Investigating the Association between LncRNA NR2F2-AS1, miR-320b, and BMI1 in Gastric Cancer: Insights into Expression Profiles as Potential Biomarkers for Disease Management.

AIM: This study aims to investigate the potential role of lncRNA NR2F2-AS1 in the development of gastric cancer by affecting the levels of miR-320b and BMI1. BACKGROUND: Gastric cancer is a high-mortality malignancy, and understanding the underlying molecular mechanisms is crucial. Non-coding RNAs play an important role in gene expression, and their dysregulation can lead to tumor initiation and progression. OBJECTIVE: This study aims to determine the pathological role of LncRNA NR2F2-AS1 in gastric cancer progression and its association with the clinicopathological characteristics of patients. METHODS: Bioinformatics databases were used to predict the expression levels and interactions between the studied factors to achieve this objective. The expression pattern of NR2F2-AS1/miR- 320b/BMI1 in 40 pairs of tumor and adjacent normal tissues was examined using RT-PCR, IHC, and western blot. The correlation, ROC curve, and survival analyses were also conducted for the aforementioned factors. RESULTS: The results showed an increase of more than 2-fold for BMI-1 and lncRNA NR2F2-AS1 in lower stages, and the elevation continued with the increasing stage of the disease. This correlated with significant downregulation of miR-320b and PTEN, indicating their association with gastric cancer progression and decreased patient survival. LncRNA NR2F2-AS1 acts as an oncogene by influencing the level of miR-320b, altering the amount of BMI1. A reduction in the amount of miR-320b against lncRNA NR2F2-AS1 and BMI1 directly correlates with a reduced overall survival rate of patients, especially if this disproportion is more than 3.0. ROC curve analysis indicated that alteration in the lncRNA NR2F2-AS1 level showed more than 98.0% sensitivity and specificity to differentiate the lower from higher stages of GC and predict the early onset of metastasis. CONCLUSION: In conclusion, these results suggest that NR2F2-AS1/miR-320b/BMI1 has the potential to be a prognostic as well as diagnostic biomarker for gastric cancer.

Clinical significance of long non-coding RNA ZEB2-AS1 and EMT-related markers in ductal and lobular breast cancer.

BACKGROUND: Breast cancer is considered the most prevalent type of cancer in women and accounts for a high rate of death. A body of research has demonstrated that lncRNAs have a regulatory function in human diseases, especially cancers. ZEB2-AS1 is known as an oncogenic lncRNA in various types of cancers, and its deregulation may contribute to cancer development and progression. Therefore, we aimed to reveal the association of ZEB2-AS1 expression with epithelial-mesenchymal transition (EMT) markers, as a hallmark of cancer progression, in a clinical setting. METHODS: A recent study suggested that ZEB2-AS1 is significantly involved in EMT. Here we intended to explore the roles of lncRNA ZEB2-AS1 in breast cancer (BC) using bioinformatics tools and laboratory settings. We first evaluated the expression of ZEB2-AS1 mRNA in tumor and healthy control tissues by lnCAR database. Furthermore, ZEB2-AS1 expression level, ZEB2, E-cadherin, and vimentin was measured via qRT-PCR in 30 paired ductal and lobular carcinoma tissues from breast cancer patients and the normal adjacent ones. The correlation between the lncRNA ZEB2-AS1 expression and clinicopathological characteristics of the breast cancer patients was evaluated. RESULTS: ZEB2-AS1 showed an upregulation in breast cancer tissues (p = .04) compared to normal adjacent samples. In addition, its level was higher in breast cancer patients with advanced Stages (III & IV) (n = 18) compared to early Stages (I & II) (n = 12) (p = .04). Moreover, ZEB2 (p = .01) and vimentin (p = .02) expression were upregulated in the BC sample, but the expression level of E-cadherin (p = .02) was downregulated when compared with the adjacent normal tissues. By comparison of the expression of EMT-markers between different stages of breast cancer, overexpression of ZEB2 (p = .04) and vimentin (p = .04) and down expression of E-cadherin (p = .03) was observed in advance stages. CONCLUSIONS: Collectively, our findings suggest that ZEB2-AS1 expression is significantly upregulated in tumor tissues, especially in advanced stages and ZEB2-AS1 is associated with the aggressiveness of tumors by functioning as putative oncogenic lncRNA. In addition, a combination of ZEB2-AS1 and these EMT markers in breast cancer potentiates these genes as biomarkers for tumor progression.

