Methylation Status Of CpG Islands Research Articles

A Long Polymorphic GT Microsatellite within a Gene Promoter Mediates Non-Imprinted Allele-Specific DNA Methylation of a CpG Island in a Goldfish Inter-Strain Hybrid.

It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.

Abstract 3549: Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway

Abstract Objective: Clear cell renal cell carcinoma (ccRCC), a common histological subtype of renal cell carcinoma (RCC) are categorized by their aggressive nature and comprise 90% of metastatic RCCs. Despite recent advances, management of the disease in the advanced metastatic phase is a significant challenge. Expression of miR-141-3p (miR-141) is low and function as tumor suppressor in various cancers. However, its association with long non-coding RNAs (lncRNAs) in RCC is not well understood. This study shows that miR-141 interacts with lncRNAs, and plays vital role in the regulation of stemness and epithelial-to-mesenchymal transition (EMT) in RCC. Experimental Design: Human renal cancer cell lines (ACHN and Caki-1), normal renal epithelial cells, RPTEC and tumor tissues were used for this study. We analyzed the expression of miR-141 in tissue samples and cell lines, and studied the function of miR-141 in kidney cancer progression using in vitro and in vivo models. Statistical analysis was performed to determine the clinical significance of miR-141 in kidney cancer patients. Results: Reduced expression of miR-141 was observed in ccRCC clinical specimens and cell lines. To elucidate the epigenetic role of miR-141 silencing in RCC, methylation status of CpG islands in its putative promoter region was analyzed. Result showed significant increase in miR-141 expression after 5-Aza-CdR treatment indicating that promoter hypermethylation is responsible for its inactivation. Ectopic expression of miR-141 reduced cell proliferation, clonogenicity, migration, invasion and induced apoptosis and cell cycle arrest compared to controls. An increase in cleaved PARP, cleaved caspase-3 and epithelial marker (CLDN1) was observed with a concomitant decrease in stemness (KLF4, Nanog) and EMT markers (FN1, VIM). We further investigated the biological significance of miR-141 in RCC. Loss of miR-141 function in RPTEC cells induced pro-cancerous characteristics. In addition, we examined lncRNAs (CDKN2B-As1, PCAT1 and PVT1) that bind to miR-141 and are overexpressed in RCC clinical samples compared to controls. Reduced expression of these lncRNAs was observed in RCC cells with overexpression of miR-141, supporting the notion that miR-141 interacts with these lncRNAs in RCC. Finally, in vivo experiment in nude mice revealed that intra-tumoral administration of miR-141 in the established tumors significantly suppressed tumor growth compared to controls. Furthermore, statistical analysis of patient samples showed that miR-141 may serve as a RCC diagnostic biomarker. Conclusion: Our results demonstrate that miR-141 overexpression inhibits RCC progression and inhibits epithelial-to-mesenchymal transition. These studies also show that miR-141 may be a useful RCC biomarker for early detection and monitoring RCC progression. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Nadeem S. Bhat, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3549.

Downregulation of MGMT promotes proliferation of intrahepatic cholangiocarcinoma by regulating p21.

Intrahepatic cholangiocarcinoma (ICC) is one of the most devastating cancers of the gastrointestinal tract. It is crucial to determine the accurate prognostic factors and find new therapeutic strategies. Meanwhile, O6-methylguanine-DNA methyltransferase (MGMT) is associated with malignant tumor progression. Thus, further studies are needed to investigate whether MGMT plays a similar role in ICC. Quantitative real-time PCR, western blot, and immunohistochemistry staining were used to detect the expression of MGMT in ICC tissues. The correlations between MGMT expression and clinicopathologic features were analyzed. The cell-proliferation assay and colony-formation assay were applied to evaluate proliferation ability, while methylation-specific PCR were used to detect the methylation status of the MGMT promoter CpG island in ICC tissues and cells. Our study found that the expression of MGMT was decreased in ICC tissues when compared with paired normal tissues. In addition, we demonstrated that MGMT expression was positively correlated with overall survival rates and tumor histological grade. Silencing of MGMT significantly promoted cell proliferation in ICC. Further research showed that silencing of MGMT induced cells to enter S phase by inhibiting p21, p27, and Cyclin E expression, ultimately promoting ICC proliferation. We also demonstrated that the MGMT promoter was highly methylated in ICC, and the levels of MGMT and p21 mRNA increased after DNA demethylation. In addition, the levels of MGMT and p21 protein were positively correlated in ICC tissues. MGMT may play a critical role in carcinogenesis and the development of ICC, and provides a new marker of clinical prognosis and target for ICC treatment.

