Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content.

BACKGROUND: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. METHODS: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40). RESULTS: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). CONCLUSION: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Genome-wide expression profiling in colorectal cancer focusing on lncRNAs in the adenoma-carcinoma transition.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a fundamental role in colorectal cancer (CRC) development, however, lncRNA expression profiles in CRC and its precancerous stages remain to be explored. We aimed to study whole genomic lncRNA expression patterns in colorectal adenoma-carcinoma transition and to analyze the underlying functional interactions of aberrantly expressed lncRNAs. METHODS: LncRNA expression levels of colonic biopsy samples (20 CRCs, 20 adenomas (Ad), 20 healthy controls (N)) were analyzed with Human Transcriptome Array (HTA) 2.0. Expression of a subset of candidates was verified by qRT-PCR and in situ hybridization (ISH) analyses. Furthermore, in silico validation was performed on an independent HTA 2.0, on HGU133Plus 2.0 array data and on the TCGA COAD dataset. MiRNA targets of lncRNAs were predicted with miRCODE and lncBase v2 algorithms and miRNA expression was analyzed on miRNA3.0 Array data. MiRNA-mRNA target prediction was performed using miRWALK and c-Met protein levels were analyzed by immunohistochemistry. Comprehensive lncRNA-mRNA-miRNA co-expression pattern analysis was also performed. RESULTS: Based on our HTA results, a subset of literature-based CRC-associated lncRNAs showed remarkable expression changes already in precancerous colonic lesions. In both Ad vs. normal and CRC vs. normal comparisons 16 lncRNAs, including downregulated LINC02023, MEG8, AC092834.1, and upregulated CCAT1, CASC19 were identified showing differential expression during early carcinogenesis that persisted until CRC formation (FDR-adjusted p < 0.05). The intersection of CRC vs. N and CRC vs. Ad comparisons defines lncRNAs characteristic of malignancy in colonic tumors, where significant downregulation of LINC01752 and overexpression of UCA1 and PCAT1 were found. Two candidates with the greatest increase in expression in the adenoma-carcinoma transition were further confirmed by qRT-PCR (UCA1, CCAT1) and by ISH (UCA1). In line with aberrant expression of certain lncRNAs in tumors, the expression of miRNA and mRNA targets showed systematic alterations. For example, UCA1 upregulation in CRC samples occurred in parallel with hsa-miR-1 downregulation, accompanied by c-Met target mRNA overexpression (p < 0.05). CONCLUSION: The defined lncRNA sets may have a regulatory role in the colorectal adenoma-carcinoma transition. A subset of CRC-associated lncRNAs showed significantly differential expression in precancerous samples, raising the possibility of developing adenoma-specific markers for early detection of colonic lesions.

Gene promoter and exon DNA methylation changes in colon cancer development - mRNA expression and tumor mutation alterations.

BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.

[Role and alterations of DNA methylation during the aging and cancer]. / A DNS-metiláció szerepe és megváltozása az öregedés és a daganatos betegségek kialakulása során.

Besides the genetic research, increasing number of scientific studies focus on epigenetic phenomena - such as DNA methylation - regulating the expression of genes behind the phenotype, thus can be related to the pathomechanism of several diseases. In this review, we aim to summarize the current knowledge about the evolutionary appearance and functional diversity of DNA methylation as one of the epigenetic mechanisms and to demonstrate its role in aging and cancerous diseases. DNA methylation is also characteristic/also appear to prokaryotes, eukaryotes and viruses. In prokaryotes and viruses, it provides defence mechanisms against extragenous DNA. DNA methylation in prokaryotes plays a significant role in the regulation of transcription, the initiation of replication and in Dam-directed mismatch repair. In viruses, it participates not only in defence mechanisms, but in the assembly of capsids as well which is necessary for spreading. In eukaryotes, DNA methylation is involved in recombination, replication, X chromosome inactivation, transposon control, regulation of chromatin structure and transcription, and it also contributes to the imprinting phenomenon. Besides the above-mentioned aspects, DNA methylation also has an evolutionary role as it can change DNA mutation rate. Global hypomethylation appearing during aging and in cancerous diseases can lead to genetic instablility and spontaneous mutations through its role in the regulation of transposable elements. Local hypermethylated alterations such as hypermethylation of SFRP1, SFRP2, DKK1 and APC gene promoters can cause protein expression changes, thus contribute to development of cancer phenotype. DNA methylation alterations during aging in cancerous diseases support the importance of epigenetic research focusing on disease diagnostics and prognostics. Orv Hetil. 2018; 159(1): 3-15.