Long non-coding RNA NR2F2-AS1: its expanding oncogenic roles in tumor progression.

Long non-coding RNA (LncRNA) is a new type of non-coding RNA whose transcription is more than 200 nucleotides in length and can be up to 100 kb. The crucial regulatory function of lncRNAs in different cellular processes is now notable in many human diseases, especially in different steps of tumorigenesis, making them clinically significant. This research tried to collect all evidence obtained so far regarding Nuclear Receptor subfamily 2 group F member 2 Antisense RNA 1 (NR2F2-AS1) to explore its role in carcinogenesis and molecular mechanism in several cancers. Collecting evidence value an oncogenic role for NR2F2-AS1, whose dysregulation changes the status for cancerous cells to gain the supremacy toward cellular proliferation, dissemination, and ultimately migration. The NR2F2-AS1 acts as competitive endogenous RNA (ceRNA) and contains several microRNA response elements (MREs) for different microRNAs involved in various pathways such as PI3K/AKT, Wnt/ß-catenin, and TGF-ß. This clinically makes NR2F2-AS1 a remarkable lncRNA which contributes to cancer progression and invasion and perhaps could be a candidate as a prognostic marker or even a therapeutic target.

The Inhibitory Effect of Sulforaphane on The Proliferation of Acute Myeloid Leukemia Cell Lines through Controlling miR-181a.

OBJECTIVE: The present study investigated the role of miR-181a as a small non-coding RNA molecule in acute myeloid leukemia (AML) pathogenesis and reflected on the effects of Sulforaphane (SFN) on AML progression. MATERIALS AND METHODS: This experimental study had two parts. In vivo study, the miR-181a levels was measured in patients with symptoms of AML and compared to healthy controls (HCs) to investigate its role in AML pathogenesis. Afterward, an in vitro study was performed to examine the effects of SFN on the growth, apoptosis and proliferation rate of AML cell lines. Finally, the effect of SFN on miR-181a was evaluated as a major miRNA involved in hematopoiesis. RESULTS: The results of this study showed an increasing trend (2.9-fold, P=0.0019) in miR-181a expression levels in AML patients as compared with HCs. The data associated with MTT assay and flow cytometry (FCM) additionally demonstrated the anti-proliferative effects of SFN against AML cell lines, with a reduction in miR-181a levels. As well, no significant difference was noted between 24 hours and 48 hours treatments by SFN. It was deduced that modulation of miR-181a expression levels could be one of the mechanisms associated with the anti-proliferative effects of SFN against AML. CONCLUSION: MiR-181a levels contribute to AML pathogenesis and thus they can be considered as a strategy in controlling AML progression in patients. Accordingly, SFN can arrest cell proliferation and induce apoptosis in AML cell lines through retardation expression of miR-181a and affecting miR-181a pathway, which already clarified its role in the differentiation of hematopoietic stem cells and indicates another mode of anti-cancer action of sulforaphane.

The outstanding role of miR-132-3p in carcinogenesis of solid tumors.

MicroRNAs are a group of short non-coding RNAs (miRNAs), which are epigenetically involved in gene expression and other cellular biological processes and can be considered as potential biomarkers for cancer detection and support for treatment management. This review aims to amass the evidence to reach the molecular mechanism and clinical significance of miR-132 in different types of cancer. Dysregulation of miR-132 level in various types of malignancies, including hepatocellular carcinoma, breast cancer, colorectal cancer, gastric cancer, lung cancer, prostate cancer, osteosarcoma, pancreatic cancer, and ovarian cancer have reported, significantly decrease in its level, which can be indicated to its function as a tumor suppressor. miR-132 is involved in cell proliferation, migration, and invasion through cell cycle pathways, such as PI3K, TGFß or hippo signaling pathways, or on oncogenes such as Ras, AKT, mTOR, glycolysis. miR-132 could be potentially a candidate as a valuable biomarker for prognosis in various cancers. Through this study, we proposed that miR-132 can potentially be a candidate as a prognostic marker for early detection of tumor development, progression, as well as metastasis.

Synthesizing efficacious genistein in conjugation with superparamagnetic Fe3O4 decorated with bio-compatible carboxymethylated chitosan against acute leukemia lymphoma.

