Prognostic potential of <Emphasis Type="Italic">KLOTHO</Emphasis> and <Emphasis Type="Italic">SFRP1</Emphasis> promoter methylation in head and neck squamous cell carcinoma

Hypermethylation in the CpG island promoter regions of tumor suppressors is known to play a significant role in the development of HNSCC and the detection of which can aid the classification and prognosis of HNSCC. This study aims to profile the methylation patterns in a panel of key genes including CDKN2A, CDKN2B, KLOTHO (KL), RASSF1A, RARB, SLIT2, and SFRP1, in a group of HNSCC samples from Saudi Arabia. The extent of methylation in these genes is determined using the MethyLight assay and correlated with known clinicopathological parameters in our samples of 156 formalin-fixed and paraffin-embedded HNSCC tissues. SLIT2 methylation had the highest frequency (64.6%), followed by RASSF1A (41.3%), RARB (40.7%), SFRP1 (34.9), KL (30.7%), CKDN2B (29.6%), and CKDN2A (29.1%). KL and SFRP1 methylation were more predominant in nasopharyngeal tumors (P = 0.001 and P = 0.031 respectively). Kaplan Meier analysis showed that patients with moderately differentiated tumors who display SFRP1 methylation have significantly worse overall survival in comparison with other samples. In contrast, better clinical outcomes were seen in patients with KL methylation. In conclusion, our findings suggest that the detection of frequent methylation in SFRP1 and KL genes’ promoters could serve as prognostic biomarkers for HNSCC.     

LncRNA <Emphasis Type="Italic">ANRIL</Emphasis> Expression and <Emphasis Type="Italic">ANRIL</Emphasis> Gene Polymorphisms Contribute to the Risk of Ischemic Stroke in the Chinese Han Population

The aim of the present study was to explore the role of lncRNA ANRIL in the pathogenesis of ischemic stroke (IS) and coronary artery disease (CAD) and to determine the association between ANRIL variants and the genetic susceptibility of IS and CAD in the Chinese Han population. A genetic association study including 550 IS patients, 550 CAD patients, and 550 healthy controls was conducted. The expression levels of lncRNA ANRIL, CDKN2A, and CDKN2B were detected using qRT-PCR. Genotyping was performed by Sequenom MassARRAY on an Agena platform. Our study showed that IS patients had an increased lncRNA ANRIL expression (P?=?0.002) and a decreased CDKN2A expression (P?<?0.001) compared with normal controls. A significant difference with regard to the genotype distribution of rs2383207 was found between male IS patients and controls (P?=?0.011). The minor allele of rs2383207 significantly increased the IS risk under a recessive model (OR?=?1.52, 95% CI?=?1.05–2.21, P?=?0.027). The minor allele of rs1333049 was significantly associated with the risk of IS among the male patients under a recessive model (OR?=?1.56, 95% CI?=?1.04–2.35, P?=?0.031). However, no significant association was found between the ANRIL variants and the risk of CAD (all P?>?0.050). In addition, we found a decreased lncRNA ANRIL expression in IS patients who carried the GG genotype of rs1333049 compared with IS patients who carried the CC or CG genotype (P?=?0.041). In summary, we found that IS patients had an increased lncRNA ANRIL expression and a decreased CDKN2A expression compared with the controls, which might play an impellent role in pathological processes of IS. The ANRIL variants rs2383207 and rs1333049 were significantly associated with the risk of IS among males but not females in the Chinese Han population.     

Impact of <Emphasis Type="Italic">ZBTB7A</Emphasis> hypomethylation and expression patterns on treatment response to hydroxyurea

Background

We aimed to clarify the emerging epigenetic landscape in a group of genes classified as “modifier genes” of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.

Results

We examined the CpG islands’ DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients’ monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU.

Conclusions

This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5′ CpG sequences of all studied human HBB cluster “modifier genes.” Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the β-type hemoglobinopathy patients.

     

Culturing conditions highly affect DNA methylation and gene expression levels in MCF7 breast cancer cell line

The levels of DNA methylation and their role in gene expression are key factors that could affect diagnosis, prognosis, and treatment options of different diseases. In this study, the methylation levels of 22 genes that are mostly correlated to breast cancer were determined using EpiTect methyl II PCR array. This analysis was performed to determine the effect of cells’ passage number and the use of antibiotics in the culturing media on gene methylation levels in MCF7 cell line. DNA methylation levels of PTGS2, ADAM23, HIC1, and PYCARD were found to be significantly different among different passages. While the DNA methylation levels of CCNA1, RASSF1, and THBS1 were found to be affected by the use of 1% of penicillin/streptomycin in the culture media. Gene expression analysis after demethylation using 5-Aza-2′-deoxycytidine showed that the gene expression levels of the hypermethylated genes varied between different passage numbers. This study shows that the presence of antibiotic within cultured media and cell line’s passage number could greatly affect the methylation levels that need to be considered in future studies on cell lines.     

