Abstract 835: Downregulation Of Cardiomyocyte-enriched Micrornas Contributes To Altered Gene Expression In Heart Failure

Background: MicroRNAs (miRNAs) are a novel class of non-coding RNAs that regulate gene expression posttransciptionally. Altered miRNA expression has been implicated in diverse human diseases such as cancer. Accumulating evidence suggests the importance of miRNAs in the heart. However, the contribution of miRNAs to heart disease remains incompletely understood. Methods and Results: We measured the expression of 261 miRNAs in heart failure resulting from transgenic overexpression of calcineurin. 59 miRNAs were confidently detected in the heart, and 11 miRNAs belonging to 6 families (miR-1, -15, -30, -133, -195, -208) were downregulated compared to non-transgenic control (Welch’s t-test nominal p<0.05, false discovery rate <0.001). The results were validated by qRTPCR. There was no upregulated miRNA. Four of these miRNAs (miR-1, -30, -133, -208) were enriched in a purified cardiomyocyte preparation, compared to non-myocytes. Downregulation of these four miRNAs was reproduced in purified failing versus non-failing cardiomyocytes. This excluded artifactual downregulation from reduced myocyte fraction in failing hearts. The remaining two miRNAs (miR-15, and -195) were exclusively expressed in non-cardiomyocytes and did not changed in failing cardiomyocytes. Next, we used Affymetrix expression profiling to show that the predicted targets of these downregulated miRNAs were disproportionately upregulated compared to the entire transcriptome (Fisher’s exact p < 0.001). This suggests an association between downregulation of these miRNAs and upregulation of predicted target genes in heart failure. One particularly intriguing target of the predominant cardiac microRNA miR-1 is calmodulin, a key regulator of calcium signaling. We showed that calmodulin and downstream calmodulin signaling to NFAT is regulated by miR-1 in cultured cardiomyocytes. Conclusion: Our results indicate that altered expression of cardiomyocyte-enriched miRNAs may contribute to abnormal gene expression in heart failure. The regulation of calmodulin and calcium signaling by miR-1 suggests a mechanism by which miR-1 may regulate heart function.

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Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure

AJP Renal Physiology ◽

10.1152/ajprenal.00329.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. F953-F963 ◽

Cited By ~ 70

Author(s):

David P. Basile ◽

Katherine Fredrich ◽

Morufu Alausa ◽

Carlos P. Vio ◽

Mingyu Liang ◽

...

Keyword(s):

Gene Expression ◽

Renal Failure ◽

Acute Renal Failure ◽

Ischemia Reperfusion ◽

Regenerative Process ◽

Matrix Gla Protein ◽

Specific Marker ◽

Altered Expression ◽

Altered Gene ◽

Ischemic Acute Renal Failure

Recovery from ischemic acute renal failure (ARF) involves a well-described regenerative process; however, recovery from ARF also results in a predisposition to a progressive renal disease that is not well understood. This study sought to identify alterations in renal gene expression in postischemic, recovered animals that might play important roles in this progressive disorder. RNA isolated from sham-operated control rats or rats 35 days after recovery from bilateral ischemia-reperfusion (I/R) injury was compared using a cDNA microarray containing ∼2,000 known rat genes. A reference hybridization strategy was utilized to define a 99.9% interval and to identify 16 genes that were persistently altered after recovery from I/R injury (12 were upregulated and 4 were downregulated). Real-time PCR verified the altered expression of six of eight genes that had been positively identified. Several genes that were identified had not previously been evaluated within the context of ARF. S100A4, a specific marker of fibroblasts, was identified in a population of interstitial cells that were present postischemic injury. S100A4-positive cells were also identified in tubular cells at earlier time points postischemia. Genes associated with calcification, including osteopontin and matrix Gla protein, were also enhanced postischemic injury. Several proinflammatory genes were identified, including complement C4, were enhanced in postischemic tissues. Conversely, renal kallikrein expression was specifically reduced in the postischemic kidney. In summary, genes with known inflammatory, remodeling, and vasoactive activities were identified in rat kidneys after recovery from ARF, some of which may play a role in altering long-term renal function after recovery from ARF.

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Molecular Alterations in Bone Marrow Mesenchymal Stroma Cells of AML Patients

Blood ◽

10.1182/blood.v124.21.699.699 ◽

2014 ◽

Vol 124 (21) ◽

pp. 699-699

Author(s):

Eva von der Heide ◽

Sebastian Vosberg ◽

Martin Neumann ◽

Liliana H. Mochmann ◽

Alva Rani James ◽

...

Keyword(s):

Gene Expression ◽

Genetic Alterations ◽

P Value ◽

Pathogenic Role ◽

Altered Expression ◽

Target Region ◽

Molecular Alterations ◽

Altered Gene Expression ◽

Altered Gene ◽

Mesenchymal Stroma Cells

Abstract It is increasingly recognized that the tumor microenvironment plays a pivotal role in cancer initiation and progression. In mouse models it was shown that a genetically altered bone marrow (BM) micro milieu was sufficient to induce leukemia (Raaijmakers, Nature 2010); however, the pathogenic role and contribution of the BM stroma in leukemia initiation and during disease progression warrants further investigation. To address this, we have performed gene expression, methylation, RNAseq, whole exome sequencing (WES) in BM mesenchymal stroma cells (BM-MSC) and leukemic cells from AML patients (pts) to unravel underlying molecular alterations. We collected BM hematopoietic cells (BM-HC) as well as plastic-adherent BM-MSC from aspirates from AML pts and healthy donors (HD). BM-MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33-. We investigated gene expression profiles (Affymetrix) of BM-MSC from newly diagnosed AML pts (n=20) and compared these to BM-MSC from HD (n=4). BM-MSC from AML pts displayed an altered expression signature with 191and 175genesbeingsignificantly 2-fold over- and under-expressed. KEGG analysis of differentially expressed genes in BM-MSC from AML pts exhibited enrichment for TGF-ß signalling, whereas downregulated genes were enriched for cytokine receptor interactions. Several of these candidates were validated in a larger set of BM-MSC samples by RT-PCR. One putative stroma-leukemia interaction molecule, lumican (LUM) was highly overexpressed in BM-MSC (n=60) from AML pts compared to HD (n=5; p value =0.019) indicating that LUM may affect the BM niche in AML. To explore the altered expression pattern in AML BM-MSC compared to HD BM-MSC, global methylation analyses (Illumina Infinium HumanMethylation 450 bead chip arrays) were performed in 5 AML pts where we had collected BM-HC and BM-MSC at 3 sequential time points [initial diagnosis (ID), remission (CR), relapse (REL); n=30] as well as in BM-HC and BM-MSC from HDs (n=6). A significantly different methylation profile was evident comparing AML BM-HC to the corresponding AML BM-MSC samples, the latter showing a homogenous pattern during the course of disease. When AML BM-MSC were compared to a set of HD BM-MSC, we identified 2416 differentially methylated CpG sites (p value <0.01) indicating that an epigenetic deregulation contributes to the altered gene expression profile observed in AML BM-MSC. These 30 AML BM-MSC/BM-HC samples were subsequently analyzed by WES to unravel genetic alterations in the compartments of the mesenchymal and hematopoietic cell fractions. In WES (HiSeq2000, 100bp paired-end), we obtained an average of 100 reads for the target region; more than 90% of the exome target region was covered at least 30-fold. When the AML BM-HC CR sample was used as germline control, a median of 3 SNVs were detected in AML BM-MSC samples. The only BM-MSC-specific alteration present in one AML patient at all time points (ID, CR, REL) was a mutation in the plectin gene (PLEC). This mutation in the ROD domain of this cytoskeletal linker protein is located in the hot spot for mutations described in epidermolysis bullosa. This mutation was validated by Sanger sequencing, however in a larger cohort of 50 AML ID BM-MSC, no additional PLEC mutation at the same position was found. The set of AML BM-MSC (n=15) samples further allowed us to identify lesions (SNVs, Indels) in the corresponding BM-HC (n=15). When we used the AML BM-MSC as germline control we identified in total 43 lesions in the AML BM-HC fractions, which were not found when the corresponding BM-HC CR sample was used as germline control. This unraveled pre-leukemic lesions present in the AML BM-HC at remission: importantly using this approach, lesions in ASXL1 (Y591*) and DNMT3A (R882H), and in another patient a DNMT3A (M880V) mutation were revealed. In conclusion, the altered gene expression profile and methylation signature of AML BM-MSC provide novel insights into the pathogenic role of the leukemic BM microenvironment. Genetic alterations explored by WES revealed only very few genetic hits that will require further functional exploration. However, the low number of genetic alterations suggests that the transcriptional and epigenetic alterations are directed by extrinsic factors. At the same time, AML BM-MSC provides a non-hematopoietic derived germline control that allows to unravel pre-leukemic lesions in BM-HC. Disclosures No relevant conflicts of interest to declare.

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342. Downregulation of Cardiomyocyte-Enriched Micrornas Contributes to Altered Gene Expression in Heart Failure

Molecular Therapy ◽

10.1016/s1525-0016(16)39745-3 ◽

2008 ◽

Vol 16 ◽

pp. S128-S129

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Altered Gene Expression ◽

Altered Gene

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Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00707.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. H268-H276 ◽

Cited By ~ 44

Author(s):

Charles Steenbergen ◽

Cynthia A. Afshari ◽

John G. Petranka ◽

Jennifer Collins ◽

Karla Martin ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Human Heart ◽

Compensatory Hypertrophy ◽

Unknown Etiology ◽

Inhibitory Protein ◽

Human Heart Failure ◽

Altered Gene Expression ◽

Tnf Α ◽

Altered Gene

Dilated cardiomyopathy, a disease of unknown etiology and pathogenesis, is associated with heart failure and compensatory hypertrophy. Although cell and animal models suggest a role for altered gene expression in the transition to heart failure, there is a paucity of data derived from the study of human heart tissue. In this study, we used DNA microarray profiling to investigate changes in the expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We observed altered gene expression consistent with a proapoptotic shift in the TNF-α signaling pathway. Specifically, we found decreased expression of TNF-α- and NF-κB-induced antiapoptotic genes such as growth arrest and DNA damage-inducible ( GADD) 45β, Flice inhibitory protein ( FLIP), and TNF-induced protein 3 ( A20). Consistent with a role for apoptosis in heart failure, we also observed a significant decrease in phosphorylation of BAD at Ser-112. This study identifies several pathways that are altered in human heart failure and provides new targets for therapy.

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H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions

10.1101/684712 ◽

2019 ◽

Author(s):

Yichao Cai ◽

Ying Zhang ◽

Yan Ping Loh ◽

Jia Qi Tng ◽

Mei Chee Lim ◽

...

Keyword(s):

Gene Expression ◽

Tumor Growth ◽

Target Genes ◽

Gene Repression ◽

Xenograft Tumor ◽

Regulate Gene Expression ◽

Chromatin Interactions ◽

Genomic Regions ◽

Xenograft Tumor Growth ◽

Regulate Gene

AbstractGene repression and silencers are poorly understood. We reasoned that H3K27me3-rich regions (MRRs) of the genome defined from clusters of H3K27me3 peaks may be used to identify silencers that can regulate gene expression via proximity or looping. MRRs were associated with chromatin interactions and interact preferentially with each other. MRR component removal at interaction anchors by CRISPR led to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. The MRR knockout cells also showed changes in phenotype associated with cell identity, and altered xenograft tumor growth. MRR-associated genes and long-range chromatin interactions were susceptible to H3K27me3 depletion. Our results characterized H3K27me3-rich regions and their mechanisms of functioning via looping.

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Correlation between miRNA-targeted-gene promoter methylation and miRNA regulation of target genes

F1000Research ◽

10.12688/f1000research.2-21.v3 ◽

2013 ◽

Vol 2 ◽

pp. 21 ◽

Cited By ~ 2

Author(s):

Y-h Taguchi

Keyword(s):

Gene Expression ◽

Promoter Methylation ◽

Target Genes ◽

Regulation Of Gene Expression ◽

Gene Promoter ◽

Gene Expression Omnibus ◽

Seed Region ◽

Mirna Regulation ◽

Regulate Gene Expression ◽

Mirna Targeting

Background miRNA regulation of target genes and promoter methylation are known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied.Methods Gene expression and promoter methylation profiles of 270 distinct human cell lines were obtained from Gene Expression Omnibus. P-values that describe both miRNA-targeted-gene promoter methylation and miRNA regulation of target genes were computed using the MiRaGE method proposed recently by the author.Results Significant changes in promoter methylation were associated with miRNA targeting. It was also found that miRNA-targeted-gene promoter hypomethylation was related to differential target gene expression; the genes with miRNA-targeted-gene promoter hypomethylation were downregulated during cell senescence and upregulated during cellular differentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs. miRNA-targeted-gene promoter methylation may also be related to the seed region features of miRNA.Conclusions It was found that promoter methylation was correlated to miRNA targeting. Furthermore, miRNA-targeted-gene promoter hypomethylation was especially enhanced in promoters of genes targeted by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs) and was suggested to be highly related to some seed region features of miRNAs.

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P1614Soluble guanylate cyclase as a therapeutic target in heart failure: myocardial gene expression in response to sGC stimulation in pressure overload

European Heart Journal ◽

10.1093/eurheartj/ehz748.0373 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

J Ruedebusch ◽

A Benkner ◽

N Nath ◽

L Kaderali ◽

K Klingel ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Expression Pattern ◽

Heart Function ◽

Gene Expression Pattern ◽

Pressure Overload ◽

Left Ventricular ◽

Marker Genes ◽

Cgmp Pathway

Abstract Background Heart Failure (HF) is associated with endothelial dysfunction and reduced bioavailability of NO with insufficient stimulation of sGC and reduced production of cGMP. Therefore, the impairment of the NO-sGC-cGMP pathway results in vasoconstriction, platelet aggregation, inflammation, fibrosis and most importantly maladaptive cardiac hypertrophy. The restoration of the NO-sGC -cGMP pathway is an attractive pharmacological target for HF therapy. Purpose Riociguat is an NO independent stimulator of the sGC that sensitizes the sGC to endogenous NO and directly stimulates sGC to produce cGMP. We therefore hypothesized that Riociguat prevents pathological effects occurring during HF. Methods Pressure overload was induced by transverse aortic constriction (TAC) in 8 weeks old male C57Bl6/N mice. Three weeks after TAC when cardiac hypertrophy has developed either Riociguat (RIO; 3 mg/kg) or a Solvent was administered daily for 5 more weeks (n=12 per group). Animals with sham surgery and same drug regime served as controls. The heart function in all groups was evaluated weekly by small animal echocardiography. Eight weeks after surgery, the transcriptome of the left ventricles (LV) of sham and TAC mice were analysed by RNA Sequencing. Differentially expressed genes (DEG) were categorised using Ingenuity Pathway Analysis (IPA). Results TAC resulted in a steady decrease of left ventricular fractional shortening (FS) in the mice until week 3. When Riociguat treatment commenced, the systolic LV function of the TAC+Rio group recovered significantly whereas the solvent group showed a further decline until week 8 (FS 21.4±3.4% vs. 9.5±2%, p<0.001). Both sham groups (Sham+Sol and Sham+Rio) showed no changes in the heart function over timer. Regarding the hypertrophic response to LV pressure overload, Riociguat treatment attenuated significantly the increase of the left ventricular mass (LVM 208.3±15.8mg vs. 148.9±11.8mg, p<0.001) after TAC. In line with the reduced LVM, histological staining showed a significantly reduced fibrosis and myocyte cross sectional area in the TAC+Rio group compared to TAC+Sol group. Regarding the myocardial transcriptome, the treatment with Riociguat resulted in less changes of gene expression pattern after TAC (TAC+Sol vs. Sham+Sol 3160 DEG; TAC+Rio vs. Sham+Rio 2237 DEG). The expression of heart failure marker genes like ANP (Nppa), BNP (Nppb), β-Myosin Heavy Chain (Myh7) and the Collagens 1 and 3 (Col1a1, Col1a2, Col3a1) were significantly decreased in TAC+Rio, when compared to TAC+Sol. IPA analysis revealed that the activation of biological pathways in response to TAC, like actin cytoskeleton- and Integrin signalling, renin-angiotensin or cardiac hypertrophy signalling was attenuated when Riociguat was administered. Conclusion Riociguat attenuates pressure overload induced LV remodelling resulting in less hypertrophy, improved heart function and less alteration of gene expression pattern.