BACKGROUND: Genistein (C15H10O5) is a soy isoflavone with anti-cancer properties such as inhibition of cell growth, proliferation and tumor invasion, but effective dosage against hematopoietic malignant cells was not in non-toxic range. This property cause to impede its usage as chemotherapeutic agent. Therefore, this hypothesis raised that synthesizing biocompatible nanoparticle could assist to prevail this struggle. METHODS: Genistein covalently attached on Fe3O4 nanoparticles decorated with carboxymethylated chitosan to fabricate Fe3O4-CMC-genistein in alkaline circumstance. This obtained nanoparticles were evaluated by TEM, DLS, FTIR, XRD and VSM and its anti-cancer effect by growth rate and MTT assays as well as flow cytometer on ALL cancer cell lines. RESULTS: Different evaluations indicated that the drug delivery vehicle had a mean diameter size around 12ƞm with well bounded components. This system presented high degree of magnetization and superparamagnetic properties as well as good water solubility. In comparison with pure genistein, significant growth inhibition on hematopoietic cancer cells in lower dose of genistein nano-conjugated onto Fe3O4-CMC. It increased long lasting effect of genistein in cancer cells also. CONCLUSION: This delivery system for genistein could be remarkably promised and futuristic as biocompatible chemotherapeutic agent against hematopoietic malignant cells.

Effects of Sorafenib and Arsenic Trioxide on U937 and KG-1 Cell Lines: Apoptosis or Autophagy?

OBJECTIVE: Acute myeloid leukemia (AML) is a clonal disorder of hemopoietic progenitor cells. The Raf serine/threonine (Ser/Thr) protein kinase isoforms including B-Raf and RAF1, are the upstream in the MAPK cascade that play essential functions in regulating cellular proliferation and survival. Activated autophagy-related genes have a dual role in both cell death and cell survival in cancer cells. The cytotoxic activities of arsenic trioxide (ATO) were widely assessed in many cancers. Sorafenib is known as a multikinase inhibitor which acts through suppression of Ser/Thr kinase Raf that was reported to have a key role in tumor cell signaling, proliferation, and angiogenesis. In this study, we examined the combination effect of ATO and sorafenib in AML cell lines. MATERIALS AND METHODS: In this experimental study, we studied in vitro effects of ATO and sorafenib on human leukemia cell lines. The effective concentrations of compounds were determined by MTT assay in both single and combination treatments. Apoptosis was evaluated by annexin-V FITC staining. Finally, mRNA levels of apoptotic and autophagy genes were evaluated using real-time polymerase chain reaction (PCR). RESULTS: Data demonstrated that sorafenib, ATO, and their combination significantly increase the number of apoptotic cells. We found that the combination of ATO and sorafenib significantly reduces the viability of U937 and KG-1 cells. The expression level of selective autophagy genes, ULK1 and Beclin1 decreased but LC3-II increased in U937. CONCLUSION: The expression levels of apoptotic and autophagy activator genes were increased in response to treatment. The crosstalk between apoptosis and autophagy is a complicated mechanism and further investigations seem to be necessary.

Arsenic Trioxide and Thalidomide Combination Induces Autophagy Along with Apoptosis in Acute Myeloid Cell Lines.

OBJECTIVE: Autophagy and apoptosis play key roles in cancer survival and pathogenesis and are governed by specific genes which have a dual role in both cell death and survival. Arsenic trioxide (ATO) and thalidomide (THAL) are used for treatment of many types of hematologic malignancies. ATO prevents the proliferation of cells and induces apoptosis in some cancer cells. Moreover, THAL has immunomodulatory and antiangiogenic effects in malignant cells. The aim of present study was to examine the effects of ATO and THAL on U937 and KG-1 cells, and evaluation of mRNA expression level of VEGFs genes, PI3K genes and some of autophagy genes. MATERIALS AND METHODS: In this in vitro experimental study, U937 and KG-1 cells were treated by ATO (0.4-5 µM) and THAL (5-100 µM) for 24, 48 and 72 hours. Cell viability was measured by MTT assay. The apoptosis rate and cell cycle arrest were evaluated by flow cytometry (Annexin/PI) and cell cycle flow cytometry analysis, respectively. The effect of ATO/THAL on mRNAs expression was evaluated by real-time polymerase chain reaction (PCR). RESULTS: ATO/THAL combination enhanced cell apoptosis in a dose-dependent manner. Also, ATO/THAL induced SubG1/ G1 phase arrest. mRNA expression levels of VEGFC (contrary to other VEGFs isoform), PI3K, AKT, mTOR, MEK1, PTEN, IL6, LC3 and P62 genes were upregulated in acute myeloid leukemia (AML) cells following treatment with ATO/THAL. CONCLUSION: Combined treatment with ATO and THAL can inhibit proliferation and invasion of AML cells by down-regulating ULK1 and BECLIN1 and up-regulating PTEN and IL6, and this effect was more marked than the effects of ATO and THAL alone.