Exosomes in colorectal carcinoma formation: ALIX under the magnifying glass.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 µm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer.

BACKGROUND: Colorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence. METHODS: Whole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10-10 macrodissected and 5-5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed. RESULTS: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence. CONCLUSION: Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.

Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and automated RNA isolation.

Although RNA isolation is a routine process in gene expression analysis studies, the applicability of most widely available formalin-fixed, paraffin-embedded (FFPE) samples is still limited compared to fresh frozen tissue samples due to the lower quality of the isolated RNA. Recently, novel automated isolation methods were developed in order to reduce manual sample handling and increase RNA quality and quantity. Here we present a comparison of the performance of fresh frozen and matched FFPE tissue samples obtained from the same surgically removed colonic specimens (10 normal, 10 CRC) in RT-PCR experiments. RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) compared to the manual RNeasy FFPE Mini Kit (Qiagen). Gene expression analysis of a colorectal cancer-specific marker set (with 7 genes: COL12A1, CXCL1, CXCL2, GREM1, IL1B, IL8, SLC7A5) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler® 480 System (Roche). On the basis of the gene expression of the analyzed markers, fresh frozen tumorous and normal samples could be distinguished with 100% sensitivity and 100% specificity after both isolation methods. The FFPE samples could be distinguished by similarly high specificity and sensitivity with the MagNA Pure 96 isolated samples (sensitivity: 90,0%; specificity: 90,0%) and the samples isolated with manual Qiagen method (sensitivity: 85,0%; specificity: 70,0%). According to these results, FFPE samples isolated by automated methods can serve as valuable source for retrospective gene expression studies in the field of biomarker discovery and development.

Patterns of Somatic Variants in Colorectal Adenoma and Carcinoma Tissue and Matched Plasma Samples from the Hungarian Oncogenome Program.

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Regeneration associated growth factor receptor and epithelial marker expression in lymphoid aggregates of ulcerative colitis.

OBJECTIVE: Mesenchymal-epithelial transition may have crucial role in mucosal regeneration, hence we assayed epithelial growth factor receptor (EGFR), insulin-like growth factor receptor-1 (IGF1R), hepatocyte-derived growth factor receptor (HGFR), CDX2 and cytokeratin (CK) expression in lymphoid aggregates (LA) of ulcerative colitis (UC). MATERIAL AND METHODS: Tissue microarrays (TMAs) made of biopsy samples from 20 mildly, 20 moderately and 20 severely active UC, 12 non-specific colitis (NSC) and 20 healthy colon were prepared, and immunolabelled with anti-EGFR, -IGF1R, -HGFR, -CDX2, -CK antibodies. After virtual microscopic evaluation, one-way ANOVA and correlation analysis were performed. For validation, TaqMan real-time RT-PCR was performed by using RNA from laser microdissected LA from 10 healthy colon and 10 endoscopically active UC biopsies. RESULTS: The number of LA was in tight positive correlation with the severity of inflammation (r=0.9). The number of EGFR/HGFR positive subepithelial cells was found to be significantly elevated in severe (21.6+/-2.1%/21.3+/-1.9%), moderate (14.3+/-1.7%/14.6+/-1.6%) and mild (7.2+/-1.6%/7.4+/-1.3%) inflammation compared to healthy colon mucosa (2.6+/-1.4%/2.4+/-1.03%) (p < 0.005). Some alterations were found between UC and NSC samples regarding EGFR and HGFR expression. IGF1R immunoreactive cells were only found in a trace number in all cases. Increasing trend of CDX2 and CK positive subepithelial cells was found in active UC, but it was not in significant correlation with the severity of inflammation. CONCLUSION: EGFR and HGFR positive subepithelial cells in LA may be involved in the induction of the regenerative mucosal processes. The presence of CDX2/CK positive subepithelial cells suggests that mesenchymal-to-epithelial transition may be located to lymphoid aggregates.

