Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

<p>Abstract</p> <p>Background</p> <p>Inhibitor of DNA binding/Inhibitor of differentiation 4 (<it>ID4</it>) is a critical factor for cell proliferation and differentiation in normal vertebrate development. <it>ID4</it> has regulative functions for differentiation and growth of the developing brain. The role of <it>ID1</it>, <it>ID2</it> and <it>ID3</it> are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of <it>ID3</it> expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of <it>ID4</it> in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. </p> <p>Methods</p> <p><it>ID4</it> promoter methylation, <it>ID4</it> mRNA expression and <it>ID4</it> protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of <it>ID4</it> promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of <it>ID4</it> promoter methylation with <it>ID4</it> mRNA and <it>ID4</it> protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between <it>ID4</it> promoter methylation and clinicopathological parameters. </p> <p>Results</p> <p>Frequent <it>ID4</it> promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between <it>ID4</it> promoter methylation and loss of <it>ID4</it> expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with <it>ID4</it> promoter methylation showed distinct loss of <it>ID4</it> expression on both transcription and protein level. Interestingly, <it>ID4</it> promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030). </p> <p>Conclusion</p> <p>ID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, <it>ID4</it> methylation status could serve as a prognostic biomarker in human breast cancer.</p

Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype

Background: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer.Methods: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription-PCR.Results: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours.Conclusion: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer

NF-kappaB p65-Dependent Transactivation of miRNA Genes following Cryptosporidium parvum Infection Stimulates Epithelial Cell Immune Responses

Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general

MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells

Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this investigation reveals another mode of exploitation of cellular miRNA machinery by virus to their own advantage

IL-35 Is a Novel Responsive Anti-inflammatory Cytokine — A New System of Categorizing Anti-inflammatory Cytokines

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-β, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies

PREVALENCE OF GLUTAMATE CARBOXYPEPTIDASE II C1561T, REDUCED FOLATE CARRIER 1 A80G, AND METHIONINE SYNTHASE A2756G GENE POLYMORPHISMS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS AMONG SOUTH INDIANS

Objective: Glutamate carboxypeptidase II (GCPII), reduced folate carrier 1 (RFC1), and methionine synthase (MTR) genes involved in the folate metabolic pathway may play a key role in the pathogenesis of diabetes and its complications. The present study aimed to investigate the prevalence of genetic polymorphisms of GCPII C1561T, RFC1 A80G, and MTR A2756G in individuals with type 2 diabetes mellitus (T2DM) among South Indians. Methods: The study subjects consisted of 100 healthy individuals and 200 patients with T2DM. Genetic polymorphisms (GCPII C1561T, RFCI A80G, and MTR A2756G) in the folate metabolic pathway were analyzed by polymerase chain reaction-restriction fragment length polymorphism method. Statistical analysis was performed to test the level of significance. Results: With regard to GCPII C1561T and MTR A2756G gene polymorphisms, significant differences were not found when diabetic patients (with and without complications) and controls were compared according to different statistical models (dominant, recessive, and overdominant) p&gt;0.05. A case–control genetic association analysis of RFC1 A80G gene polymorphism has shown that there was 3.7-fold increased risk for patients without complications and 4.9-fold increased risk for diabetic patients with complications. Conclusions: Our findings suggest that the GCPII C1561T and MTR A2756G gene polymorphisms were not significantly associated with diabetes and its complications. Whereas, the RFCI A80G gene polymorphism involved in folate metabolism confers increased risk for diabetes and its complications in South Indian population

ASSESSMENT OF DNA STRAND BREAKS AND TOTAL ANTIOXIDANT STATUS IN TYPE 2 DIABETIC PATIENTS WITH AND WITHOUT COMPLICATIONS- A CASE-CONTROL STUDY

 Objective: To evaluate the total antioxidant status (TAS) and the extent of DNA strand breaks (damage) as a measure of oxidative stress biomarkers in Type 2 diabetic patients (with and without complications) and controls.Materials and Methods: Blood samples were collected from 200 patients with type 2 diabetes mellitus (n=100 with complications and n=100 without complications) and 100 healthy individuals. Oxidative DNA damage was evaluated using alkaline single cell gel electrophoresis (comet assay). Total antioxidant status was assessed by Ferric Reducing Ability of Plasma (FRAP) assay.Results: TAS was found to be significantly lower in type 2 diabetic patients (with and without complications) compared to controls (p&lt; 0.001).  Similarly, patients with complications of type 2 diabetes mellitus had significantly lower TAS when compared to diabetic patients (p= 0.007). DNA damage analysis showed that the extent of damage was high in patients with diabetes mellitus (with and without complications) compared to controls (p&lt; 0.001). Fasting glucose and glycosylated haemoglobin level (HbA1c) was found to be significantly higherin diabetic patients than controls (p&lt; 0.05). Correlation analysis showed that there is no association between age, duration, sugar level, HbA1c, TAS and DNA damage in patients with Type 2 diabetes mellitus.Conclusion: Alterations in TAS and the extent of DNA damage was observed in patients with complications of diabetes mellitus indicate that oxidative stress is more in patients with complications when compared to patients without complications and healthy individuals. Therefore, further DNA damage and onset of complications in Type 2 diabetes mellitus could be prevented by counteracting the oxidative stress by therapeutic interventions using appropriate antioxidants.Key words: Hyperglycemia, Oxidative stress, DNA damage, Total antioxidant status, Type 2 diabetes mellitus, Vascular complicatio

Two-phase flow and heat transfer enhancement

2013-2014 > Academic research: refereed > Publication in refereed journal201811_a bcmaVersion of RecordPublishe

Prevalence of diabetes and cardiovascular risk factors in middle-class urban participants in India.

OBJECTIVES: To determine the prevalence of diabetes and awareness, treatment and control of cardiovascular risk factors in population-based participants in India. METHODS: A study was conducted in 11 cities in different regions of India using cluster samp