The relationship of miR-181a expression level and AML: A systematic review protocol.

INTRODUCTION: The most common type of leukemia is acute myeloid leukemia (AML) with the lowest survival rate among all of the leukemias particularly in adults. The evidence has shown that dysregulation of miRNA expression is associated with AML. Therefore, the aim of this systematic review was to clarify the role of miR-181a expression in AML. METHODS AND ANALYSIS: In the present study, observational studies of the roles of miR-181a expression in patients with AML will be included. Standards and indicators test should be performed for all patients. We will search PubMed, SCOPUS and ISI Web of Science with no restriction of language. The outcomes will be reviewed for association between miR-181a level and AML progression and the strength of this relationship with AML will be investigated. Selection of articles and data extraction will be performed by two independent reviewers. STROBE will be used for assessment of study quality. Publication bias and data synthesis will be an assessment by funnel plots and Beggs and Egger's tests using Stata software V.12.1. ETHICS AND DISSEMINATION: There are no ethical issues. TRIAL REGISTRATION NUMBER: This systematic review protocol is registered in the PROSPERO (International Prospective Register of Systematic Reviews), and registration number CRD42016040080.

The impact of variations in transcription of DICER and AGO2 on exacerbation of childhood B-cell lineage acute lymphoblastic leukaemia.

The expression of microRNA in eukaryotic cells is subject to tightly regulated processing. The altered expression of microRNAs in a number of cancers suggests their contribution to disease pathogenesis, where processing pathways may be involved in disease pathogenesis. In the present study, we evaluated changes in the profile of two main components of microRNA biogenesis, AGO2 and DICER, and assessed their correlation with disease progression in childhood acute lymphoblastic leukaemia (ALL). To achieve this aim, 25 patients afflicted with ALL were included in the study along with 25 healthy subjects as control. The expression level of AGO2 and DICER was evaluated by real-time PCR. The results revealed an increase in the expression of DICER and a decrease in AGO2 in patients. The correlation between the alteration levels of these genes with pathologic events was also studied. This increase or decrease proved to be directly correlated with the progression of the disease particularly in L1 to L2. According to the obtained results, it can be deduced that dysregulation in transcription of DICER and AGO2, involved in the formation of mature microRNAs in cytoplasm of ALL cancer cells, is a part of the pathological molecular mechanism implicated in the exacerbation of this malignancy. Therefore, the genes involved in microRNAs biogenesis that have been studied here could be considered as candidate prognostic markers especially in childhood ALL which will help towards a better understanding of the molecular basis of ALL.

Restraining the Proliferation of Acute Lymphoblastic Leukemia Cells by Genistein through Up-regulation of B-cell Translocation Gene-3 at Transcription Level.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a highly prevalent pediatric cancer accounting for approximately 78% of leukemia cases in patients younger than 15 years old. Different studies have demonstrated that B-cell translocation gene 3 (BTG3) plays a suppressive role in the progress of different cancers. Genistein is considered a natural and biocompatible compound and a new anti-cancer agent. In this study, we evaluate the effect of genistein on BTG3 expression and proliferation of ALL cancer cells. MATERIALS AND METHODS: ALL cell lines (MOLT4, MOLT17, and JURKAT) were cultured in standard conditions. Cytotoxicity of genistein was detected using MTT assay. The cells were treated with different concentrations of genistein (10, 25, 40, and 55µM) for 24, 48, and 72 hours, and then cell viability and growth rate were measured. The quantitative real-time polymerase chain reaction was applied to investigate the effect of genistein on BTG3 expression. RESULTS: The percentage of vital cells treated with genistein significantly decreased compared to the non-treated cells, showed an inverse relationship with an increasing genistein concentration. The present study suggests a dose of 40µM for genistein as a potent anticancer effect. Genistein could elevate BTG3 for 1.7 folds in MOLT4 and JURKAT and 2.7 folds in MOLT17 cell lines at transcription level conveged with 60 to 90% reduction in the proliferation rate of cancer cells. CONCLUSION: Up-regulation of BTG3 as a tumor suppressor gene can be induced by genistein. It seems that BTG3 reactivation can be introduced as another mechanism of anti-proliferative effect of genistein and could be considered as a retardant agent candidate against hematopoietic malignancy.

