Long non-coding RNAs (lncRNAs) are members of the non-protein coding RNA family longer than 200 nucleotides. They participate in the regulation of gene and protein expression influencing apoptosis, cell proliferation and immune responses, thereby playing a critical role in the development and progression of various cancers, including colorectal cancer (CRC). As CRC is one of the most frequently diagnosed malignancies worldwide with high mortality, its screening and early detection are crucial, so the identification of disease-specific biomarkers is necessary. LncRNAs are promising candidates as they are involved in carcinogenesis, and certain lncRNAs (e.g., CCAT1, CRNDE, CRCAL1-4) show altered expression in adenomas, making them potential early diagnostic markers. In addition to being useful as tissue-specific markers, analysis of circulating lncRNAs (e.g., CCAT1, CCAT2, BLACAT1, CRNDE, NEAT1, UCA1) in peripheral blood offers the possibility to establish minimally invasive, liquid biopsy-based diagnostic tests. This review article aims to describe the origin, structure, and functions of lncRNAs and to discuss their contribution to CRC development. Moreover, our purpose is to summarise lncRNAs showing altered expression levels during tumor formation in both colon tissue and plasma/serum samples and to demonstrate their clinical implications as diagnostic or prognostic biomarkers for CRC.

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.

BackgroundDNA 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.MethodsLong 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.ResultsAccording 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.ConclusionsDNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4609-x) contains supplementary material, which is available to authorized users.

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.

Napjainkban a genetikai kutatások mellett egyre inkább előtérbe kerülnek az epigenetikai vizsgálatok, ugyanis az epigenetikai jelenségek -köztük a DNS-metiláció is -részt vesznek a fenotípust meghatározó gének expressziójának szabályozásában, így számos betegség mechanizmusával összefüggenek. Jelen összefoglaló közleményünk célja az epigenetikai mechanizmusok közül a DNS-metiláció evolúció során történő megjelenésének, funkciói változatosságának, illetve az öregedésben és a rákos megbetegedésekben betöltött szerepé-nek bemutatása. A DNS-metiláció a prokarióták, az eukarióták, illetve a vírusok körében is megfigyelhető epigenetikai módosulás. A prokarióták és vírusok esetén idegen DNS-sel szembeni védelmi funkciót lát el. A DNS-metiláció prokariótáknál jelentős szereppel bír a transzkripció regulációjában, a replikáció iniciációjában, illetve a Dam-irányított hibajavítás-ban. A vírusoknál a védelmi funkció mellett a terjedésükhöz szükséges kapszid formálásában is részt vesz. Az eukarióták esetén a DNS-metiláció szerepet játszik a kromatinstruktúra és a transzkripció szabályozásában, a rekombináci-óban, a replikációban, az X-kromoszóma inaktivációjában, a transzpozonok szabályozásában és az imprinting jelenség létrehozásában. A fenti tulajdonságok mellett evolúciós szereppel is rendelkezik azáltal, hogy megváltoztatja a DNS mutációs rátáját. Az öregedés során és a rákos megbetegedésekben kialakuló globális hipometilációs eltérések genetikai instabilitáshoz és spontán mutációs eltérésekhez vezethetnek a transzpozonok szabályozásában betöltött funkciójuk révén. A lokális hipermetilációs (például az SFRP1, az SFRP2, a DKK1 és az APC promóterének hipermetilációja) változásoknak a fehérjeexpressziós változások létrehozásában, ezáltal a rák fenotípus kialakulásában van jelentős szerepe. Az elválto-zások általános jellege alapján a fenti eredmények a biológiai kor és a betegségek epigenetikai változások kimutatásán alapuló diagnosztikai és prognosztikai módszerei kutatásának fontosságát támasztják alá. Orv Hetil. 2018; 159(1): 3-15. Kulcsszavak: epigenomika, DNS-metiláció, 5-metilcitozin, öregedés, daganatos megbetegedések Role and alterations of DNA methylation during the aging and cancerBesides 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 onl...

Monitoring the therapeutic response of colorectal cancer (CRC) patients is crucial to determine treatment strategies; therefore, we constructed a liquid biopsy-based approach for tracking tumor dynamics in non-metastatic (nmCRC) and metastatic (mCRC) patients (n = 55). Serial blood collections were performed during chemotherapy for measuring the amount and the global methylation pattern of cell-free DNA (cfDNA), the promoter methylation of SFRP2 and SDC2 genes, and the plasma homocysteine level. The average cfDNA amount was higher (p < 0.05) in nmCRC patients with recurrent cancer (30.4 ± 17.6 ng) and mCRC patients with progressive disease (PD) (44.3 ± 34.5 ng) compared to individuals with remission (13.2 ± 10.0 ng) or stable disease (12.5 ± 3.4 ng). More than 10% elevation of cfDNA from first to last sample collection was detected in all recurrent cases and 92% of PD patients, while a decrease was observed in most patients with remission. Global methylation level changes indicated a decline (75.5 ± 3.4% vs. 68.2 ± 8.4%), while the promoter methylation of SFRP2 and SDC2 and homocysteine level (10.9 ± 3.4 µmol/L vs. 13.7 ± 4.3 µmol/L) presented an increase in PD patients. In contrast, we found exact opposite changes in remission cases. Our study offers a more precise blood-based approach to monitor the treatment response to different chemotherapies than the currently used markers.

Piglets from a porcine circovirus type 2 (PCV2) stable farm of low and high levels of maternally derived antibodies (MDA) against PCV2 were vaccinated either with a whole virus type or a PCV2 ORF2 antigen-based commercial subunit vaccine at three weeks of age. Two non-vaccinated groups served as low and high MDA positive controls. At four weeks post vaccination, all piglets were challenged with a PCV2d-2 type virus strain and were checked for parameters related to vaccine protection over a four-week observation period. MDA levels evidently impacted the outcome of the PCV2d-2 challenge in non-vaccinated animals, while it did not have a significant effect on vaccine-induced protection levels. The humoral immune response developed faster in the whole virus vaccinates than in the subunit vaccinated pigs in the low MDA groups. Further, high MDA levels elicited a stronger negative effect on the vaccine-induced humoral immune response for the subunit vaccine than for the whole virus vaccine. The group-based oral fluid samples and the group mean viraemia and faecal shedding data correlated well, enabling this simple, and animal welfare-friendly sampling method for the evaluation of the PCV2 viral load status of these nursery piglets.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.