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. 2021 Apr;21(4):241.
doi: 10.3892/ol.2021.12502. Epub 2021 Jan 31.

IL10 hypomethylation is associated with the risk of gastric cancer

Junjian Tang  1   2 , Ranran Pan  3 , Lele Xu  4 , Qinghua Ma  5 , Xiuru Ying  3 , Jun Zhao  3 , Haibin Zhao  6 , Li Miao  7 , Yue Xu  8 , Shiwei Duan  3 , Jinzhi Wang  8
Affiliations

IL10 hypomethylation is associated with the risk of gastric cancer

Junjian Tang et al. Oncol Lett. 2021 Apr.

Abstract

Interleukin-10 (IL10), a pleiotropic cytokine secreted by type-2 helper (Th2) T cells, contributes to the oncogenic activation or inactivation of tumor-suppressor genes. The present study investigated whether hypomethylation of IL10 CpG island (CGI) was associated with the risk of developing gastric cancer (GC) and the prognosis of patients with GC. A fragment (hg18, chr1: 206945638-206945774) at the CGI of IL10 was selected for the present methylation assay. Quantitative methylation-specific PCR was used to evaluate the methylation of IL10 CGI in 117 tumor samples from patients with GC. The results demonstrated that IL10 CGI methylation was significantly lower in the tumor tissues compared with that in the paired adjacent non-tumor tissues (median percentage of methylated reference, 29.16 vs. 42.82%, respectively; P=4×10-8). Furthermore, results from receiver operating characteristic curve analysis identified a significant area under the curve of 0.706, with a sensitivity and a specificity of 77.8 and 58.1%, respectively, between cancer tissues and paired adjacent non-tumor tissues. Furthermore, the methylation of IL10 CGI was significantly associated with patients' age at diagnosis (r=-0.201; P=0.03). Subgroup analyses demonstrated that the association between IL10 CGI hypomethylation and the risk of GC was specific for patients with low differentiation (P=1×10-7) and Borrmann types III+IV (P=1×10-7). In addition, IL10 CGI hypomethylation was significantly associated with the risk of GC for patients without smoking history (P=3×10-7) or a family history of cancer (P=2×10-7). The results from Kaplan-Meier survival analysis demonstrated that IL10 CGI hypomethylation was associated with a significantly shorter overall survival of patients with GC (P=0.041). Similar results were identified for patients with GC who did not have smoking history (P=0.037) or a family history of cancer (P=0.049). The results from this study demonstrated that IL10 CGI hypomethylation may be considered as a potential biomarker for the diagnosis and prognosis of patients with GC in the Chinese population.

Keywords: CpG island; DNA methylation; IL10; gastric cancer; quantitative methylation-specific PCR.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Target sequence on IL10 promoter region. (A) Genomic position and functional annotation of the amplified fragment. The primers for the quantitative methylation-specific PCR were underlined, and five CpG sites were identified (in grey). (B) Capillary electrophoresis of the amplified fragment (137 bp). (C) Sanger sequencing results. Top row of the sequence represents the original sequence of the fragment. Second row of the sequence represents the converted sequences. C nucleobase with corresponding converted T were in black boxes. A, adenine; C, cytosine; F, forward; G, guanine; IL10, interleukin 10; R, reverse; T, thymine.
Figure 2.
Figure 2.
Comparisons of IL10 CpG island methylation between tumor tissues and paired adjacent normal tissues. IL10, interleukin 10, PMR, percentage of methylated reference. GC, gastric cancer.
Figure 3.
Figure 3.
Diagnostic value of IL10 CGI hypomethylation between gastric cancer tissues and paired para-tumor tissues. ROC curve analysis confirmed the potential diagnostic value of IL10 CGI hypomethylation, and yielded a significant AUC of 0.706 with a sensitivity of 77.8% and a specificity of 58.1% between cancer tissues and para-tumor tissues. AUC, area under the curve; CGI, CpG island; IL10, interleukin 10; ROC, receiver operating characteristic; Sens, sensitivity; Spe, specificity.
Figure 4.
Figure 4.
Association of IL10 CGI methylation with age of patients with GC. An inverse correlation was found between IL10 methylation in tumor and age of patients with GC. The P-value was calculated by Spearman correlation test. GC, gastric cancer.
Figure 5.
Figure 5.
Stratified comparisons of IL10 CpG island methylation between tumor tissues and paired para-tumor tissues. P-values were calculated using Wilcoxon rank sum test. Stratified tests were performed according to tumor differentiation, Borrmann type, smoking history and family history of cancer. Plots are presented as median with interquartile range. A significant P-value was identified in the following subgroups: Low differentiation, III+IV Borrmann types, non-smoker patients and patients with no family history of cancer. IL10, interleukin 10; N, normal adjacent tissue; PMR, percentage of methylated reference; T, tumor tissue.
Figure 6.
Figure 6.
Association between aberrant IL10 CGI methylation and the prognosis of patients with GC. (A) Cox regression model of OS analysis demonstrated that the median OS time was 36.0 months. (B) Kaplan-Meier and log-rank analysis of the OS of patients with GC. (C) Kaplan-Meier and log-rank analysis of the survival in patients with GC with no and low differentiation. (D) Kaplan-Meier and log-rank analysis of the survival in non-smoker patients with GC. The cut-off value of IL10 CGI hypermethylation was set at 32.0%. GC, gastric cancer; OS, overall survival; CGI, CpG island; IL10, interleukin 10.
Figure 7.
Figure 7.
Association between IL10 methylation and IL10 expression. (A) An inverse correlation was found between IL10 methylation and mRNA expression in 372 samples of the stomach adenocarcinoma dataset (TCGA, PanCancer Altas) (r=−0.348, P=5×10−12). (B) Gene Expression Omnibus data mining results showed that the relative expression level of IL10 increased when OC3, SAS, SCC and HSC3 (oral squamous cell carcinoma cell lines) cells were treated with the demethylation agent 5′-AZA.
Figure 8.
Figure 8.
Association of CGI methylation with other 7 CpG sites at the IL10 locus based on bioinformatics analysis. (A) The genomic positions (GRCh38/hg38 version) of 7 CpG sites at the IL10 locus. (B) The table shows the association analysis of 7 CpG loci on the IL10 locus. The top table represents the correlation coefficients between 7 CpG sites at the IL10 locus, while the bottom table represents the P-values between 7 CpG sites at the IL10 locus.
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Grants and funding

This study was supported by the Zhejiang Medical and Health Science and Technology Project (grant no. 2017KY646), the Suzhou Science and Technology Project (grant no. SYSD2018057) and the K. C. Wong Magna Fund in Ningbo University.