Geraldo A. Passos

Identifying common and specific microRNAs expressed in peripheral blood mononuclear cell of type 1, type 2, and gestational diabetes mellitus patients

BackgroundRegardless the regulatory function of microRNAs (miRNA), their differential expression pattern has been used to define miRNA signatures and to disclose disease biomarkers. To address the question of whether patients presenting the different types of diabetes mellitus could be distinguished on the basis of their miRNA and mRNA expression profiling, we obtained peripheral blood mononuclear cell (PBMC) RNAs from 7 type 1 (T1D), 7 type 2 (T2D), and 6 gestational diabetes (GDM) patients, which were hybridized to Agilent miRNA and mRNA microarrays. Data quantification and quality control were obtained using the Feature Extraction software, and data distribution was normalized using quantile function implemented in the Aroma light package. Differentially expressed miRNAs/mRNAs were identified using Rank products, comparing T1DxGDM, T2DxGDM and T1DxT2D. Hierarchical clustering was performed using the average linkage criterion with Pearson uncentered distance as metrics.ResultsThe use of the same microarrays platform permitted the identification of sets of shared or specific miRNAs/mRNA interaction for each type of diabetes. Nine miRNAs (hsa-miR-126, hsa-miR-1307, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144, hsa-miR-199a-5p, hsa-miR-27a, hsa-miR-29b, and hsa-miR-342-3p) were shared among T1D, T2D and GDM, and additional specific miRNAs were identified for T1D (20 miRNAs), T2D (14) and GDM (19) patients. ROC curves allowed the identification of specific and relevant (greater AUC values) miRNAs for each type of diabetes, including: i) hsa-miR-1274a, hsa-miR-1274b and hsa-let-7f for T1D; ii) hsa-miR-222, hsa-miR-30e and hsa-miR-140-3p for T2D, and iii) hsa-miR-181a and hsa-miR-1268 for GDM. Many of these miRNAs targeted mRNAs associated with diabetes pathogenesis.ConclusionsThese results indicate that PBMC can be used as reporter cells to characterize the miRNA expression profiling disclosed by the different diabetes mellitus manifestations. Shared miRNAs may characterize diabetes as a metabolic and inflammatory disorder, whereas specific miRNAs may represent biological markers for each type of diabetes, deserving further attention.

Gene expression profiles in human lymphocytes irradiated in vitro with low doses of gamma rays

Abstract Fachin, A. L., Mello, S. S., Sandrin-Garcia, P., Junta, C. M., Donadi, E. A., Passos, G. A. S. and Sakamoto-Hojo, E. T. Gene Expression Profiles in Human Lymphocytes Irradiated In Vitro with Low Doses of Gamma Rays. Radiat. Res. 168, 650–665 (2007). The molecular mechanisms underlying responses to low radiation doses are still unknown, especially in normal lymphocytes, despite the evidence suggesting specific changes that may characterize cellular responses. Our purpose was to analyze gene expression profiles by DNA microarrays in human lymphocytes after in vitro irradiation (10, 25 and 50 cGy) with γ rays. A cytogenetic analysis was also carried out for different radiation doses. G0 lymphocytes were irradiated and induced to proliferate for 48 h; then RNA samples were collected for gene expression analysis. ANOVA was applied to data obtained in four experiments with four healthy donors, followed by SAM analysis and hierarchical clustering. For 10, 25 and 50 cGy, the numbers of significantly (FDR ≤ 0.05) modulated genes were 86, 130 and 142, respectively, and 25, 35 and 33 genes were exclusively modulated for each dose, respectively. We found CYP4X1, MAPK10 and ATF6 (10 cGy), DUSP16 and RAD51L1 (25 cGy), and RAD50, REV3L and DCLRE1A (50 cGy). A set of 34 significant genes was common for all doses; while SERPINB2 and C14orf104 were up-regulated, CREB3L2, DDX49, STK25 and XAB2 were down-regulated. Chromosome damage was significantly induced for doses ≥10 cGy (total aberrations) and ≥50 cGy (dicentrics/ rings). Therefore, low to moderate radiation doses induced qualitative and/or quantitative differences and similarities in transcript profiles, reflecting the type and extent of DNA lesions. The main biological processes associated with modulated genes were metabolism, stress response/DNA repair, cell growth/differentiation, and transcription regulation. The results indicate a potential risk to humans regarding the development of genetic instability and acquired diseases.

Immunosuppressive therapy modulates T lymphocyte gene expression in patients with systemic lupus erythematosus

To evaluate the T‐cell large‐scale differential gene expression in systemic lupus erythematosus (SLE) patients presenting with glomerulonephritis we studied SLE patients before and after immunosuppressive treatment. Large‐scale gene expression of peripheral blood mononuclear T cells was evaluated using cDNA microarray nylon membranes containing 5184 cDNAs. Data were analysed using the SAM and Cluster and Treeview software. When untreated patients were compared to healthy individuals, 38 genes, most of them located on chromosomes 1, 3, 6, 17 and 19, were repressed, and when untreated patients were compared to treated ones, 154 genes, located on chromosomes 1, 6, 7, 12 and 17, were induced. In terms of biological function of coded proteins, the differentially expressed genes were associated with apoptosis, cell cycle, chromosomal scaffold, cytokine/chemokine, DNA repair/replication, Golgi/mitochondrial proteins, mRNA processing, signalling molecules and tumour suppressors. Two autoantigen genes related to RNA splicing (small nuclear riboprotein 70 000 MW‐U1 SNR, and splicing factor 3a, 60 000 MW), and the tetranectin‐plasminogen‐binding protein were repressed. The Fc fragment of immunoglobulin G low affinity IIb, apoptotic protease activating factor‐1, two subunits of cytochrome c, caspase 8, complement C5a, HLA‐DRA, HLA‐DQB1, transforming growth factor‐β receptor II, small nuclear ribonucleoprotein polypeptide N (Sm protein N) genes, heterogeneous nuclear riboprotein‐C, and argininosuccinate lyase genes, among others, were induced. A total of 10 genes were repressed in untreated patients and induced in treated ones, among them tumour necrosis factor (ligand) superfamily member 9, tumour protein p53, mannosidase alpha class IA, and CD22. Although some of these differentially expressed genes are typically expressed in B cells, CD22 and CD32 have also been reported in T cells and may provide regulatory signals to B cells. Assessment of differential gene expression may provide hybridization signatures that may identify susceptibility, diagnostic and prognostic markers of SLE.

MicroRNA expression profiling and functional annotation analysis of their targets in patients with type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing pancreatic β-cells, leading to elimination of insulin production. The exact cause of this disorder is still unclear. Although the differential expression of microRNAs (miRNAs), small non-coding RNAs that control gene expression in a post-transcriptional manner, has been identified in many diseases, including T1DM, only scarce information exists concerning miRNA expression profile in T1DM. Thus, we employed the microarray technology to examine the miRNA expression profiles displayed by peripheral blood mononuclear cells (PBMCs) from T1DM patients compared with healthy subjects. Total RNA extracted from PBMCs from 11 T1DM patients and nine healthy subjects was hybridized onto Agilent human miRNA microarray slides (V3), 8x15K, and expression data were analyzed on R statistical environment. After applying the rank products statistical test, the receiver-operating characteristic (ROC) curves were generated and the areas under the ROC curves (AUC) were calculated. To examine the functions of the differentially expressed (p-value<0.01, percentage of false-positives <0.05) miRNAs that passed the AUC cutoff value ≥ 0.90, the database miRWalk was used to predict their potential targets, which were afterwards submitted to the functional annotation tool provided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.7, using annotations from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. We found 57 probes, corresponding to 44 different miRNAs (35 up-regulated and 9 down-regulated), that were differentially expressed in T1DM and passed the AUC threshold of 0.90. The hierarchical clustering analysis indicated the discriminatory power of those miRNAs, since they were able to clearly distinguish T1DM patients from healthy individuals. Target prediction indicated that 47 candidate genes for T1DM are potentially regulated by the differentially expressed miRNAs. After performing functional annotation analysis of the predicted targets, we observed 22 and 12 annotated KEGG pathways for the induced and repressed miRNAs, respectively. Interestingly, many pathways were enriched for the targets of both up- and down-regulated miRNAs and the majority of those pathways have been previously associated with T1DM, including many cancer-related pathways. In conclusion, our study indicated miRNAs that may be potential biomarkers of T1DM as well as provided new insights into the molecular mechanisms involved in this disorder.

Autoimmune regulator (Aire) controls the expression of microRNAs in medullary thymic epithelial cells

The autoimmune regulator (Aire) is a transcription factor that controls the ectopic expression of a large set of peripheral tissue antigen (PTA) genes in medullary thymic epithelial cells (mTECs). Recent evidence has demonstrated that Aire releases stalled RNA polymerase II (RNA Pol II) from blockage at the promoter region of its target genes. Given that, in addition to messenger RNAs (mRNA), RNA Pol II also transcribes microRNAs (miRNAs), we raised the hypothesis that Aire might play a role as an upstream controller of miRNA transcription. To test this, we initially analyzed the expression profiles of 662 miRNAs in control and Aire-silenced (siRNA) murine mTEC 3.10 cells using microarrays. The bioinformatics programs SAM and Cluster-TreeView were then used to identify the differentially expressed miRNAs and their profiles, respectively. Thirty Aire-dependent miRNAs were identified in the Aire-silenced mTECs, of which 18 were up- and 12 were down-regulated. The down-regulated miR-376 family was the focus of this study because its members (miR-376a, miR-376b and miR-376c) are located in the genome within the Gm2922 open-reading frame (ORF) gene segment on the chromosome 12F1. The T-boxes (TTATTA) and G-boxes (GATTGG), which represent putative RNA Pol II promoter motifs, were located in a portion spanning 10 kb upstream of the ATG codon of Gm2922. Moreover, we found that Gm2922 encodes an mRNA, which was also down-regulated in Aire-silenced mTECs. These results represent the first evidence that Aire can play a role as a controller of transcription of miRNAs located within genomic regions encompassing ORF and/or mRNA genes.

Microarray-based gene expression analysis of human osteoblasts in response to different biomaterials

Several biomaterials have been widely used in bone regeneration/substitution procedures in orthopedic and oral surgery. However, how these biomaterials alter osteoblast gene expression is poorly understood. We therefore attempted to address this question by using cDNA microarray technique to identify genes that are differentially regulated in osteoblasts exposed to biomaterials comprehending the biocompatibility spectrum of bioactive (bioglass and hydroxyapatite), bioinert (Ti and stainless steel), and biotolerant (polymethylmethacrylate). By using a cDNA microarray containing 687 human IMAGE sequences, we identified in primary cultures of osteoblastic cells differentiated from the human bone marrow and exposed to these biomaterials, genes whose expression was significantly upregulated or downregulated. Among the differentially expressed genes we have found those involved with cell cycle regulation, cell differentiation and proliferation, apoptosis, cell adhesion, bone mineralization and skeletal development. These results can be relevant to a better understanding of the molecular mechanism underlying the behavior of osteoblasts in bone regenerative procedures.

Promiscuous gene expression in the thymus: The root of central tolerance

The thymus is a complex organ with an epithelium formed by two main cell types, the cortical thymic epithelial (cTECs) and medullary thymic epithelial cells (mTECs), referred to as stroma. Immature thymocytes arising from the bone marrow, macrophages and dendritic cells also populate the thymus. Thymocytes evolve to mature T cells featuring cell differentiation antigens (CDs), which characterize the phenotypically distinct stages, defined as double-negative (DN), double positive (DP) and single positive (SP), based on expression of the coreceptors CD4 and CD8. The thymus is therefore implicated in T cell differentiation and during development into T cells thymocytes are in close association with the stroma. Recent evidence showed that mTECs express a diverse set of genes coding for parenchymal organ specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has led to the reconsideration of the role of the thymus in central T cell tolerance to self-antigens, which prevents autoimmunity. The evidence of PGE is causing a reanalysis in the scope of central tolerance understanding. We summarize the evidence of PGE in the thymus, focusing particularly the use of cDNA microarray technology for the broad characterization of gene expression and demarcation of PGE emergence during thymus ontogeny.

Frequency of the Δccr5 deletion allele in the urban Brazilian population

Abstract Studies on screening genes conferring resistance to HIV-1 and AIDS onset have shown a direct relationship between a 32 base pair (bp) deletion in the CCR5 β-chemokine receptor gene (Δccr5 mutant allele) and long survival of HIV-1 infected individuals bearing this mutation. These findings led to an interest in studies of Δccr5 allele distribution in human populations. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 193-bp product from the normal CCR5 allele and a 161-bp product from the 32-bp deletion allele. In an investigation of the urban Brazilian population we detected a 93% frequency of normal CCR5/CCR5 homozygous individuals and a 7% frequency of CCR5/Δccr5 heterozygous individuals. The frequency of the Δccr5 mutant allele in this population is 0.035; however, no homozygous Δccr5 individual has been detected thus far. This is the first evidence for the contribution of the Δccr5 allele to the genetic background of the urban Brazilian population, which is characterized by intense ethnic admixture. These findings open perspectives for further studies on the relationship between Δccr5 allele frequency and AIDS onset in high-risk HIV-1 exposures individuals.

Recurrent 22q11.2 deletion in a sibship suggestive of parental germline mosaicism in velocardiofacial syndrome.

Deletions of chromosome 22q11.2 are recognized as the main cause of a number of clinical phenotypes, including velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS). Velocardiofacial syndrome is a relatively common developmental disorder that is characterized by craniofacial anomalies and conotruncal heart defects. Most 22q11.2 deletions occur sporadically, although the deletion may be transmitted in some cases. The present performed a molecular analysis in one family including a patient with clinical diagnosis of VCFS and his sister with a suggestive phenotype. Six polymorphic 22q11.2 markers (i.e. D22S420, D22S264, D22S941, D22S306, D22S425 and D22S257) were used for genotype analysis of the DNA from the patients and unaffected relatives. The results revealed a 22q11.2 deletion in the patient and his sister from one of six markers (i.e. D22S941). Genotype analysis demonstrated that the deletion in this sib was of maternal origin. The results suggest that the mother probably has gonadal mosaicism. The other relatives present normal DNA profiles for all markers. These results have implications for genetic counseling because of a risk of transmission by germ cells carrying the deletion, even when parents present with a normal DNA profile in their blood cells.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4×44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease.