A total of 44 pairs of tumor tissues and distant non-tumor tissues were collected from June 2017 to September 2018. These samples were obtained from 44 patients with NSCLC at The First Affiliated Hospital of Fujian Medical University (Fuzhou, China). Patient clinicopathological information was collected, including age, sex, family history. The average age of the patients was 62.91±9.05 years (mean ± SD; age range, 42–87 years), including 23 female and 21 male patients. There were 32 cases of adenocarcinoma, 12 cases of squamous cell carcinoma and 2 inflammatory pseudotumors. The research protocol was approved by The Ethics Committee of the First Affiliated Hospital of Fujian Medical University (Fuzhou, China; approval number, 2017-KY-068). All subjects provided written informed consent. Samples were taken during resection and the sampling procedure was performed under the guidance of a pathologist to distinguish between tumor tissues and distant non-tumor tissues (>10 cm away from the lesions).

Primer sequences were designed based on the region of the TNFRSF10C and TNFRSF10D genes rich in CG sites. The primer sequences were as follows: TNFRSF10C forward, 5′-AGGGTGCGATTTAGGATTTAG-3′ and reverse, 5′-CGATAACGACGACGAACTT5′; TNFRSF10D forward, 5′-CGACGATGAAGACGACGAAT-3′ and reverse, 5′-AAACCAAACCATAACTCCTAAACC-3′. The conditions for qMSP were as follows: Initial denaturation at 95°C for 10 min, denaturation at 95°C for 20 sec, annealing at 56°C for 20 sec and extension at 72°C for 30 sec for 45 cycles. The melting curve program included 95°C for 15 sec, 60°C for 60 sec and then from 60°C to 95°C at a speed of 0.11°C/sec.

DNA was extracted from patient tissue samples according to the instructions of the QIAamp DNA FFPE Tissue kit (Qiagen GmbH). The extracted samples were measured for purity and concentration using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). The DNA concentration of all samples was >25 ng/µl, and the purity of DNA was 1.8–2.0 based on the absorbance ratio at 260 and 280 nm. The DNA was subjected to bisulfite modification using the EZ DNA Methylation-Gold™ kit according to the manufacturer's instructions (Zymo Research Corp.). The primers and the bisulfite-modified DNA were then subjected to qMSP. The total reaction system was 10 µl, containing 5 µl SYBR mixture, 4 µl double-distilled H₂O, 0.5 µl each of the forward and reverse primers and 0.5 µl modified DNA. To avoid errors in sample loading, ACTB was used as an internal reference for each sample. The primer sequences of ACTB were as follows: Forward, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and reverse, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. A total of 100% M-Sss I (New England BioLabs, Inc.) treated sperm DNA was used as the positive control and nuclease-free water was used as the negative control for each group. The qMSP product was then subjected to Qsep100 DNA fragment analysis (BiOptic, Inc.) and visualized. Three randomly picked qMSP products of each gene were Sanger sequenced for validation.

ACTB is a housekeeping gene that is often stably expressed in most healthy and tumor cells; thus, it is commonly used as an internal reference for quantitation of mRNA expression levels and qMSP assays (23). Completely methylated DNA referred to human sperm DNA treated with methylated-SssI, which allowed all C sites in the DNA to be methylated, including CG sites, and thus was used as a positive control. The formula for quantifying methylation levels was used to calculate the relative methylation level by the comparison between the target gene and the ACTB gene (24,25). For each sample, the relative methylation value was determined using the 2^−∆∆Cq method. The sample methylation reference percentage (PMR) was calculated using the following formula: PMR=2^−∆∆Cq ×100%; ∆∆Cq=sample DNA (Cq_gene-Cq_ACTB)-fully methylated DNA (Cq_gene-Cq_ACTB).

