancreatic tissues of mice treated with antibody compared to controls and during different stages of pancreatic cancer progression. Expression of miR-375 decreased approximately 100 fold in PanIN and PDAC tissues compared to control tissues, but this decrease was not seen in PanIN tissues of mice treated with anti-ALK IgG. Reports have shown miR-375's role in pancreas development and the regulation of glucose homeostasis, and that its expression decreased in certain cancers. These data suggest miR-375 is necessary for normal physiological pancreatic activity and has a possible tumor suppressive role, and that anti-ALK IgG can help maintain this function. MiR- 22, 141, 148a, 148b, and 301a also had similar expression patterns in PanIN tissues and controls when pancreata were from mice treated with antibody. Another group of microRNAs, including miR- 10, 16, 21, 100, 155, and 199 showed little change in expression in antibody or control treated mice. These changes in microRNA expression suggest a response to antibody treatment at the genetic level, which may provide further insights into the functional mechanism of anti-ALK IgG's efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 721.

Abstract 2073: Changes in microRNA expression in response to short-term chemotherapeutic exposure is associated with transient anticancer drug resistance

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that act as posttranscriptional repressors of gene expression by binding the 3’ untranslated region of target genes. There is emerging evidence showing that miRNAs regulate diverse biologic processes including multidrug resistance (MDR). Our previous profiling study revealed that certain miRNAs are differentially expressed in multidrug resistant human T-cell leukemia cell line CEM/VM-1-5 compared to drug-sensitive parental cell line CCRF-CEM. To investigate whether these expression-altered miRNAs have a role in MDR, we ectopically expressed three miRNAs that are highly upregulated in CEM/VM-1-5 cells (hsa-miR-135b, −146b, and −196b) in CCRF-CEM cells with the use of lentiviral vectors. We found that overexpression of these miRNAs, individually or in combination, resulted in only slight increases in resistance of CCRF-CEM cells to teniposide, the agent used to select CEM/VM-1-5 cells. Moreover, we were unable to sensitize CEM/VM-1-5 cells to etoposide by suppression of hsa-miR-135b expression using LNA antisense oligonucleotide. Our data implied that these miRNAs do not confer drug resistance directly. However, it is still not clear whether these miRNAs play any role in the development of MDR. Therefore, we asked whether the expression of these miRNAs changed in CCRF-CEM cells after short exposure (3 to 48 hours) to various chemotherapeutic drugs at concentrations below their IC50-values. We observed substantial increases in hsa-miR-135b and −196b expression in cells treated with etoposide, doxorubicin and topotecan, but not in cells treated with vinblastine. These results suggested that the upregulation of these two miRNAs might be the consequence of DNA damage. Time-course and dose-response studies showed that the elevation of hsa-miR-135b and −196b correlated positively with the drug exposure time and drug concentration. Furthermore, the levels of miRNAs gradually decreased after the withdrawal of etoposide from the media following 48 h exposure to etoposide. Strikingly, the elevated expression of the miRNAs accompanied increased drug resistance. CCRF-CEM cells exposed to etoposide for 48 h were over 50-fold more resistant than control cells. The acquired drug resistance, like elevated expression of miRNAs, did not appear to be stable and became attenuated with increasing duration of the drug-free incubation. Together, our results suggest that miRNAs might play a significant role in cellular response to genotoxic agents and could be involved in the development of cancer drug resistance, but their roles in conferring resistance remain to be determined. That said, targeting miRNA expression could be a potential approach to prevent multidrug resistance. (Supported in part by 5RO1CA040570 from NCI to WTB, and in part by UIC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2073.

Abstract 3033: Profiling of global changes in microRNA levels during adipocyte differentiation by real-time PCR