Molecular pathogenesis of Helicobacter pylori infection: the role of bacterial virulence factors.

Helicobacter pylori is one of the most common pathogens affecting humankind, infecting approximately 50% of the world's population. Of those infected, many will develop asymptomatic gastritis, but 10% develop gastric or duodenal ulcers. The clinical outcome of the infection may involve a combination of bacterial factors, host factors and environmental factors. In the process of development of gastritis, ulceration and cancer, several cellular and molecular steps follow each other. Infection, acid survival, adhesion, cytotoxicity, epithelial cell turnover changes, inflammation, regeneration or pathological alteration towards erosions, ulceration, and cancer can be observed on the cellular level. Bacterial factors like urease, AmiE, AmiF, hydrogenase and arginase are needed for survival in the acidic gastric environment. The bacterial flagellae are essential to move the bacteria towards the epithelial surface. Adhesive factors like BabA, SabA and ureaseA are necessary for adhesion against MHC-II complexes and Le antigens. The bacteria VacA and CagA are cytotoxic factors. The Cag type IV secretion system delivers these proteins inside the epithelial cells. After disruption of epithelial cell junctions, the bacteria can pass through the gastric wall facing direct immune response from neutrophils, lymphocytes, mast cells and dendritic cells. This review describes and summarizes our present molecular biological information and knowledge about the Helicobacter infective component, cell functions and processes. The possible role of host counter responses and interactions with gastric epithelia and immune cells are also detailed.

[Identification of methylation related genes from laser capture microdissected colon samples during investigation of adenoma-carcinoma sequence]. / Metilációs szabályozás alatt álló gének azonosítása lézerrel kimetszett vastagbéldaganat-sejtekben az adenoma-carcinoma sorrend vizsgálata során.

UNLABELLED: Changes of the DNA methylation pattern are proven to be an important process during tumorigenesis. This event can occur in several manners in the tumor microenvironment and there are still not any effective and high-throughput methods for genome-wide analysis of this phenomenon. AIMS: Our aim was to identify colorectal cancer development and progression specific marker genes regulated by DNA methylation using gene expression analysis. In this study we present a gene expression-based method combined with a cell culture model, which can be used for a genome-wide analysis of the methylation events during the colorectal tumorigenesis. MATERIALS AND METHODS: Genes, which expression increased after the demethylation were determined in HT-29 colon adenocarcinoma cells treated with 10 microM 5-aza-2'-deoxycitidine. In parallel, 5000 epithelial cells were collected with laser microdissection (LCM) from normal, adenoma and tumorous colonic samples. The genes with gradually decreasing expression along the adenoma-carcinoma sequence were identified. By comparing the two groups, the transcripts, which are supposed to be regulated by methylation, could be determined. Finally, the identified gene set was validated on independent samples using RT-PCR. CONCLUSION: The regulation of the identified genes showing decreased expression during the adenoma-carcinoma sequence, can be associated with DNA methylation. On the basis of our results, the set of genes including tumorsuppressors can be determined genome-widely, which can be key factors in the formation and the prognosis of the disease. The identified genes showing colorectal cancer specific methylation pattern can be potential therapeutic targets in the future.

[Age-related microscopic and molecular changes of the human colon, and their role in the development of colorectal cancer in elderly people]. / Az öregedés mikroszkópos és molekuláris jelei a vastagbélben, valamint ezek lehetséges szerepe az idoskori vastagbélrák kialakulásában.

The gastrointestinal effect of aging, the recognition of its molecular background and the mapping its connections with several diseases like sporadic colorectal cancer of elder people are a new and promising area of molecular gastroenterology. Nowadays, it is a well-known fact that some age-related molecular changes (e.g.: DNA methylation, telomere shortening) can be detected in several types of colorectal cancers. The known epidemiologic and molecular biologic features of sporadic colorectal cancer are not enough to explain the genetic, gene expression or epigenetic changes that may be involved in the increase of the disease over 45-50 age years. The connections of these alterations to the process of aging are also unclear. The understanding and custom-tailored modification of these mechanisms are of great clinical importance regarding of prevention and modern therapeutic strategies. In this review, we aimed to summarize the age-related microscopic and molecular changes of the human colon, as well as their role in the development of colorectal cancer of the elder people.