A recombinant plasmid pGL3-TNFRSF10C containing the selected fragment (−121 to +363 bp of TNFRSF10C, 485 bp) was constructed. The target plasmid pGL3-TNFRSF10C was co-transfected with the pRL-SV40 plasmid carrying the Renilla luciferase gene to provide an internal control. Cells co-transfected with the empty pGL3-Basic plasmid and the pRL-SV40 plasmid were used as negative references in each experiment. The pGL3-Promoter plasmid and the pRL-SV40 plasmid were also co-transfected as a positive control each time. The plasmids were separately transferred into 293T cells (10% FBS, 37°C, 5% CO₂; China Center for Type Culture Collection) using FuGENE^® HD Transfection Reagent (Promega Corporation), according to the manufacturer's protocol. The target plasmid (1 µg) and the Renilla luciferase plasmid (0.1 µg) were transfected into 293T cells at a ratio of 10:1 using FuGENE^® HD. After 24 h of transfection, luciferase activity was detected using a dual luciferase assay kit (Promega Corporation), according to the manufacturer's protocol. The luciferase reaction intensity was measured at a wavelength of ~560 nm, using a SpectraMax 190 microplate reader (Molecular Devices) by adding the Luciferase Assay Reagent II. After the fluorescence reaction intensity was measured, the Stop&Glo Reagent was added to the same sample to quench the aforementioned reaction, and the Renilla luciferase reaction was simultaneously initiated for the second measurement. Luciferase activity was finally calculated using the Dual Luciferase Reporter Assay system (Promega Corporation) and normalized to Renilla luciferase activity.

The embryonic cells A549 and NCI-H1299 were purchased from China Center for Type Culture Collection, and the cells were treated with 5 µmol/l 5′-azadeoxycytidine (5-Aza; A3656, Sigma-Aldrich; Merck KGaA) for 24 h at 37°C (25). RNA was extracted by TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) before and after 5-Aza treatment, and cDNA was obtained by using a PrimeScript RT kit (Takara Biotechnology Co., Ltd.). The quantitative PCR primers sequences (Sangon Biotech Co., Ltd.) were as follows: TNFRSF10C forward, 5′-TGCACAGAGGGTGTGGATTAC-3′ and reverse, 5′-ATTCCGGAAGGTGCCTTCTTT-3′; ACTB forward, 5′-AGCACAGAGCCTCGCCTTT-3′ and reverse, 5′-AGGGTGAGGATGCCTCTCTT-3′. The Real-time PCR system was configured using the Takara TB Green kit (Takara Biotechnology Co., Ltd.), and the mRNA expression levels of TNFRSF10C were detected using a StepOne Plus real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for qPCR were as follows: Initial denaturation at 95°C for 10 min, 45 cycles of denaturation at 95°C for 20 sec, annealing at 56°C for 20 sec, extension at 72°C for 30 sec and a final extension at 72°C for 10 min. Relative expression levels were calculated using the 2^−ΔΔCq method (25) and normalized to internal reference gene β-actin. A negative reference was also prepared using nuclease-free water.

A total of 312 pairs of lung cancer tumor tissues and distant non-tumor tissues were obtained from TCGA database (https://tcga.xenahubs.net) for the analysis of differential TNFRSF10C and TNFRSF10D gene methylation levels. The correlation between methylation and mRNA expression levels of the TNFRSF10C gene (49 samples) were analyzed and charted using cBioPortal (http://www.cbioportal.org). TNFRSF10C DNA methylation and TNFRSF10C expression data from the lung adenocarcinoma (LUAD) project were obtained by downloading the data of 568 TCGA lung cancer samples from National Cancer Institute GDC Data Portal (https://portal.gdc.cancer.gov/repository).

Statistical analysis was performed using SPSS version 20.0 (IBM Corp). Nonparametric Wilcoxon signed-rank tests were used to determine the differences in methylation between tumor tissues and distant non-tumor tissues. The methylation level is expressed as the median. The nonparametric Wilcoxon rank-sum test was used to analyze the methylation differences between tumor tissues and distant non-tumor tissues in TCGA database. The correlation between TNFRSF10C methylation levels and mRNA expression levels was determined using Spearman's test. Luciferase reporter gene activities were analyzed using the Kruskal Wallis test with Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Corresponding primers for the CG-rich region of TNFRSF10C and TNFRSF10D were selected for analysis (Fig. 1A). The target fragments of both genes were detected using capillary electrophoresis amplification, and the amplified fragment was confirmed using Sanger sequencing (Fig. 1B and C). There were 7 and 15 cytosines (including 5 CG and 1 CG) in the primer sequences of TNFRSF10C and TNFRSF10D, respectively. The qMSP melting curves indicated that the qMSP products of both genes were homogeneous (data not shown).