Methylation Status of Promoter Region of Tumor Necrosis Factor Alpha Gene in Subjects with Healthy Gingiva and Chronic Periodontitis - A Pilot Study.

Periodontitis is a multi-factorial disease with bacterial origin. The progression is influenced by systemic disease, smoking, genetic factors. In the recent past epigenetic influences have been indentified on the candidate genes which code for proteins that play a role in the pathogenesis of the periodontal disease. Epigenetic mechanisms involve DNA methylation and histone modification. Till date, there is no existing data associating methylation status of the promoter region (-239, -245) of the TNF alpha gene and chronic periodontitis. The objective of this study was to compare the methylation status of CpG islands in the promoter region of TNF alpha (-239, -245) gene in the peripheral blood among subjects with healthy gingiva and chronic periodontitis. A case control study design involving 50 subjects (25 healthy and 25 subjects with chronic periodontitis) was performed. DNA was isolated from peripheral blood of all the subjects and bisulfate modification with methylation specific polymerase chain reaction (MS-PCR) was performed. The methylation status of the selected region of the Tumor necrosis factor alpha gene for both the test and control groups was evaluated and assessed. The mean Ct value for the methylation of control group was 24.36 and in the periodontitis group the mean Ct value was found to be 28.09. The higher the Ct value, the lower is the amount of methylation observed. The periodontitis group had a higher mean Ct value as compared to the control group; indicating a lower amount of methylation in the promoter region of the periodontitis group as compared to the control group. A lower level of TNF alpha gene promoter (-239,-245) methylation was observed in the periodontitis group as compared to the controls and this observation appears to support a de-methylation of the TNF alpha promoter in the periodontitis subjects. Further studies evaluating the transcript levels of TNF alpha needs to be performed to confirm the observations of this study.

Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and β chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.

DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations

SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.

The hypermethylation of the CDKN2A and CHFR promoter region is a key regulatory mechanism of CDKN2A and CHFR expression in esophageal squamous cell carcinoma

Background: The aim of this study was to evaluate the correlation between CDKN2A and CHFR expression and the methylation status of the CpG island in the promoter region of esophageal squamous cell carcinoma (ESCC) cell lines, radioresistant ESCC cell lines and ESCC clinical tissue specimens. Methods: To determine whether the methylation of CHFR and CDKN2A occurs in ESCC and enhanced in radioresistant ESCC cell lines, the DNA methylation of the following was analyzed by pyrosequencing and quantitative real-time polymerase chain reaction (PCR): (I) 28 ESCC tissues; (II) the ESCC cell lines KYSE-150 and KYSE-180; and (III) the radioresistant ESCC cell lines KYSE-150-radioresistance and KYSE-180-radioresistance. Results: The methylation of CDKN2A and CHFR was found to be enhanced in the radioresistant ESCC cell lines KYSE-150-radioresistance and KYSE-180-radioresistance (P CDKN2A and CHFR was found to decrease correspondingly (P CDKN2A and CHFR was found to be higher in the ESCC tissue and paracarcinoma tissue than in the adjacent benign tissue. The overall survival rates for ESCC patients were significantly correlated with the levels of CDKN2A and CHFR expression (P Conclusions: Our findings suggest that the hypermethylation of the CDKN2A and CHFR promoter region is a key regulatory mechanism of CDKN2A and CHFR expression in ESCC. Hypermethylated CDKN2A and CHFR are potential biomarkers for predicting the radioresistance of ESCC.

Prognostic implications and interaction of L1 methylation and p53 expression statuses in advanced gastric cancer