[Potential role of estrogens in colorectal tumour development]. / Az ösztrogének lehetséges szerepe a vastagbéldaganatok kialakulásában.

Colorectal cancer (CRC) is one of the most common types of cancers worldwide. The incidence of sporadic CRC is lower in individuals below 50 years and increases with age, furthermore, it shows typical clinical, macroscopic and molecular differences between females and males. According to the results of epidemiological and molecular biology studies, the estradiol-regulating signaling pathway plays an important role in the development and prognosis of CRC, predominantly through estrogen receptor beta (ERß), which is dominant in the colonic epithelium. Estradiol has multiple gastrointestinal effects, which were confirmed by in vitro and in vivo studies on histologically intact and cancerous cells as well. In contrast to estrogen receptor alpha (ERα), the activation of ERß inhibits cell proliferation and enhances apoptosis, nevertheless, the expression of estrogen receptor beta can change both during physiological ageing and in colorectal disorders. The ERß-mediated antitumour effects of estradiol may be exerted through inhibition of cell proliferation, stimulation of apoptosis, inhibition of metastasis formation and its anti-inflammatory activity. Based on the results of cell culture and animal studies, selective modulators of estrogen receptor beta (selective estrogen receptor modulator [SERM]) and phytoestrogens can be new, additional therapeutic options in the treatment of colorectal diseases characterized by chronic inflammation and uncontrolled cell proliferation. Orv Hetil. 2020; 161(14): 532-543.

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced γ-H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Promoter Hypomethylation and Increased Expression of the Long Non-coding RNA LINC00152 Support Colorectal Carcinogenesis.

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p < 0.001) compared to normal samples, which was confirmed by real-time PCR and in situ hybridization. LINC00152 promoter hypomethylation detected in colorectal cancer (p < 0.01) was strongly correlated with increased LINC00152 expression (r=-0.90). Silencing of LINC00152 significantly suppressed cell growth, induced apoptosis and decreased cyclin D1 expression (p < 0.05). Whole transcriptome analysis of LINC00152-silenced cells revealed significant down-regulation of oncogenic and metastasis promoting genes (e.g. YES proto-oncogene 1, PORCN porcupine O-acyltransferase), and up-regulation of tumour suppressor genes (e.g. DKK1 dickkopf WNT signalling pathway inhibitor 1, PERP p53 apoptosis effector) (adjusted p < 0.05). Pathway analysis confirmed the LINC00152-related activation of oncogenic molecular pathways including those driven by PI3K/Akt, Ras, WNT, TP53, Notch and ErbB. Our results suggest that promoter hypomethylation related overexpression of LINC00152 can contribute to the pathogenesis of colorectal cancer by facilitating cell progression through the up-regulation of several oncogenic and metastasis promoting pathway elements.

Polymorphisms of the ApoE, HSD3B1, IL-1beta and p53 genes are associated with the development of early uremic complications in diabetic patients: results of a DNA resequencing array study.

Genetic polymorphisms of the genes involved in angiogenesis, the inflammatory cascade or apoptosis control can influence the chronic complications of diabetic patients. Parallel evaluation of multiple genetic polymorphisms became available with the development of DNA resequencing arrays. We aimed to develop a 16-gene, 18,859-nucleotide resequencing array to analyze the genetic background of uremic and gastrointestinal complications. DNA was isolated from 10 ml of peripheral blood of 41 non-uremic and 37 uremic patients with type II diabetes mellitus (DM); 32 suffering from gastric erosion complications. An Affymetrix Customseq Resequencing array was developed containing a total of 37 PCR products of selected genes. Confirmatory analysis was performed for 5 known polymorphisms by RFLP and for 4 others by capillary sequencing. Statistical analysis was performed using the Fisher's exact test. Correlations between the DNA resequencing array and the confirmatory methods were 96% for RFLP and 99.4% for capillary sequencing. The genetic polymorphisms of the ApoE, HSD3B1, IL-1beta and p53 genes were found to be significantly different (p<0.05) between the uremic and non-uremic diabetes group. In regards to the gastric erosion complications of the diabetic uremic patients, the A17708T polymorphism of the p53 intron 10 was found to have a statistically significant (p<0.05) role. In conclusion, DNA sequencing arrays can contribute to a multiparameter genetic analysis yielding highly correlating results using a single method in patients suffering type II DM.