Effect of Long-term Administration of Oral Magnesium Sulfate and Insulin to Reduce Streptozotocin-Induced Hyperglycemia in Rats: the Role of Akt2 and IRS1 Gene Expressions.

The effects of long-term oral administration of magnesium sulfate and insulin on hyperglycemia were investigated using Akt2 and IRS1 gene expression methods in streptozotocin-induced diabetic rats. Fifty rats were randomly divided into five experimental groups: 1, non-diabetic control (NDC); 2, Mg2+-treated non-diabetic control (Mg-NDC); 3, chronic diabetic (CD); 4, Mg2+-treated chronic diabetic (Mg-CD); and 5, insulin-treated chronic diabetic (Ins-CD). Streptozotocin was used to induce diabetes. The Mg-CD and Mg-NDC groups received 10 g/l of MgSO4 added to drinking water. The Ins-CD group received 2.5 U/kg of insulin twice a day. Blood glucose level and body weight were measured every week. The intraperitoneal glucose tolerance test (IPGTT) was performed after 16 weeks. MgSO4 administration improved the blood glucose level and IPGTT. It also increased Akt2 and IRS1 genes as well as protein expression. Insulin lowered the blood glucose level and increased IRS1 gene and protein expression, but did not affect Akt2 gene and protein expression. Glucose reduction after Mg therapy may be mediated, at least partially, via IRS1 and Akt2 genes and protein stimulation. In insulin-treated rats, insulin resistance was not significant due to the absence of Akt2 gene expression.

Alleviating the progression of acute myeloid leukemia (AML) by sulforaphane through controlling miR-155 levels.

Acute myeloid leukemia (AML) has the highest rate of mortality among the leukemias. Disruption in miRNAs level is involved in the pathogenesis of the disease. The miR-155 has a role in primary differentiation of myeloid progenitor. Meanwhile, there is little knowledge about the effects of sulforaphane against leukemia. The present study tried to evaluate pathologic effect of miR-155 in patients in various subgroups of AML, and then pioneered in assessing miR-155 levels by the effect of sulforaphane in different AML cell lines. The miR-155 level was significantly higher in patients with AML compared to the controls. Interestingly, the increase in miR-155 was converged with raising the subtype of AML (from M1 to M5). The miR-155 levels increased by 1.2 times in patients with M1, but this increase reached 2.5 times in the patients in the M5 subgroup. Sulforaphane reduced the number of live cells and increased the mortality rate of AML cells particularly by induction of apoptosis. However, the anti-proliferative effect of this agent was more dominant and could dose-dependently lessen miR-155 levels in myeloid leukemia cells. More or less, about 80% reduction in miR-155 expression was almost observed after 48 h treatment with 60 µM sulforaphane in all four studied cell lines. The obtained results indicated that miR-155 might function as an oncomir in AML and can potentially be considered as a prognosis biomarker for AML. The anti-cancer effects of sulforaphane can be correlated with reduction of miR-155 levels. These findings suggested that sulforaphane could induce more differentiation in myeloid progenitor cells through controlling the miR-155, thereby mitigating the progress of AML.

Would Phoenix Dactyflera Pollen (palm seed) be considered as a treatment agent against Males' infertility? A systematic review.

INTRODUCTION: Oxidative stress is a key factor involved in male infertility, which is due to an unnatural increase in environmental free radicals. In the majority of cases, this has a negative effect on a male's ability to impregnate a female. Currently, it is believed that spermatozoa can be protected against the damages induced by oxidative stress by saturating sperm with antioxidants. The antioxidant role of phoenix dactylifera pollen is capable of collecting the reactive oxygen and neutralizing it in and out of body cells. The present research provides a review of the antioxidant roles of phoenix dactylifera pollen on male infertility. METHODS: This research is based on English-Language studies and articles found by comprehensively reviewing electronic databases, websites, books, and academic articles over the last 10 years. RESULTS: The phenolic compounds of phoenix dactylifera pollen, due to the existing polyphenols, are strong chelators of heavy metals. Therefore, they are effective in eliminating environmental hydroxyl radicals. Moreover, these plants have high capacities of eliminating hydroxyl free radicals, picrylhydrazyl, diphenyl and phoenix dactylifera pollen and also inhibiting glutathione-S-transferase (GST). CONCLUSION: Currently, the use of herbal antioxidants to neutralize reactive oxygen species (ROS) and reduce the negative effects of oxidative stress on body cells and tissues has attracted researchers' attention. Various substances, such as flavonoids and catechins, perform their antioxidant role by increasing the concentration of glutathione peroxidase. The final product of this process is an increase in the number of motile sperm, which can have significant effects on fertility.

Effect of anti-inflammatory (IL-4, IL-10) cytokine genes in relation to risk of cervical carcinoma.

OBJECTIVES: Cervical cancer is rated the second most common malignant tumor globally and is etiologically linked to human papillomavirus infection. Interleukin-4 (IL-4) and IL-10 are cytokines with anti-inflammatory properties. The purpose of this study was to determine the relationship of different alleles of IL-4 and IL-10 genes with risk of cervical cancer among passive smokers and users of oral contraceptives. MATERIALS AND METHODS: We investigated the association of cervical cancer with 2 anti-inflammatory cytokine genes IL-4 and IL-10 using a case-control study. The study sample comprised 200 cases of cervical cancer and an equal number of matched controls who were investisgated by variable number of tandem repeat and Restriction Fragment Length Polymorphism analysis. RESULTS: In this study we observed that the Rp1/Rp2 genotype of IL-4 marginally increased the risk of developing cervical cancer [odds ratio (OR), 1.3; 95% confidence intervals (CI), 0.45-3.64; P=0.8]. In case of passive smokers we also found a marginal increase in the risk for cervical cancer with AC and combined AC+CC genotypes (OR, 1.7; 95% CI, 0.90-3.34; P=0.1; and OR,1.7; 95% CI, 0.90-3.17; P=0.1, respectively). However, a nonsignificant association was observed between use of oral contraceptives and risk of cervical cancer with anti-inflammatory cytokine genotypes. CONCLUSIONS: This study suggests that passive smokers among North Indian women having IL-4 Rp1/Rp2 and IL-10 (AC) genotypes had an increased risk for developing cervical cancer.

Frequency of P16INK4a and P14ARF genes methylation and its impact on bladder cancer cases in north Indian population.

INTRODUCTION: Amongst the genitourinary cancers, carcinoma of the urinary bladder is one of the leading causes of death in India. Hypermethylation of the CpG islands of gene promoter is one of the earliest and most frequent epigenetic alterations leading to cancer as well as in its development. Several studies have suggested that tumour suppressor genes play a key role in the development of cancer. Methylation in the CDKN2A has been associated with various malignant diseases, but information with respect to urinary bladder cancer is lacking in north Indian population. MATERIALS AND METHODS: We analyzed the methylation of P16INK4a and P14ARF in 80 tissues and matched blood samples of patients suffering from bladder cancer and 80 blood samples of cancer-free individuals by MS-PCR. RESULTS: In tissue and matched blood samples of bladder cancer patients, the incidence of P14ARF hypermethylation significantly increased (OR=0.31, 95%CI =0.12-0.8, P=0.01) and (OR=0.0, 95%CI=0.0-0.62, P=0.006) respectively with an increase in age. Clinicopathological analysis revealed that P14ARF hypermethylation in tissue and blood samples was significantly associated with invasive stage (> or =T2) (OR=0.21, 95%CI = 0.08-0.51, P=0.0002) and (OR = 0.09, 95%CI = 0.03-0.37, P= 0.00001) respectively. Muscle invasive tumour stage (> or =T2) showed significant association with increased risk of P16INK4alpha promoter hypermethylation in tissue and blood samples of patients (OR = 0.38, 95%CI = 0.17-0.82, P= 0.01) and (OR = 0.13, 95%CI = 0.05-0.36, P= 0.00005) respectively. CONCLUSION: These results suggest that the CpG island hypermethylation status of the defined panel of genes may be a useful biomarker in patients suffering from bladder cancer.

Hypermethylation-mediated partial transcriptional silencing of DAP-kinase gene in bladder cancer.

Death-associated protein kinase (DAP-kinase) is a novel serine/threonine kinase whose expression is required for interferon-gamma-induced apoptosis. This study evaluated the methylation pattern and its impact on the expression of the DAP-kinase gene in transitional cell carcinoma of the bladder as hypermethylation is one of the earliest and most frequent alterations leading to cancer. The frequency of hypermethylation of the gene promoter was 37.8%. On correlation with clinicopathological features, methylation was seen mostly in superficial tumours in the group aged > 60 years (42.9 vs 33.3% of those 60 years and with a smoking habit showed increased downregulation of mRNA compared with non-smokers of this age group (similar pattern of methylation). Hypermethylation can decrease the expression of DAP-kinase and may be one of the reasons for conversion of normal cells to malignant cells, as the frequency of methylation at the early stage (superficial) of tumours was elevated. Methylation of DAP-kinase can be considered as one of the prognosis indicators for progression and development of bladder cancer.

Overexpression of IL-13 in patients with bladder cancer.

In this case-control study, expression pattern of IL-13 as a possible anti-inflammatory cytokine in patients with bladder carcinoma was investigated by using RT-PCR and ELISA. Highly significant difference in mRNA and protein expression of IL-13 between patients with bladder cancer and controls was observed (p = .000). The increased upexpression was mostly observed in lower stages (Ta+T1) of cancer than in higher (64.1% in lower vs 48.7% in higher stages). To address whether age, smoking, and alcohol drinking have any effect in IL-13 expression, it was seen that in spite of lower mean average of mRNA and protein expression in the old, smokers, and alcohol drinkers, no significant effect of these factors on expression of IL-13 was observed. The overexpression of IL-13 as a potent immunosuppressive cytokine was found in patients with bladder cancer and may be playing a role as an anti-inflammatory mediator in carcinoma of bladder. IL-13 may help to restore the disturbed T(H)1/T(H)2 balance in patients with bladder carcinoma, and can be considered as therapeutic agent in this disease.

Methylation patterns of Rb1 and Casp-8 promoters and their impact on their expression in bladder cancer.

Aberrant methylation of the promoter CpG island of human genes is an alternative gene inactivation mechanisms that contributes to the carcinogenesis of human tumors. We tried to determine the methylation status and its impact on the expression of two tumor related genes Casp-8 and Rb1 in 103 bladder tumor tissues and 48 control paraffin-embedded tissues by using MSP-PCR and SQRT-PCR. Of the patients, 19.4% for Casp-8 and 28.2% for Rb1 showed methylation in bladder cancer. There were significant differences between patients and healthy controls in methylation of Rb1 (p = 0.001) and Casp-8 (p = 0.008) and especially when both genes methylated (p = 0.004). Methylation of Casp-8 has mostly been taken places in patients with age >60 years (p = 0.013) whereas methylation of Rb1 has taken place in age >60 (p = 0.018) as well as in patients age <60 (p = 0.027). Patients with methylated of both genes with stage T2 showed an increasing risk of 4.75 fold (95% CI = 2.87-7.85, p = 0.00) and for stage T3, 23.50 fold (95% CI = 6.05-91.21, p = 0.00) of bladder cancer. Smoking showed a high significant effect on methylation (p = 0.00 in compare to non-smoker patients), especially in those with pack-years more than 44.7 (OR = 3.53, 95% CI = 1.69-7.35, p = 0.001). The risk of bladder cancer was marginally associated in drinker patients (OR = 1.78, 95% CI = 1.42-2.24, p = 0.010) featuring both genes methylated, especially in those patients consumed alcohol units>30 per week (OR = 4.57, 95% CI = 2.38-8.80, p = 0.000). Significant reduction in expression has been detected in patients with methylated Rb1 (p = 0.00) and Casp-8 (p = 0.03). These results suggest that age, smoking and drinking will increase the probability of methylation of these genes and consequently increased risk of developing of bladder cancer to higher stages of disease. Interestingly, it has been deduced that methylation by itself maybe significantly have a role on reducing the expression ofRb1, but it seems that methylation along with risk factors lead to decrease the expression of Casp-8. Methylation of Rb1 can be considered as one of prognosis indicator for progression and development bladder cancer.