Transcript Expression Profiles and MicroRNA Regulation Indicate an Upregulation of Processes Linked to Oxidative Stress, DNA Repair, Cell Death, and Inflammation in Type 1 Diabetes Mellitus Patients.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing ß-cells leading to impaired insulin secretion and hyperglycemia. T1D is accompanied by DNA damage, oxidative stress, and inflammation, although there is still scarce information about the oxidative stress response and DNA repair in T1D pathogenesis. We used the microarray method to assess mRNA expression profiles in peripheral blood mononuclear cells (PBMCs) of 19 T1D patients compared to 11 controls and identify mRNA targets of microRNAs that were previously reported for T1D patients. We found 277 differentially expressed genes (220 upregulated and 57 downregulated) in T1D patients compared to controls. Analysis by gene sets (GSA and GSEA) showed an upregulation of processes linked to ROS generation, oxidative stress, inflammation, cell death, ER stress, and DNA repair in T1D patients. Besides, genes related to oxidative stress responses and DNA repair (PTGS2, ATF3, FOSB, DUSP1, and TNFAIP3) were found to be targets of four microRNAs (hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424). The expression levels of these mRNAs and microRNAs were confirmed by qRT-PCR. Therefore, the present study on differential expression profiles indicates relevant biological functions related to oxidative stress response, DNA repair, inflammation, and apoptosis in PBMCs of T1D patients relative to controls. We also report new insights regarding microRNA-mRNA interactions, which may play important roles in the T1D pathogenesis.

Assessment of DNA damage and mRNA/miRNA transcriptional expression profiles in hyperglycemic versus non-hyperglycemic patients with type 2 diabetes mellitus.

The development of type 2 diabetes mellitus (T2D) is associated with a number of genetic and environmental factors. Hyperglycemia, a T2D hallmark, is related to several metabolic complications, comorbidities and increased DNA damage. However, the molecular alterations of a proper glucose control are still unclarified. In this study, we aimed to evaluate DNA damage (comet assay), as well as to compare the transcriptional expression (mRNA and miRNA analyzed by the microarray technique) displayed by peripheral blood mononuclear cells (PBMCs) from three distinct groups: hyperglycemic T2D patients (T2D-H, n=14), non-hyperglycemic T2D patients (T2D-N, n=15), and healthy non-diabetic individuals (n=16). The comet assay revealed significantly (p<0.05) higher levels of DNA damage in T2D-H group compared to both T2D-N and control groups, while a significant difference was not observed between the control and T2D-N groups. After bioinformatics analysis, the differentially expressed mRNAs were subjected to functional enrichment analysis (DAVID) and inflammatory response was among the enriched terms found when comparing T2D-N with controls and T2D-H with T2D-N. Concerning the gene set enrichment and gene set analyses, among the differentially expressed gene sets, three were of interest: regulation of DNA repair (T2D-H versus T2D-N), superoxide response (T2D-H versus control group), and response to endoplasmic reticulum stress (T2D-H versus control group). We also identified miRNAs related with T2D and hyperglycemia not yet associated with these conditions in the literature. Some of the differentially expressed mRNAs were among the predicted targets of the differentially expressed miRNAs. Our results showed the association of hyperglycemia with increased DNA damage and aberrant expression of miRNAs and genes related to several biological processes, such as inflammation, DNA repair, ROS production and antioxidant defense, highlighting the importance of proper glycemic control. Moreover, the transcriptional expression of miRNAs provided novel information for understanding the regulatory mechanisms involved in the T2D progression.

One-week intervention period led to improvements in glycemic control and reduction in DNA damage levels in patients with type 2 diabetes mellitus.