Abstract We have used a novel real time PCR approach to profile global changes in microRNA expression during adipocytic differentiation of 3T3-L1 fibroblast cells. This profiling strategy makes use of a novel two stage screen resulting in a substantial increase in throughput and decrease in reagent use. In the first step, cDNA is prepared from individual samples of undifferentiated fibroblast, and cells at early and late stages of Adipocyte differentiation. A pooled cDNA sample prepared by pooling equimolar amounts of individual cDNAs was used in an initial profiling experiment to identify microRNAs that are expressed at detectable levels in the experimental system. Profiling was performed in triplicate with real time PCR assays for 591 mouse miRNAs (miRBase v12.0). In the second stage of screening, individual cDNA samples were each analyzed in triplicate using the assays for miRNAs that showed expression in the pooled cDNA sample. Expression data was normalized against a panel of small non-coding RNAs and was expressed as x-fold changes relative to expresion in undifferentiated 3T3L1 cells. Our results indicate that 192 (∼32%) miRNAs described in miRBase v12.0 were detectable in resting and differentiated 3T3-L1 cells, and of these, 38 (or 20%) showed differential regulation during adipogenesis (x-fold change&amp;gt;3.0). When compared to data from previous array-based profiling studies, our screen identified additional differentially regulated miRNAs. In addition to profiling changes in mature miRNA levels, miRNA precursor stem loop transcripts were also quantified to gain insight into post transcriptional regulation (if any) of miRNA biogenesis during adipocyte differentiation. The high specificity and sensitivity of the miScript RT-PCR system, together with a novel sample-pooling strategy makes this system a method of choice for miRNA expression profiling. Pooled primary profiling can substantially reduce the total number of PCR reactions which need to be performed and increases assay throughput. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3033.

Abstract 1987: RNA-seq of the bronchial airway transcriptome identifies novel gene and microRNA expression changes associated with smoking and lung cancer

Abstract Prior studies have shown that cigarette smoke creates a field of molecular injury in the airway epithelium. Using microarrays, our group has previously identified gene and microRNA expression differences in bronchial airway epithelial cells that are associated with smoking and lung cancer. We hypothesize that RNA-seq of the airway transcriptome will enhance our understanding of the response to tobacco smoke exposure and lung cancer pathogenesis by identifying mRNA splice variants, non-coding RNA, and novel RNA not interrogated by microarrays. We profiled pooled bronchial airway epithelial cell brushings (n=3 patients/pool) obtained during bronchoscopy from healthy never and current smoker volunteers and smokers with and without lung cancer undergoing surgery for suspicion of lung cancer. The high MW fraction (&amp;gt;200 bp) was amplified using a combination of oligo d(T) and random hexamers (NuGEN, San Carlos, CA) followed by library preparation with Illumina's mRNA Seq Library Prep Kit and sequencing using Illumina's Genome Analyzer generating ∼27-30 million 36 bp reads/sample. The low MW fraction (15-40 bp) was processed using ABI's Small RNA Library Sequencing Kit and sequenced using ABI's SOLiD system generating ∼50-90 million 35 bp reads/sample. The high MW reads were aligned to rRNA (5-13%), the human genome (33-40%), and to computationally generated splice junctions (1%) using BowTie. Differential gene and isoform expression was evaluated using Cufflinks and the R package Genominator. Low MW reads were aligned using both RNA2MAP and Geospiza to a filter containing rRNA, tRNA, and repeats (4-14%), to miRBase (4-10%), and the human genome minus miRBase sequences (4-8%). Expression values were calculated for microRNAs. We compared Affymetrix Exon 1.0 ST, HGU133A2, and Invitrogen miRNA microarrays to RNA-seq and found strong correlation for genes/miRNAs interrogated by microarrays, with most smoking- and cancer related changes in transcript expression being identified by sequencing and not by arrays. Genes found differentially expressed only by sequencing were validated by RT-PCR. RNA-seq also reveals differentially expressed isoforms, potential novel miRNAs, and several isoforms of known miRNAs which are being further investigated. RNA-seq provides a comprehensive and high-resolution view of the airway transcriptome and will provide insights into the molecular field of injury induced by smoking and the pathogenesis of smoking-related lung disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1987.

Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. To determine changes in expression and role of microRNAs in IPF. RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

E-Cadherin Expression Is Regulated by miR-192/215 by a Mechanism That Is Independent of the Profibrotic Effects of Transforming Growth Factor-β

