Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

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.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Role of allelic genes of matrix metalloproteinases and their tissue inhibitors in the risk of peptic ulcer disease development

Peptic ulcer disease is a chronic disease of the gastrointestinal tract, mainly manifesting itself in the formation of the fairly persistent ulcer defect of the mucous membrane of the stomach and/or duodenum. Association analysis of common polymorphisms of matrix metalloproteinases genes MMP-1 (rs1799750, rs494379), MMP-2 (rs2285052), MMP-3 (rs3025058), MMP-9 (rs3918242, rs17576), and MMP-12 (rs2276109) and their tissue inhibitors TIMP-2 (rs8179090) and TIMP-3 (rs9619311) was carried out in 353 patients with a gastric ulcer or duodenal ulcer and in 325 unrelated healthy individuals from the Republic of Bashkortostan. Associations of polymorphic variants rs1799750 and rs494379 of gene MMP-1, rs3025058 of gene MMP-3, rs3918242 and rs17576 of gene MMP-9, and rs9619311 of gene TIMP-3 with the risk of peptic ulcer disease in Russians and Tatars were revealed.     

Novel miRNA genes hypermethylated in breast cancer

MicroRNAs play an important role in the regulation of expression of many genes involved in cancer pathogenesis. One of the causes of miRNA level deregulation in tumors is the methylation of CpG islands in the promoter regions of the genes that encode them. Hypermethylation may lead to the suppression of miRNA gene expression and, as a consequence, to a decrease in their inhibitory effect on target gene mRNAs. A search for new miRNA genes hypermethylated in breast cancer has been carried out in the present study. The methylation of five miRNA genes associated with breast cancer (miR-132, miR-1258, miR-107, miR-130b, miR-137) has been as studied using a representative set of 41 breast cancer samples by methylation-specific PCR. Three new genes, MIR-132, MIR-137 and MIR-1258, with a high frequency of hypermethylation (41, 37 and 34%, respectively) have been identified in breast cancer. The methylation of these genes in the breast tissues of ten donors without cancer pathology in anamnesis was only found in single cases. These results enable the involvement of three miRNAs (miR-132, miR-137, miR-1258) and the methylation of the genes that encode them in the pathogenesis of breast cancer to be suggested.     

Altered expression of the <Emphasis Type="Italic">SEMA3B</Emphasis> gene in epithelial tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.     

Adherence to Mediterranean diet is associated with methylation changes in inflammation-related genes in peripheral blood cells

Epigenetic processes, including DNA methylation, might be modulated by environmental factors such as the diet, which in turn have been associated with the onset of several diseases such as obesity or cardiovascular events. Meanwhile, Mediterranean diet (MedDiet) has demonstrated favourable effects on cardiovascular risk, blood pressure, inflammation and other complications related to excessive adiposity. Some of these effects could be mediated by epigenetic modifications. Therefore, the objective of this study was to investigate whether the adherence to MedDiet is associated with changes in the methylation status from peripheral blood cells. A subset of 36 individuals was selected within the Prevención con Dieta Mediterránea (PREDIMED)-Navarra study, a randomised, controlled, parallel trial with three groups of intervention in high cardiovascular risk volunteers, two with a MedDiet and one low-fat control group. Changes in methylation between baseline and 5 years were studied. DNA methylation arrays were analysed by several robust statistical tests and functional classifications. Eight genes related to inflammation and immunocompetence (EEF2, COL18A1, IL4I1, LEPR, PLAGL1, IFRD1, MAPKAPK2, PPARGC1B) were finally selected as changes in their methylation levels correlated with adherence to MedDiet and because they presented sensitivity related to a high variability in methylation changes. Additionally, EEF2 methylation levels positively correlated with concentrations of TNF-α and CRP. This report is apparently the first showing that adherence to MedDiet is associated with the methylation of the reported genes related to inflammation with a potential regulatory impact.     

