Project description:Investigation of content of microvesicles exocytosed by cardiomyocytes. The aims of the study were to identify DNA and RNA content in microvesicles from cardiomyocytes. DNA and RNA were purified from microveicles from cultured cardiomyocytes. Illumina microarrays were used to detect DNA and mRNA molecules and identify them.
Project description:Introduction: Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in target cells. We have recently shown that cultured cardiomyocytes release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the released exosome characteristics. Material and Methods: Exosomes were isolated from media collected from cultured cardiomyocyte (HL-1) cells with or without growth factor treatment (TGF-beta2 and PDGF-BB), with a series of differential centrifugations. The exosomes were characterized with dynamic light scattering (DLS) and Western blot and analysed with Illumina whole genome microarray gene expression. Results: An average size of 50-80 nm in diameter with no difference between treatment groups was found. Analysis of the mRNA content revealed 623 transcripts in the control group, 691 in the TGF-beta2-treated group and 362 in the PDGF-BB-treated group. 235 transcripts were common for all three groups. Conclusion: We conclude that there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. To study if the transcriptional content in exosomes derived from untreated and growth factor-treated cultured cardiomyocytes (HL-1) differ, and if so, can this difference be explained, 4 control (untreated) exosome samples, 4 TFG-beta2-treated cardiomyocyte-derived exosome samples and 4 PDGF-BB-treated cardiomyocyte-derived exosomes were studied.
Project description:BACKGROUND: In previous studies using candidate gene approaches, low sperm count (oligospermia) has been associated with altered sperm mRNA content and DNA methylation in both imprinted and non-imprinted genes. We performed a genome-wide analysis of sperm DNA methylation and mRNA content to test for associations with sperm function. METHODS AND RESULTS: Sperm DNA and mRNA were isolated from 21 men with a range of semen parameters presenting to a tertiary male reproductive health clinic. DNA methylation was measured with the Illumina Infinium array at 27,000 CpG loci. Unsupervised clustering of methylation data differentiated the 21 sperm samples by their motility values. Recursively partitioned mixture modeling (RPMM) of methylation data resulted in four distinct methylation profiles that were significantly associated with sperm motility (P=0.01). Linear models of microarray analysis (LIMMA) was performed based on motility and identified 9,189 CpG loci with significantly altered methylation (Q<0.05) in the low motility samples, with many loci located in genes associated with subfertility and epigenetic regulation. In the low motility samples, the majority of disrupted CpG loci (80%) were hypomethylated. Of the aberrantly methylated CpGs, 194 were associated with imprinted genes almost equally distributed into hypermethylated (predominantly paternally expressed) and hypomethylated (predominantly maternally expressed) groups. Sperm mRNA was measured with the Human Gene 1.0 ST Affymetrix GeneChip Array. LIMMA analysis based on motility identified 20 candidate transcripts as differentially expressed in low motility sperm, including HDAC1 (NCBI 3065), SIRT3 (NCBI 23410), and DNMT3A (NCBI 1788). Altered expression of these epigenetic regulatory genes was associated with RPMM DNA methylation class. CONCLUSIONS: Using integrative genome-wide approaches to study epigenetic and gene expression patterns in human sperm we identified CpG methylation profiles and mRNA alterations associated with low sperm motility, and that low motility sperm may have aberrant genome-wide hypomethylation due to excess HDAC1 activity.
Project description:BACKGROUND: In previous studies using candidate gene approaches, low sperm count (oligospermia) has been associated with altered sperm mRNA content and DNA methylation in both imprinted and non-imprinted genes. We performed a genome-wide analysis of sperm DNA methylation and mRNA content to test for associations with sperm function. METHODS AND RESULTS: Sperm DNA and mRNA were isolated from 21 men with a range of semen parameters presenting to a tertiary male reproductive health clinic. DNA methylation was measured with the Illumina Infinium array at 27,000 CpG loci. Unsupervised clustering of methylation data differentiated the 21 sperm samples by their motility values. Recursively partitioned mixture modeling (RPMM) of methylation data resulted in four distinct methylation profiles that were significantly associated with sperm motility (P=0.01). Linear models of microarray analysis (LIMMA) was performed based on motility and identified 9,189 CpG loci with significantly altered methylation (Q<0.05) in the low motility samples, with many loci located in genes associated with subfertility and epigenetic regulation. In the low motility samples, the majority of disrupted CpG loci (80%) were hypomethylated. Of the aberrantly methylated CpGs, 194 were associated with imprinted genes almost equally distributed into hypermethylated (predominantly paternally expressed) and hypomethylated (predominantly maternally expressed) groups. Sperm mRNA was measured with the Human Gene 1.0 ST Affymetrix GeneChip Array. LIMMA analysis based on motility identified 20 candidate transcripts as differentially expressed in low motility sperm, including HDAC1 (NCBI 3065), SIRT3 (NCBI 23410), and DNMT3A (NCBI 1788). Altered expression of these epigenetic regulatory genes was associated with RPMM DNA methylation class. CONCLUSIONS: Using integrative genome-wide approaches to study epigenetic and gene expression patterns in human sperm we identified CpG methylation profiles and mRNA alterations associated with low sperm motility, and that low motility sperm may have aberrant genome-wide hypomethylation due to excess HDAC1 activity. See "summary" above
Project description:Aims: Cardiomyocytes apoptosis is the predominant pathological feature following myocardial infarction (MI) and significantly impacts disease progression and outcomes. Zinc finger NFX1-type containing 1 (ZNFX1), an RNA helicase family member, remains relatively understudied in molecular biology and its role in cardiovascular diseases remains unclear. This study aims to explore the involvement of ZNFX1 in MI and uncover its mechanisms underlying cardiomyocyte apoptosis. Methods and results: Male C57BL/6 mice were administered AAV9-ZNFX1 or AAV9-sh-ZNFX1 with the cTNT promoter via tail vein injection for the purpose of cardiomyocyte-specific ZNFX1 overexpression or knockdown. Concurrently, left anterior descending coronary artery ligation (LAD) was performed to induce MI. Echocardiography was utilized to evaluate the cardiac function. Triphenyl tetrazolium chloride (TTC), Hematoxylin and eosin (HE) and Masson's trichrome staining were used to evaluate the cardiac infarction and cardiac remodeling. TUNEL assay, flow cytometry, western blot and quantitative reverse transcription PCR (qRT-PCR) were employed to evaluate apoptosis. The cardiomyocyte viability was quantified using the CCK-8 assay. RNA sequencing (RNA-seq) and RNA immunoprecipitation assays (RIP) were employed to investigate the interaction between ZNFX1 and apoptosis-associated genes. The expression of ZNFX1 was decreased in MI myocardium and hypoxia-treated cardiomyocytes. Overexpression of ZNFX1 significantly attenuated cardiac dysfunction, reduced infarct size, inhibited collagen deposition and alleviated cardiac hypertrophy which was ascribed to MI in mice, whereas knockdown of ZNFX1 produced the opposite effects. RNA-seq identified apoptosis as a possible regulated pathway of ZNFX1, overexpression of ZNFX1 represses the cardiomyocyte apoptosis that gives rise to MI while knockdown of ZNFX1 deteriorates it. Mechanically, ZNFX1 binds with mRNA of apoptosis genes that contain highly structured 3'UTR and prompts their decay. Conclusion: ZNFX1 plays a protective role in MI by degrading mRNA of apoptosis-related genes, which often possess highly structured 3'UTRs. This highlights ZNFX1 as a potential novel molecular target for treating cardiac dysfunction associated with MI. Keywords: ZNFX1, apoptosis, mRNA stability, helicase activity, UPF1, myocardial infarction
Project description:Introduction: Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in target cells. We have recently shown that cultured cardiomyocytes release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the released exosome characteristics. Material and Methods: Exosomes were isolated from media collected from cultured cardiomyocyte (HL-1) cells with or without growth factor treatment (TGF-beta2 and PDGF-BB), with a series of differential centrifugations. The exosomes were characterized with dynamic light scattering (DLS) and Western blot and analysed with Illumina whole genome microarray gene expression. Results: An average size of 50-80 nm in diameter with no difference between treatment groups was found. Analysis of the mRNA content revealed 623 transcripts in the control group, 691 in the TGF-beta2-treated group and 362 in the PDGF-BB-treated group. 235 transcripts were common for all three groups. Conclusion: We conclude that there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.