Epimutations of the <Emphasis Type="Italic">KCNQ1OT1</Emphasis> imprinting center of chromosome 11 in early human embryolethality

Disturbance of the epigenetic status of the H19 and KCNQ1OT1 imprinting centers of chromosome 11 and the CDKN1C imprinted gene in early human embryolethality have been studied. This is the first study to detect hypomethylation of KCNQ1OT1 in the maternal homolog in placental tissues from some (9.5%) spontaneous abortions with impaired cell proliferation during the first trimester of pregnancy. Tissue specificity of the aberrant methylation status of the imprinting center has been found. A hypothesis on the postimplantation origin of epimutations in somatic cells of the developing embryo is put forward. The selective role of epimutations of imprinted genes in early human ontogeny as compared to uniparental disomy is considered; estimation of the epigenetic risk entailed in using assisted reproductive technologies is discussed.     

A cross-talk between DNA methylation and H3 lysine 9 dimethylation at the KvDMR1 region controls the induction of Cdkn1c in muscle cells

The cdk inhibitor p57^kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.     

<Emphasis Type="Italic">FAT4</Emphasis> hypermethylation and grade dependent downregulation in gastric adenocarcinoma

Gastric cancer is one of the major causes of death due to cancer in the world. It is a multi-factorial disease with epigenetic factors being also involved in its development. FAT4 is a tumor suppressor gene exerting an important role in cell adhesion. This study aimed at analyzing FAT4 expression and promoter methylation in gastric cancer. FAT4 expression was studied in 30 tumoral tissues and their non-tumoral counterparts using Taqman real time PCR method. Promoter methylation was assessed using bisulfite conversion method followed by sequencing. Tumor tissues showed reduced FAT4 expression (P = 0.04). FAT4 downregulation was associated with tumor grade, with higher repression at advanced grades. Significant increase of promoter methylation was observed in tumoral tissues. Reduced expression of FAT4 and increased methylation of its promoter may be one of the effective processes in turning a healthy stomach tissue into a tumor tissue.     

Distinct Histone Modifications Modulate <Emphasis Type="Italic">DEFB1</Emphasis> Expression in Human Vaginal Keratinocytes in Response to <Emphasis Type="Italic">Lactobacillus</Emphasis> spp.

Vaginal commensal lactobacilli are considered to contribute significantly to the control of vaginal microbiota by competing with other microflora for adherence to the vaginal epithelium and by producing antimicrobial compounds. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic communication in the vaginal ecosystem remain poorly understood. Here, we showed that both DNA methylation and histone modifications were associated with expression of the DEFB1 gene, which encodes the antimicrobial peptide human β-defensin-1, in vaginal keratinocyte VK2/E6E7 cells. We investigated whether exposure to Lactobacillus gasseri and Lactobacillus reuteri would trigger the epigenetic modulation of DEFB1 expression in VK2/E6E7 cells in a bacterial species-dependent manner. While enhanced expression of DEFB1 was observed when VK2/E6E7 cells were exposed to L. gasseri, treatment with L. reuteri resulted in reduced DEFB1 expression. Moreover, L. gasseri stimulated the recruitment of active histone marks and, in contrast, L. reuteri led to the decrease of active histone marks at the DEFB1 promoter. It was remarkable that distinct histone modifications within the same promoter region of DEFB1 were mediated by L. gasseri and L. reuteri. Therefore, our study suggested that one of the underlying mechanisms of DEFB1 expression in the vaginal ecosystem might be associated with the epigenetic crosstalk between individual Lactobacillus spp. and vaginal keratinocytes.     

Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression,and activation of their potential target genes in clear cell renal cell carcinoma

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31–48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher’s exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher’s exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman’s correlation coefficient r _s ranging 0.31–0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (r _s ranging 0.35–0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Cytochromes <Emphasis Type="Italic">c</Emphasis> of the anaerobic methacrylate reducer <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> AM-1

Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1.