OBJECTIVEIncreased deposition of extracellular matrix (ECM) within the kidney is driven by profibrotic mediators including transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF). We investigated whether some of their effects may be mediated through changes in expression of certain microRNAs (miRNAs).RESEARCH DESIGN AND METHODSProximal tubular cells, primary rat mesangial cells, and human podocytes were analyzed for changes in the expression of key genes, ECM proteins, and miRNA after exposure to TGF-β (1–10 ng/μl). Tubular cells were also infected with CTGF-adenovirus. Kidneys from diabetic apoE mice were also analyzed for changes in gene expression and miRNA levels.RESULTSTGF-β treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells. TGF-β treatment also modulated the expression of certain miRNAs, including decreased expression of miR-192/215 in tubular cells, mesangial cells, which are also decreased in diabetic kidney. Ectopic expression of miR-192/215 increased E-cadherin levels via repressed translation of ZEB2 mRNA, in the presence and absence of TGF-β, as demonstrated by a ZEB2 3′-untranslated region luciferase reporter assay. However, ectopic expression of miR-192/215 did not affect the expression of matrix proteins or their induction by TGF-β. In contrast, CTGF increased miR-192/215 levels, causing a decrease in ZEB2, and consequently increased E-cadherin mRNA.CONCLUSIONSThese data demonstrate the linking role of miRNA-192/215 and ZEB2 in TGF-β/CTGF–mediated changes in E-cadherin expression. These changes appear to occur independently of augmentation of matrix protein synthesis, suggesting that a multistep EMT program is not necessary for fibrogenesis to occur.

Kidney microRNA expression profile in Ang II‐dependent Hypertension

This study was conducted to determine if there are changes in microRNAs expression in the kidneys during angiotensin (Ang)‐II dependent hypertension. microRNAs are small non‐coding RNAs that cause posttranscriptional repression of their targets with emerging recognition as key players in kidney disease. C57BL/6J male mice (8–9 week) were subjected to sham operation (n=3) or Ang II infusions (400 ng/kg/min, n=3) for 12 days during which systolic blood pressure (SBP) was monitored by tail‐cuff method. RNA was isolated from the kidneys and microRNA expression was determined by microarray. SBP was increased by Ang II (151±3 mmHg) vs. controls (119±0.2, p&lt;0.05). When compared to controls, the expression of 5 microRNAs was reduced in the kidneys of Ang II‐infused mice (p&lt;0.05, results expressed as AU): miR‐22 (4,734 ± 1,327 vs. 2,144 ± 637), miR‐133a (34±5 vs. 22±2), miR‐1187 (619±227 vs. 140±66), miR‐574‐5p (501±193 vs. 148±53), and miR‐669f (110 ±43 vs. 25±13). In turn, the expression of 8 microRNAS was increased: miR‐155 (91±17 vs. 151±28), miR‐128 (145±15 vs. 196±4), miR‐183 (49±6 vs. 75±8), miR‐139‐5p (194±9 vs. 294±33), miR‐132 (84±5 vs. 187±39), miR‐200C (804±147 vs. 1,385±280), miR‐25 (895+89 vs. 1,223±103), and miR‐676 (54±6 vs. 98±11). In conclusion, these results demonstrate that there are significant changes in the microRNA expression profile of the mouse kidney during Ang II‐dependent hypertension.Supported by NIH grants P20RR017659, HL26371, and 1K99DK083455.

Maternal obesity affects MicroRNA expression in fetal muscle at mid‐gestation

INTRODUCTIONObesity is increasing at an alarming rate. Previous findings in our laboratory have shown that maternal obesity leads to increased adipogenesis in fetal muscle. Expression of adipogenic genes lead to adipogenesis. MicroRNAs play a crucial role in the regulation of gene expression. Using the pregnant sheep as a model, the objective was to assess microRNA expression in fetal muscle due to maternal obesity.METHODSNon pregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n = 5) or obesogenic (OB, 150% of NRC, n = 5) diet from 60 days before to 75 days after conception, when fetal longissimus dorsi (Ld) muscle was collected. MicroRNAs were extracted, purified and subjected to microRNA microarray analyses using the known microRNAs from human, rat, mouse and cattle.RESULTS67 microRNAs were identified in fetal LD muscle based on the generation of strong hybridization signals. Among those, 3 microRNAs, including hsa‐miR‐381, hsa‐let‐7g and bta‐miR‐376d, were differentially expressed between the Con and OB groups, which was confirmed by real‐time quantitative PCR. Hsa‐miR‐381 was 51.7 ± 17.3% higher (P &lt; 0.05) in the OB fetal muscle compared to Con, while Hsa‐let‐7g (21.5 ± 4.9% (P = 0.06) and Bta‐miR‐376d (26.0 ± 8.8% (P &lt; 0.05) were lower.CONCLUSIONMaternal obesity induced differential expression of microRNAs in fetal LD muscle. Since microRNAs regulate gene expression, changes in microRNA expression potentially play important roles in the development of fetal muscle, including adipogenesis, in the setting of maternal obesity. (Supported by NIH 1R03HD057506 and Wyoming INBRE P20RR016474)