[Free circulating DNA based colorectal cancer screening from peripheral blood: the possibility of the methylated septin 9 gene marker]. / Szabad DNS-alapú vastagbéldaganat-szurés perifériás vérbol: a metilált szeptin-9 génmarker lehetoségei.

DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development.

[Tissue microarray (TMA) validated progression markers in colorectal cancer using antibody microarrays]. / Vastagbéldaganatok ellenanyag-microarray-vel kimutatott, TMA-validált progressziós markerei.

BACKGROUND: The exact molecular biological background of colorectal cancer development and progression are not hitherto known. Using microarray systems, hundreds or thousands of parameters could be examined simultaneously for answering the mentioned questions. AIM: To identify possible protein markers of colorectal cancer development and progression using antibody array, and the validation of these markers on tissue microarrays done with colorectal cancer samples. Furthermore, to determine colorectal cancer diagnostic marker combination in protein level. MATERIALS AND METHODS: Surgically resected samples from ten Dukes B and six Dukes D stage patients containing both diseased and un-involved parts of the colon were freshly frozen. The samples were homogenized and the extracted proteins were used for Clontech AB500 arrays. Twelve selected genes were validated on tissue microarrays. RESULTS: The expression of 67 proteins was altered (p < 0.05) between the normal colon and cancerous samples. These genes are related to apoptosis (n = 5), cell cycle regulation (n = 7), transcription (n = 4), DNA replication (n = 6) and other cell functions, such as transport and cell adhesion (n = 45). Twelve potential markers were immunohistochemically validated on morphological level by using tissue microarrays (CYCA1, HSP60, TOP1, APC, CBP, ERK, EGFR, C-myc, Cald, DARPP32, MRE11A, AR, EPS8). CONCLUSION: Based on these results, validated colorectal cancer development related protein markers are involved in a wide spectrum of cell functions such as apoptosis, cell cycle regulation, and signal transduction. A set of six proteins has been determined, which helps to differentiate between normal specimen, early and late stage colorectal cancer with high sensitivity and specificity.

Comparison of Circulating miRNAs Expression Alterations in Matched Tissue and Plasma Samples During Colorectal Cancer Progression.

MicroRNAs (miRNAs) have been found to play a critical role in colorectal adenoma-carcinoma sequence. MiRNA-specific high-throughput arrays became available to detect promising miRNA expression alterations even in biological fluids, such as plasma samples, where miRNAs are stable. The purpose of this study was to identify circulating miRNAs showing altered expression between normal colonic (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and colorectal cancer (CRC) matched plasma and tissue samples. Sixteen peripheral plasma and matched tissue biopsy samples (N n = 4; ADT n = 4; ADTV n = 4; CRC n = 4) were selected, and total RNA including miRNA fraction was isolated. MiRNAs from plasma samples were extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen). Matched tissue-plasma miRNA microarray experiments were conducted by GeneChip® miRNA 3.0 Array (Affymetrix). RT-qPCR (microRNA Ready-to-use PCR Human Panel I + II; Exiqon) was used for validation. Characteristic miRNA expression alterations were observed in comparison of AD and CRC groups (miR-149*, miR-3196, miR-4687) in plasma samples. In the N vs. CRC comparison, significant overexpression of miR-612, miR-1296, miR-933, miR-937 and miR-1207 was detected by RT-PCR (p < 0.05). Similar expression pattern of these miRNAs were observed using microarray in tissue pairs, as well. Although miRNAs were also found in circulatory system in a lower concentration compared to tissues, expression patterns slightly overlapped between tissue and plasma samples. Detected circulating miRNA alterations may originate not only from the primer tumor but from other cell types including immune cells.

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection.

During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of further methylation analysis.