Polymorphism of the genes for antioxidant defense enzymes and their association with the development of chronic obstructive pulmonary disease in the population of Bashkortostan

In this study, frequencies of the polymorphic variants of the genes encoding antioxidant enzymes, GSTM1, GSTT1, GSTP1, CAT, GPX1, NQO1, SOD1, and SOD3 were examined in three ethnic groups of healthy subjects from the Republic of Bashkortostan (Russians, Tatars, and Bashkirs). An association of these markers with the development of chronic obstructive pulmonary disease (COPD) was tested. Interethnic differences relative to the distribution of the polymorphic variants of the GSTP1 locus Ile105Val and the NQO1 locus 609C/T were revealed. Relative to the genotype distribution at the Ile105Val locus of the GSTP1 gene, ethnic group of Bashkirs was found to be statistically significantly different from Tatars (χ² = 8.819; d.f. = 2; P = 0.012). Relative to the genotype frequency distribution pattern at the NQO1 locus 609C/T, the group of Bashkirs differed from Russians (χ² = 8.913; d.f. = 2; P = 0.012). An association of genotype Val/Val of the GSTP1 Ile105Val locus with the risk of COPD in Russians (χ² = 5.25; P = 0.022; P _cor = 0.044; OR = 4.09), and of the GSTP1 haplotype *D in Tatars, was demonstrated (χ² = 11.575; P = 0.0014; P _cor = 0.0042; OR = 3.178). Genotype TT of the CAT ?262C/T locus marked resistance to the COPD development in Russians (χ² = 6.82; P = 0.0098; P _cor = 0.0196; OR = 0.31; 95%CI, 0.119 to 0.77). The risk for COPD in the ethnic group of Tatars was associated with the CAT haplotype (?262)C/(1167)T (χ² = 6.038; P = 0.0147; P _cor = 0.044; OR = 1.71). Analysis of the NQO1 haplotypes at the 465C/T and 609C/T loci showed that haplotype 465C/609T was associated with COPD in Russians (χ² = 4.571; P = 0.0328; P _cor = 0.01; OR = 1.799). It was demonstrated that Gly allele of the Arg213Gly polymorphic locus of the SOD3 gene marked the risk for COPD in the ethnic group of Tatars (OR = 2.23; 95%CI, 1.22 to 4.1). Thus, GSTP1, CAT, NQO1, and SOD3 polymorphisms play an important role in the development of COPD among the population of Bashkortostan.     

Isolation,functional characterization and stress responses of raffinose synthase genes in sugar beet

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K _m values of BvRS1 for galactinol and sucrose and the K _m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.     

The crucial role of SEMA3F in suppressing the progression of oral squamous cell carcinoma

Background

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancy. Semaphorin 3F (SEMA3F) is highly conserved but present at a lower level in various cancers than in healthy tissues. While it has been reported that SEMA3F is involved in cancer cell proliferation, migration and invasion, its function in OSCC remains unknown.

Methods

The expression of SEMA3F in OSCC tissues and OSCC-derived cells was analyzed using qRT-PCR and western blotting. Using SAS and HSC2 cells, we also monitored the effect of SEMA3F on OSCC cell proliferation, migration and invasion using MTT, colony formation and transwell assays. The function of SEMA3F in OSCC tumor formation was also assessed in vivo.

Results

SEMA3F was significantly downregulated in OSCC tissues and OSCC-derived cells. SEMA3F shows growth inhibitory activity in SAS and HSC2 cells and may act as a tumor suppressor. It can inhibit the migration and invasion potential of OSCC cells. Our results also demonstrate that SEMA3F can suppress the growth of OSCC cells in vivo.

Conclusions

This study revealed that SEMA3F plays a role as a tumor suppressor in OSCC cell proliferation, migration and invasion. Our finding provides new insight into the progression of OSCC. Therapeutically, SEMA3F has some potential as a target for OSCC treatment, given sufficient future research.

     

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.     

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).