MicroRNAs and Heart Failure Diagnosis

See related article, pages 1035–1039 Heart disease is the greatest noninfectious health hazard ever to confront the human race. It is the leading cause of death in industrialized nations, and, accordingly, it is responsible for a huge economic burden to society. It is estimated that 5 million Americans have heart failure (HF), a syndrome with mortality of approximately 50% at 5 years.1 Now, evidence indicates that the epidemic of HF is extending to the developing world. The road to advances in the treatment of HF is littered with failures, and many new means of diagnosing and tracking disease progression have disappointed. Therefore, there is an urgent need to identify novel mechanisms, markers, and therapeutic targets in HF pathogenesis. MicroRNAs (miRNAs or miRs) are among the most exciting areas in medicine and science today. MicroRNAs are endogenous, highly conserved, ≈22-nucleotide-long noncoding RNAs that regulate the stability and subsequent translation of nascent mRNA transcripts. Generally, miRs inhibit protein translation and/or promote mRNA degradation by base-pairing with 3′ untranslated regions within a transcript. MicroRNAs were first discovered in 1993 in Caenorhabditis elegans and are known to be critical to early vertebrate development and in an increasing number of basic molecular processes. Over the last few years, miRs have emerged as important mechanisms in human disease, including cardiovascular disease, diabetes, and cancer.2–6 Now, there is burgeoning interest in testing whether miRs could serve as markers for disease progression or prognostication. Indeed, in the cancer literature, strategies are emerging to use miR profiles for diagnosis and risk stratification. Currently, miR profiling is being evaluated in prostate cancer, head and neck squamous cell carcinomas, breast cancer, acute lymphoblastic leukemia, malignant mesothelioma, and other tumors.2,7–10 Most clinical studies to date on miRs have relied on tissue-based measurement of miR abundances, but miRs are …

Lack of change in microRNA expression in adult mouse liver following treatment with benzo(a)pyrene despite robust mRNA transcriptional response

Benzo(a)pyrene (BaP) is a mutagenic and carcinogenic environmental contaminant. Metabolic activation of BaP is required for it to exert its mutagenic effects. Metabolism occurs via BaP interaction with the aryl hydrocarbon receptor (AHR) resulting in induction of phase 1 enzymes. Exposure to BaP is expected to cause differential regulation of AHR-responsive genes as well as pathways responding to DNA damage induced by its metabolites. MicroRNAs (miRNAs) are short non-coding molecules that control mRNA levels and protein translation. MiRNA regulation may also be affected by chemical insult. Here we investigate the correlation between hepatic mRNA and miRNA response to BaP in vivo. Mature male mice were orally exposed to 3 daily doses of 150 mg/kg BaP. DNA microarrays were used to profile gene and miRNA expression in the liver 4 and 24 h following the final dose. Despite widespread changes in gene expression (>400 genes) in pathways consistent with the known effects of BaP, we found no changes in miRNA. This was confirmed on two microarray platforms and by qRT-PCR. Analysis of positive controls (2 distinct reference pools) indicated that the Agilent technology could identify differences in miRNA. The effects of sample storage at −80 °C were also compared. We found little effect of prolonged freezing on the technical correlation between samples or on differential expression. Our results are consistent with the lack of response of miRNA in rodent liver to dioxin, another potent AHR-agonist. We conclude that hepatic miRNA in vivo is not directly responsive to BaP exposure.

Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

BackgroundTelomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.Methodology/Principal FindingsIn this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.Conclusions/SignificanceTo our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.

Involvement of MicroRNAs in the Cytotoxic Effects Exerted by Proinflammatory Cytokines on Pancreatic β-Cells

OBJECTIVEPancreatic β-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated β-cell cytotoxicity.RESEARCH DESIGN AND METHODSWe used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in β-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated β-cell failure by modifying their expression in insulin-secreting MIN6 cells.RESULTSWe found that IL-1β and TNF-α induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1β exposure. Moreover, anti–miR-34a and anti–miR-146a treatment protected MIN6 cells from cytokine-triggered cell death.CONCLUSIONSOur data identify miR-21, miR-34a, and miR-146a as novel players in β-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice.