(Open access funding provided by Semmelweis University)
(2020-1.1.2-PIACI-KFI-2021-00298) Támogató: Nemzeti Kutatás, Fejlesztés és Innovációs
Iroda
(Higher Education Institutional Excellence Programme of the Ministry of Human Capacities
in Hungary, within the framework of the molecular biology thematic program of the
Semmelweis University)
Szakterületek:
Általános- és belgyógyászat
Klinikai orvostan
Onkológia
Orvos- és egészségtudomány
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.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).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).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.