AIMS: Hyperglycemia leads to increased production of reactive oxygen species (ROS), which reduces cellular antioxidant defenses and induces several DNA lesions. We investigated the effects on DNA damage of a seven-day hospitalization period in patients with type 2 diabetes mellitus (T2DM) to achieve adequate blood glucose levels through dietary intervention and medication treatment, compared with non-diabetic individuals. METHODS: DNA damage levels were evaluated by the alkaline comet assay (with modified and without conventional use of hOGG1 enzyme, which detects oxidized DNA bases) for 10 patients and 16 controls. Real time PCR array method was performed to analyze the transcriptional expression of a set of 84 genes implicated in antioxidant defense and response to oxidative stress in blood samples from T2DM patients (n=6) collected before and after the hospitalization period. RESULTS: The seven-day period was sufficient to improve glycemic control and to significantly decrease (p<0.05) DNA damage levels in T2DM patients, although those levels were slightly higher than those in control subjects. We also found a tendency towards a decrease in the levels of oxidative DNA damage in T2DM patients after the hospitalization period. However, for all genes analyzed, a statistically significant difference in the transcriptional expression levels was not observed. CONCLUSIONS: The study demonstrated that although the transcriptional expression of the genes studied did not show significant alterations, one-week of glycemic control in hospital resulted in a significant reduction in DNA damage levels detected in T2DM patients, highlighting the importance of an adequate glycemic control.

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 ß-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.

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.

Transcriptional Regulation of the p53 Tumor Suppressor Gene in S-Phase of the Cell-Cycle and the Cellular Response to DNA Damage.

The p53 tumor suppressor induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors and thus participates in maintaining genome stability. Numerous studies have demonstrated that p53 transcription is activated before or during early S-phase in cells progressing from G(0)/G(1) into S-phase through the combined action of two DNA-binding factors RBP-Jκ and C/EBPß-2. Here, we review evidence that this induction occurs to provide available p53 mRNA in order to prepare the cell for DNA damage in S-phase, this ensuring a rapid response to DNA damage before exiting this stage of the cell cycle.

Elevated transcription of the p53 gene in early S-phase leads to a rapid DNA-damage response during S-phase of the cell cycle.

p53 induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors, and thus participates in maintaining genome stability. Under stress conditions, p53 must be activated to prohibit the replication of cells containing damaged DNA. However, in normal, non-stressed cells, p53 activity must be inhibited. Previous studies have demonstrated that p53 transcription is activated before or during early S-phase in cells progressing from G(0)/G(1) into S-phase. Since this is not what would be predicted from a gene involved in growth arrest and apoptosis, in this study, we provide evidence that this induction occurs to provide sufficient p53 mRNA to ensure a rapid response to DNA damage before exiting S-phase. When comparing exponentially growing Swiss3T3 cells to those synchronized to enter S-phase simultaneously and treated with the DNA damaging agent camptothecin, we found that with cells in S-phase, p53 protein levels increased earlier, Bax and p21 transcription was activated earlier and to a greater extent and apoptosis occurred earlier and to a greater extent. These findings are consistent with p53 transcription being induced in S-phase to provide for a rapid DNA-damage response during S-phase of the cell cycle.

ChIP (chromatin immunoprecipitation) analysis demonstrates co-ordinated binding of two transcription factors to the promoter of the p53 tumour-suppressor gene.

p53 is a tumour-suppressor protein that plays a role in many cellular processes, including regulation of the cell cycle, DNA repair, transcriptional regulation of genes, chromosomal segregation, cell senescence and apoptosis. The protein's role as a transcription factor has shown that deregulated transcription, whether increased or decreased, has the potential to contribute to the formation of human cancers. It was previously reported that binding of two transcription factors, C/EBPbeta and RBP-Jkappa, to a regulatory site on the p53 promoter regulates its activity, in vitro, in a cell cycle-dependent manner. C/EBPbeta is a CCAAT enhancer-binding protein that is a member of the basic leucine zipper transcription factor (bZIP) family that plays an important role in mediating cell proliferation, differentiation and can also be involved in inflammatory responses, metabolism, cellular transformation, oncogene-induced senescence and tumorigenesis. RBP-Jkappa participates in the transcriptional regulation of target genes by interacting with the cytoplasmic domain of the Notch receptors. When RBP-Jkappa is released, transcriptional repression of its target genes occurs through the recruitment of co-repressor complexes and prevents transcription from occurring. Our reports, here and previously published, show that repression of p53 by RBP-Jkappa and activation of p53 by C/EBPbeta through differential binding of these two factors indicates a type of co-operative regulation in p53 expression. Here, we demonstrate through the use of chromatin immunoprecipitation (ChIP) assays that the co-ordinated binding of these two factors to the p53 promoter occurs in vivo and serves to regulate p53's activity during the cell cycle.