BackgroundTP53 is frequently mutated across various tissue types of cancers. In normal cells, long interspersed nuclear element-1 (LINE-1, L1) is mostly repressed by DNA methylation in its 5′ untranslated region but is activated by DNA demethylation process during tumorigenesis. p53 is indispensable for maintaining genomic stability and plays its role in controlling genomic stability by repressing retrotransposon activity. However, it is unclear whether p53 regulates expression or methylation of L1 differently depending on the mutational status of TP53. Four hundred ninety cases of advanced gastric cancer (AGC) were analyzed for their statuses in p53 expression and L1 methylation using immunohistochemistry and pyrosequencing, respectively. Whether L1 methylation and expression statuses were differently affected by types of TP53 mutants was analyzed in gastric cancer cell line.ResultsBy p53 immunohistochemistry, tumors were classified into 4 groups according to the intensity and extent of stained tumor nuclei. L1 methylation level was significantly higher in p53 expression group 1 than in the other groups in which L1 methylation level was similar (P < 0.001). Although L1 methylation and p53 expression statuses were associated with patient survival, multivariate analysis revealed that L1 methylation was an independent prognostic parameter. In in vitro analysis of AGS cells with the introduction of wild type or mutant types of TP53, L1 methylation level and activity were different depending on types of TP53 mutation.ConclusionsFindings suggest that L1 methylation level is affected by TP53 mutation status; although, L1 methylation status was an independent prognostic parameter in patients with AGC. Further study is required to elucidate the mechanism of how wild type or mutant p53 affects L1 activity and methylation status of L1 CpG island.

Organochlorine and organophosphorous pesticides may induce colorectal cancer; A case-control study

ObjectivesAmong the numerous agents, genetic factors and environmental elements such as pesticides have an important role in colorectal cancer (CRC) incidence. The present study aimed to investigate the probable-role of some organochlorine pesticides (OCPs) and organophosphorous pesticides (OPPs) in patients with CRC. MethodsIn this case-control study, 42 patients with CRC and 30 healthy subjects were selected. The serum levels of some OCPs (α-HCH, β-HCH, γ-HCH, 2,4 DDE, 4,4 DDE, 2,4DDT and 4,4DDT) were measured by gas chromatography (GC) method. Serum levels of malondialdehyde (MDA), and total antioxidant capacity (TAC) as well as the enzyme activity of acetylcholinesterase (AChE) and arylesterase activity of Paraoxonase-1 (PON-1) were evaluated in all participants. The methylation specific PCR (MSP) assay was used for determining the methylation status of CpG island of p16 and MGMT genes in CRC patients. ResultsThe mean serum levels of each OCPs were significantly higher in the patient group compared to the control group (P < 0.001). The AChE and arylesterase activity of PON-1 in the patient group were significantly lower than the control group (P < 0.001). The mean serum levels of MDA and TAC in the serum of the patient group were significantly higher than the control group (P < 0.001 and P < 0.002, respectively). The current findings demonstrated significantly hypermethylation of p16 promoter in CRC patients. ConclusionRegarding the higher levels of OCPs in CRC patients, along with hypermethylation of the p16 promoter gene, diminishing in AChE and PON-1 activity and increasing in oxidative stress factors, the role of OCPs and OPPs in the CRC progression in the South-East of Iran may be assumed.

DNA methylation-reprogrammed oxytocin receptor underlies insensitivity to oxytocin in pre-eclamptic placental vasculature.

Pre‐eclampsia is associated with inadequate placental blood flow and placental ischaemia. Placental vascular tone is essential for maintaining adequate placental blood flow. Oxytocin is increased in placental system at late pregnancy and onset of labour, and presented strongly concentration‐dependent contractions in placental vascular, suggesting that oxytocin could be involved in regulating placental vascular tone and circulation. However, information about the reactivity of oxytocin in pre‐eclamptic placental vasculature is limited. This study used a large number of human placentas to reveal the pathophysiological changes and its underlying mechanisms of oxytocin‐induced vasoconstrictions in placental vessels under pre‐eclamptic condition. Present study found that oxytocin‐induced contractions were significantly decreased in human pre‐eclamptic placental vasculature, associated with a deactivated transcription of oxytocin receptor gene. The deactivated oxytocin receptor gene transcription was ascribed to a relatively higher DNA methylation status of CpG islands in oxytocin receptor gene promoter. This study was first to reveal that a hyper‐methylation of CpG islands in oxytocin receptor gene promoter, leading to a relatively low pattern of oxytocin receptor expression, was responsible for the decreased sensitivity of oxytocin in pre‐eclamptic placental vessels.

Promoter Methylation Regulates ApoA-I Gene Transcription in Chicken Abdominal Adipose Tissue.

As a central constituent of HDL (high-density lipoprotein), apolipoprotein A-I (ApoA-I) has a vital function in lipid metabolism. Our previous studies confirmed that ApoA-I was differentially expressed in the adipose tissue of the abdomen of lean and fat broilers. The aim of the current work was to evaluate whether the transcription of ApoA-I in chicken abdominal adipose tissue was regulated by DNA methylation. The methylation status of ApoA-I promoter CpG island (PCGI) and promoter non-CpG island (PNCGI) as well as the ApoA-I expression level in adipose tissue of lean and fat broilers were determined using Sequenom MassARRAY and real-time PCR. The correlation analysis results showed that the methylation level of PCGI and the ApoA-I mRNA expression level were negatively correlated. Bisulfite sequencing PCR was used to assess the methylation level of ApoA-I promoter in the ICP1 cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR: an inhibitor of DNA methyltransferase). The result showed that 5-Aza-CdR caused a reduction in the methylation level of the ApoA-I promoter, thereby causing an increase in expression of the ApoA-I mRNA. Meanwhile, luciferase reporter assays indicated that in vitro methylation of the ApoA-I promoter containing CpG island with CpG methyltransferase led to transcriptional repression. Furthermore, the noticeableactivation of NRF1 on ApoA-I transcription waslargely enhanced by the demethylation of the ApoA-I PCGI region. These observations indicated that the differential expression of ApoA-I gene in the adipose tissue of broilers could be mediated by transcription regulation, at least in part by DNA methylation in its PCGI region.

FTO and IRX3 Genes are Not Promising Markers for Obesity in Labrador Retriever Dogs

Abstract Obesity is a serious problem in numerous dog breeds, but knowledge of its hereditary background is scarce. On the contrary, numerous DNA polymorphisms associated with human obesity have been identified, with the strongest effect being demonstrated for FTO gene. We used targeted next-generation sequencing (tNGS) to search for polymorphisms in the region harboring FTO and IRX3 in 32 Labrador dogs. Moreover, we investigated the selected regions of FTO and IRX3, orthologous to the human regions associated with obesity, in 165 Labradors. For all dogs, the following information was available: age, sex, gonadal status, body weight, and body conformation score (BCS). The use of tNGS revealed 12,217 polymorphisms, but none of these obtained significance when lean and obese dogs were compared. Study of two SNPs in the 5’-flanking region of FTO in 165 dogs – creating two upstream reading frames (uORFs) – also showed no association with body weight and BCS but suggested the need for improvement in FTO annotation. No polymorphism was found in the 5’UTR of IRX3. Additionally, no differences of CpG islands methylation status between lean and obese dogs were found. Our study suggests that FTO and IRX3 are not useful markers of obesity in Labrador dogs.

Hypomethylation-mediated activation of cancer/testis antigen KK-LC-1 facilitates hepatocellular carcinoma progression through activating the Notch1/Hes1 signalling.

ObjectivesKita‐Kyushu lung cancer antigen‐1 (KK‐LC‐1) is a cancer/testis antigen reactivated in several human malignancies. So far, the major focus of studies on KK‐LC‐1 has been on its potential as diagnostic biomarker and immunotherapy target. However, its biological functions and molecular mechanisms in cancer progression remain unknown.Materials and MethodsExpression of KK‐LC‐1 in HCC was analysed using RT‐qPCR, Western blot and immunohistochemistry. The roles of KK‐LC‐1 on HCC progression were examined by loss‐of‐function and gain‐of‐function approaches. Pathway inhibitor DAPT was employed to confirm the regulatory effect of KK‐LC‐1 on the downstream Notch signalling. The interaction of KK‐LC‐1 with presenilin‐1 was determined by co‐immunoprecipitation. The association of CpG island methylation status with KK‐LC‐1 reactivation was evaluated by methylation‐specific PCR, bisulphite sequencing PCR and 5‐Aza‐dC treatment.ResultsWe identified that HCC tissues exhibited increased levels of KK‐LC‐1. High KK‐LC‐1 level independently predicted poor survival outcome. KK‐LC‐1 promoted cell growth, migration, invasion and epithelial‐mesenchymal transition in vitro and in vivo. KK‐LC‐1 modulated the Notch1/Hes1 pathway to exacerbate HCC progression through physically interacting with presenilin‐1. Upregulation of KK‐LC‐1 in HCC was attributed to hypomethylated CpG islands.ConclusionsThis study identified that hypomethylation‐induced KK‐LC‐1 overexpression played an important role in HCC progression and independently predicted poor survival. We defined the KK‐LC‐1/presenilin‐1/Notch1/Hes1 as a novel signalling pathway that was involved in the growth and metastasis of HCC.

MicroRNA-596 acts as a tumor suppressor in gastric cancer and is upregulated by promotor demethylation.

BACKGROUNDIn the present study, we investigated a suppressive role of microRNA-596 (miR-596) in gastric cancer (GC). Moreover, the downregulation of miR-596 in GC cell lines was associated with an increase of miR-596 promoter methylation. We also established that miR-596 controls the expression of peroxiredoxin 1 (PRDX1), which has never been reported before, suggesting that this interaction could play an important role in GC progression.AIMTo study the potential role and possible regulatory mechanism of miR-596 in GC.METHODSThe expression levels of miR-596 and PRDX1 in gastric cancer tissues and cell lines were detected by quantitative real-time PCR (qRT-PCR). Western blot and luciferase reporter assay were used to detect the effect of miR-596 on PRDX1 expression. Then, the proliferation, metastasis, and invasion of GC cell lines transfected with miR-596 mimics were analyzed, respectively, by Cell Counting Kit-8 proliferation assay, wound healing assay, and transwell invasion assay. Meanwhile, the methylation status of the promoter CpG islands of miR-596 in GC cell lines was detected by methylation-specific PCR (MSP).RESULTSExpression of miR-596 was decreased and PRDX1 was upregulated in GC tissues and cell lines. Overexpression of miR-596 decreased the expression of PRDX1 and luciferase reporter assays detected the direct binding of miR-596 to the 3'-untranslated region (UTR) of PRDX1 transcripts. Furthermore, we found that overexpression of miR-596 remarkably suppressed cell proliferation, migration, and invasion in GC cells. We further analyzed miR-596 promoter methylation by MSP and qRT-PCR, and found the downregulation of miR-596 was associated with promoter methylation status in GC cell lines. Moreover, DNA demethylation and reactivation of miR-596 after treatment with 5-Aza-2’-deoxycytidine inhibited the proliferative ability of GC cells.CONCLUSIONMiR-596 has a tumor suppressive role in GC and is downregulated partly due to promoter hypermethylation. Furthermore, PRDX1 is one of the putative target genes of miR-596.

Methylation status of KLF4 CpG islands in endometrial cancer

Introduction: Endometrial carcinoma is one of the most frequent gynaecological malignant disease in developed countries. Despite its relatively frequent occurrence, role of epigenetic changes such as methylation status of this tumor are not well characterised. In this study we focused on a tumor suppressor KLF4 gene (Kruppel Like Factor 4, zinc finger transcription factor), which is involved in the regulation of proliferation, differentiation, apoptosis and somatic cell reprogramming as it controls G1-to-S transition of the cell cycle.

Hypermethylation of ERа-A gene and high serum homocysteine level are correlated with cognitive impairment in white matter hyperintensity patients.

To investigate the methylation status in promoter region of estrogen receptor alpha (ERа)-A gene and its relation with plasma homocysteine (Hcy) level and cognitive impairment in white matter hyperintensity (WMH) patients. 210 patients aged 65 and older were selected. The methylation status of CpG islands in ERа-A gene promoter was analyzed by nested methylation-specific PCR. Serum Hcy and estradiol levels were measured by enzyme-linked immunosorbent assay. Cognitive function were evaluated using minimum mental state examination, the montreal cognitive assessment, Stroop color-word test, symbol digit modalities, trail making test B and instrumental activities of daily living (IADL). The severity of WMH was evaluated with the Fazekas scale by brain magnetic resonance imaging. We found a significant association between the severity of WMH and CpG island methylation of ERа-A gene (P < 0.05). Multiple regression analysis showed that serum Hcy level, methylation of ERа-A gene and WMH severity were significant determining factors for cognitive impairment (P < 0.05). The spearman rank correlation analysis showed a significant correlation of methylation of ERа-A gene with serum Hcy level, WMH severity, cognitive function and IADL status (P < 0.05). Methylation of ERа-A gene promoter has a high frequency in WMH patients with cognitive impairment and is correlated with high plasma Hcy level.

Expression and CpG island methylation pattern of MMP-2 and MMP-9 genes in patients with congenital factor XIII deficiency and intracranial hemorrhage

ABSTRACTObjectives: Congenital factor XIII (FXIII) deficiency is a rare severe bleeding disorder. Intracranial hemorrhage (ICH) is the leading cause of mortality and morbidity in FXIII deficiency. However, its pathogenesis is not well understood yet. In this study, we investigated the expression and CpG island methylation status of matrix metalloproteinase-2 (MMP-2) and MMP-9 in patients with FXIII deficiency and ICH.Methods: Forty patients with FXIII deficiency including twenty patients with ICH, and twenty without ICH were recruited as case and control groups, respectively. Methylation status was determined by bisulfite sequencing polymerase chain reaction (PCR), and gene expression was assessed by quantitative real-time PCR.Results and discussion: We found an unmethylated pattern for both MMP-2 and MMP-9 genes in the case group. Both genes were partially methylated in the control group, while the percentage of methylated CpGs was significantly higher in MMP-9 than MMP-2 (P = 0.001). Furthermore, higher expression of MMP-9 (in both the mRNA and protein levels) was found in the case than control group (P = 0.008 and P = 0.009, respectively). On the other hand, there was no significant difference in MMP-2 expression level (neither mRNA nor protein) between the two groups (P = 0.12 and P = 0.25, respectively).Conclusion: Our findings indicated that MMP-9 over-expression might be related to ICH in FXIII deficiency, and gene methylation effectively regulates its expression. Future researches will expand our understanding of the pathogenesis of ICH in congenital FXIII deficiency.

Over-expression of TGF-β1 gene in medication free Schizophrenia

Background and purposeImmunological pathways play a crucial role in developing and precipitating neuropsychiatric disorders. Although the exact pathogenesis of schizophrenia is unknown, the possible role of genetic and biomarker involvement of the immune system is gaining attention. Here we quantified the mRNA expression of cytokines as a key role player of the immune system from the peripheral blood mononuclear cells of patients with schizophrenia and healthy controls to identify the differentially expressed genes. MethodsSixteen medication-free schizophrenia patients and 16 healthy subjects were enrolled in the current study. To investigate the desired expression level of mRNAs including TGF-β1, IL-1β, IL-23, TNF-α, NF-κB, and BDNF, quantitative real-time PCR was performed using specific oligonucleotide primers and the Applied Bio systems StepOne™ real time PCR system. DNA methylation was also analyzed through methylation-specific polymerase chain reaction (MSP). ResultsTGF-β1 was significantly up-regulated in peripheral blood mononuclear cells of patients vs. healthy individuals (P value = 0.03). In addition, we found a significant correlation between the positive symptom scale and TGF-β1 gene overexpression (r = 0.536, P = 0.039). However, we did not observe any statistically significant differences for the methylation status of CpG Islands 1 and 2 between the patients and normal group. No statistical significance was found either for gene expression of IL-1β (P = 0.32), IL-23 (P = 0.12), TNF-α (P = 0.87), NF-κB (P = 0.07), and BDNF (P = 0.33). ConclusionsAlthough the number of medication-free schizophrenia patients is extremely limited, our data highlighted the potential role of TGF-β1 as a regulatory cytokine in complex inflammatory mechanism involved in medication-free schizophrenia. In addition, we observed that increased level of TGF-β1 mRNA in this disease might not be under methylation as an epigenetic control element at the genomic level.

Anti-aging protective effect of L-carnitine as clinical agent in regenerative medicine through increasing telomerase activity and change in the hTERT promoter CpG island methylation status of adipose tissue-derived mesenchymal stem cells.

The identification of factors that reduce the senescent tendency of the mesenchymal stem cells (MSCs) upon expansion has great potential for cellular therapies in regenerative medicine. Previous studies have shown the aging protective effect of L-carnitine (LC). On the other hand, reduction in proliferation potential and age-dependent decline in number and functions of MSCs were accompanied by telomere shortening, reduction in telomerase activity and epigenetic changes. The aim of this study was to evaluate the effects of LC on aging of MSCs through telomerase activity assessment and the investigation of methylation status of the hTERT gene promoter. Telomerase activity and hTERT promoter methylation investigation was performed with PCR-ELISA TRAP assay and methylation specific PCR (MSP), respectively. Also, beta-galactosidase (SA-ß-gal) staining was used to calculate the percentage of senescent cells. The results showed that the LC could efficiently promote the telomerase activity. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with LC were seen. In conclusion, it seems that LC could improve the aging-related features due to increasing the telomerase activity, decreasing aging, and changing the methylation status of hTERT promoter; it could potentially beneficial for enhancing the application of aged-MSCs in regenerative medicine.

Methylation status of the putative Pax6 promoter in olive ridley sea turtle embryos with eye defects: An initial approach

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5′-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.