Abstract
BackgroundThe influence of natural regulatory T cells (nTregs) on the patients with colon cancer is unclear. Demethylated status of the Treg-specific demethylated region (TSDR) of the FOXP3 gene was reported to be a potential biomarker for the identification of nTregs.MethodsThe demethylation rate of the TSDR (TSDR-DMR) was calculated by using methylation-specific quantitative polymerase chain reaction (MS-qPCR) assay. The expression of TSDR-DMR and FOXP3 mRNA was investigated in various colorectal cancer cell lines. A total of 130 colon carcinoma samples were utilized to study the DMR at tumor sites (DMRT) and adjacent normal tissue (DMRN). The correlations between DMRs and clinicopathological variables of patients with colon cancer were studied.ResultsThe TSDR-DMRs varied dramatically among nTregs (97.920 ± 0.466%) and iTregs (3.917 ± 0.750%). Significantly, DMRT (3.296 ± 0.213%) was higher than DMRN (1.605 ± 0.146%) (n = 130, p = 0.000). Higher DMRN levels were found in female patients (p = 0.001) and those with distant metastases (p = 0.017), and were also associated with worse recurrence-free survival in non-stage IV patients (low vs. high, p = 0.022). However, further Cox multivariate analysis revealed that the FOXP3-TSDR status does not have prognostic value.ConclusionMS-qPCR assays of FOXP3-TSDR can efficiently distinguish nTregs from non-nTregs. Abnormal recruitment of nTregs occurs in the local tumor microenvironment. Infiltration of tissue-resident nTregs may have a negative role in anti-tumor effects in patients with colon cancer; however, this role is limited and complicated.
Highlights
The influence of natural regulatory T cells on the patients with colon cancer is unclear
The methylation-specific quantitative polymerase chain reaction (MS-qPCR) system could efficiently differentiate natural regulatory T cells (nTregs) from non-nTregs using the forkhead box P3 (FOXP3)-Treg-specific demethylated region (TSDR) demethylation assay Human CD4+CD25+CD127lo nTregs and CD4+CD25− CD45RA+ naïve T cells from a male donor were purified after separation from peripheral blood mononuclear cells (PBMCs) by fluorescence-activated cell sorting (FACS), and induced Tregs (iTregs) were differentiated from the population of naïve T cells (Figure 1A)
Our data confirmed that the MS-qPCR assay coupled with the specific primer sets could efficiently distinguish FOXP3+ nTregs from non-nTregs
Summary
The influence of natural regulatory T cells (nTregs) on the patients with colon cancer is unclear. Demethylated status of the Treg-specific demethylated region (TSDR) of the FOXP3 gene was reported to be a potential biomarker for the identification of nTregs. FOXP3 is not expressed exclusively in natural Tregs (nTregs) [9,10,11,12,13]. It was shown that the Treg-specific demethylated region (TSDR) is significantly demethylated in human nTregs, while it is completely methylated in induced Tregs (iTregs) and other non-suppressive T cells that express FOXP3 [14,15]. TSDR is a CpG dinucleotide-rich and highly conserved region within the conserved non-coding sequences
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