ABSTRACT
BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. RESULTS: We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4 × 108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. CONCLUSION: Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.
Subject(s)
B-Lymphocytes/metabolism , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , MicroRNAs/genetics , Sequence Analysis, RNA , Aged, 80 and over , Aging/genetics , Base Sequence , Female , Humans , Male , MicroRNAs/metabolism , Middle Aged , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies. RESULTS: We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. CONCLUSIONS: Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.
Subject(s)
Amnion/metabolism , DNA Methylation , Hypertension/genetics , Pre-Eclampsia/genetics , Prenatal Exposure Delayed Effects/genetics , CpG Islands , Epigenesis, Genetic , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Pregnancy , Promoter Regions, Genetic , Regression AnalysisABSTRACT
Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT-1 and ferroportin, are known, it is presumed that many more are involved and as yet unknown. Excessive exposure to manganese, usually in industrial settings such as mining or welding, can lead to neurotoxicity and a condition known as manganism that closely resembles Parkinson's disease. Elucidating transcriptional changes following manganese exposure could lead to the development of biomarkers for exposure. This unit presents a protocol for RNA sequencing in the worm Caenorhabditis elegans to assay for transcriptional changes following exposure to manganese. This protocol is adaptable to any environmental exposure in C. elegans. The protocol results in counts of gene transcripts in control versus exposed conditions and a ranked list of differentially expressed genes for further study.
Subject(s)
Caenorhabditis elegans/metabolism , Gene Expression Regulation/drug effects , Manganese/toxicity , RNA/metabolism , Animals , Base Sequence , RNA/geneticsABSTRACT
BACKGROUND: We investigated the clinical and molecular significance of minimal peritubular capillary (PTC) and isolated glomerular C4d+ staining using microarrays. METHODS: Two hundred fifty-five clinically indicated transplant biopsies were included in the analyses. C4d staining was performed on paraffin sections using a polyclonal rabbit anti-C4d antibody. Gene expression profiles in a subset of patients were studied using Affymetrix HuGene 1.0ST arrays. RESULTS: Immunohistochemistry for C4d of 255 biopsies showed 51% C4d negative, 4% minimal PTC C4d+, 15% focal or diffuse PTC C4d+, and 31% isolated glomerular C4d+ biopsies. Patients with minimal and focal/diffuse PTC C4d+ staining had higher frequency of donor-specific anti-HLA antibodies (DSA) (67% and 82%) and antibody mediated rejection (AMR) (66% and 89%) when compared with C4d-negative biopsies (25% and 19%, respectively) (P<0.001). The glomerulitis, interstitial inflammation, and peritubular capillaritis scores were also significantly higher in minimal (0.88, 1.25, and 1.5) and focal/diffuse PTC C4d+ biopsies (0.65, 1.41, and 1.5), compared with C4d-negative biopsies (0.25, 079, and 0.34), respectively. There were no differences in the DSA frequency, AMR rate, or Banff scores between isolated glomerular C4d+ and C4d-negative patients. Although both minimal and focal/diffuse C4d+ biopsies showed increased expression of genes related to the immune response, interferon-gamma and rejection-induced, cytotoxic T cell and constitutive macrophage-associated pathogenesis-based transcripts, there was no activation of immune-response-related genes in isolated glomerular C4d+ biopsies. CONCLUSION: Minimal PTC C4d+ staining but not isolated glomerular C4d+ staining is associated with AMR, circulating DSAs and immune-response-related gene activation.
Subject(s)
Biomarkers/analysis , Complement C4b/analysis , Kidney Transplantation/immunology , Kidney/immunology , Kidney/surgery , Peptide Fragments/analysis , Adult , Biopsy , Capillaries/immunology , Capillaries/pathology , Chi-Square Distribution , Complement C4b/genetics , Female , Gene Expression Profiling/methods , Gene Expression Regulation , Graft Rejection/immunology , Graft Rejection/pathology , HLA Antigens/immunology , Histocompatibility , Humans , Immunohistochemistry , Isoantibodies/blood , Kaplan-Meier Estimate , Kidney/blood supply , Kidney/pathology , Kidney Glomerulus/immunology , Kidney Glomerulus/pathology , Kidney Transplantation/mortality , Male , Middle Aged , Odds Ratio , Oligonucleotide Array Sequence Analysis , Peptide Fragments/genetics , RNA, Messenger/analysis , Treatment OutcomeABSTRACT
Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD) mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W), display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD) mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD) mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD) mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.