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Aspects of Altered Gene Expression in Heart Failure and Myocardial Hypertrophy

Chronic Heart Failure ◽

10.1007/978-3-642-85913-7_14 ◽

1998 ◽

pp. 249-260

Author(s):

Michael Böhm ◽

Erland Erdmann

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Myocardial Hypertrophy ◽

Altered Gene Expression ◽

Altered Gene

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siRNA Mediated Knockdown of the Chimeric Transcription Factor E2A-PBX1 Identifies Potential Target Genes.

Blood ◽

10.1182/blood.v110.11.3179.3179 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3179-3179

Author(s):

Chun-Liang Chen ◽

Harkness Connell ◽

Ira Racoma ◽

Brandi Regula ◽

Katherine Foley ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Prognostic Significance ◽

Pediatric Leukemia ◽

Altered Expression ◽

Qrt Pcr ◽

Potential Target ◽

Genome Wide ◽

Genome Wide Gene Expression

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Several chromosomal translocations have prognostic significance and are used to classify patients for risk-directed therapy. The translocation t(1;19) which results in the fusion of TCF3 (E2A) and PBX1 genes occurs in 5% of pediatric ALL. E2A-PBX1 has been shown previously to have transcriptional activity. However, very little is known about what genes have altered expression in the presence of E2A-PBX1. To explore this question, we assessed genome wide gene expression after siRNA mediated knock down of E2A-PBX1. An E2A-PBX1 containing cell line (697) was transfected with E2A-PBX1 specific siRNA utilizing an Amaxa nucleofector2. Biologic replicates were performed by transfection of independent cultures. 70–80% reduction of E2A-PBX1 at mRNA and protein levels in 697 cells was reproducibly achieved using nucleofection and a combination of E2A-PBX1 specific siRNAs. Genome wide gene expression was assessed by Affymetrix U133 2.0 Plus arrays. Hybridizations were prepared and performed according to current Affymetrix protocols in the Functional Genomics Core at the Research Institute. Microarray data was normalized with RMA. Differentially expressed genes were selected using Significance Analysis of Microarrays (SAM). 78 probe sets demonstrated change in expression in E2A-PBX1 siRNA mediated knockdown relative to both mock transfection and nontargeting siRNA controls at a FDR of ≤ 5%. The 78 probe sets represent 49 known genes and 8 ESTs. Genes of specific interest upregulated by E2A-PBX1 include WNT-16, ANKS1B(EB-1), FAT, and RORB. Other investigators have previously cloned these genes by representational differential display from E2A-PBX1 expressing cell lines. We confirmed knockdown of these messages by E2A-PBX1 siRNA using qRT-PCR. Gene expression was calculated using the ΔΔCt method and normalization to ABL. Additionally, these genes are part of the E2A-PBX1 gene expression profile derived from primary pediatric leukemia samples previously published. [Ross, Blood 2003] An additional 34 candidate genes were further verified using qRT-PCR. The direction of change in gene expression correlated with microarray results in 30/34 genes evaluated. Other classes of genes upregulated by E2A-PBX1: kinases (FGFR2, MAP3K1), phosphtases (PPPIR14C), transcription factors (FOSL2, IRX2, EBF3, BMI1, BCL6), cell cycle-related genes (FBXW7, ETV5), Ras and Rho family genes (RAB8B). While B cell surface antigen expression (HLA-DRA, CD22) was decreased by E2A-PBX1. We have utilized siRNA to E2A-PBX1 to identify potential target genes of E2A-PBX1. This data suggests E2A-PBX1 can alter expression of a variety of classes of genes. Some of these genes may be potential targets for molecularly targeted therapy.

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