Methylation levels of the TNFRSF10C and TNFRSF10D genes between tumor and distant non-tumor tissues were compared in 44 patients with NSCLC. The results demonstrated that the methylation levels of TNFRSF10C in tumor tissues were significantly higher compared with those in distant non-tumor tissues (P=0.013; Fig. 2A). Further subgroup analysis of the clinicopathological data revealed a significant association between TNFRSF10C methylation levels and NSCLC in male patients and in non-smokers (Table I). In male patients, the methylation levels of TNFRSF10C in tumor tissues were higher compared with those in distant non-tumor tissues (P=0.041; Fig. 2B), and the methylation levels in tumor tissues from male patients were significantly higher compared with those in tumor tissues from female patients (P=0.013; Fig. 2B). In patients who were non-smokers, TNFRSF10C methylation levels were higher in tumor tissues compared with distant non-tumor tissues (P=0.031; Fig. 2C). However, in female patients who were non-smokers there were no significant differences in the DNA methylation levels of TNFRSF10C between tumor tissues and distant non-tumor tissues (P=0.46; Table I). In male patients who were non-smokers, the methylation levels in tumor tissues were significantly lower compared with those in distant non-tumor tissues (P=0.03; Table I). In male patients who were smokers, no significant difference was observed in the TNFRSF10C DNA methylation levels between tumor tissues and distant non-tumor tissues (P=0.225; Table I). These results indicated that the association of TNFRSF10C hypomethylation with lung cancer was only observed in male patients who were non-smokers. Due to the limited number of patients in each subgroup, this conclusion needs to be interpreted with caution.

The levels of methylation of TNFRSF10D were not significantly different in the paired samples (P=0.198; Fig. 2D). Furthermore, subgroup analysis of the clinicopathological data did not reveal any significant associations between TNFRSF10D methylation levels and NSCLC.

To ensure that the data of the inflammatory pseudotumor did not affect the results of the experiment, the data from the two inflammatory pseudotumors were removed from analysis.

TCGA database was used to obtained methylation data for the TNFRSF10C promoter region in lung tumor tissue samples and to compare the methylation levels of TNFRSF10C between tumor tissues and distant non-tumor tissues. The results revealed that methylation levels at one of the CG sites in the TNFRSF10C promoter region was higher in tumor tissues compared with in distant non-tumor tissues samples (cg14015044, P=0.045; Fig. 3A). Using lung cancer data from the cBioPortal database, it was reported that the methylation levels of TNFRSF10C were negatively correlated with TNFRSF10C mRNA expression levels (r=−0.379, P=0.008; Fig. 3B). The prognostic data from male patients with lung cancer in TCGA database revealed that the TNFRSF10C methylation levels had no effect on overall patient survival time (data not shown).

TCGA data analysis also failed to identify any significant association between TNFRSF10D and lung cancer (data not shown). Overall, these results suggested no association between TNFRSF10D methylation levels and lung cancer.

Clinicopathological, TNFRSF10C DNA methylation and TNFRSF10C expression data from the lung adenocarcinoma (LUAD) project were downloaded from TCGA database. From the gene promoter region (−2,000 to +500 bp), 10 CG sites closest to the qMSP amplicon presented in Fig. 4A were selected, and between-group comparison and association analyses were performed. Based on the comparison between cancer and non-cancerous tissues, it was demonstrated that multiple CG sites, such as cg08309809, were significantly associated with LUAD (Fig. 4B). Further subgroup analysis revealed an association between cg08309809 and LUAD in male patients (Fig. 4C), female patients (Fig. 4D) and patients who were smokers (Fig. 4E). There was no significant association between cg08309809 and LUAD in the non-smoking group (Fig. 4F); however, this may be due to the small number of non-tumor tissues (n=2). The methylation and expression levels of multiple CG sites in the TNFRSF10C gene were significantly inversely correlated (r=−0.408~-0.146; P=1.00×10⁻¹³~2.65×10⁻⁴ for different CG sites; Fig. 4G), suggesting that TNFRSF10C promoter methylation may be associated with gene silencing.

A dual luciferase reporter plasmid containing the promoter fragment of TNFRSF10C (−121 to +363 bp) was constructed. The results demonstrated that the inserted gene fragment significantly enhanced the transcriptional activity of the reporter gene compared with the control group [fold-change (FC)=1.570, P=0.032; Fig. 5A]. The purpose of the luciferase experiment was to confirm whether the promoter fragment regulated gene expression levels. By cloning the fragment into the reporter vector, it was demonstrated that the fragment enhanced the expression levels of the reporter gene, suggesting that the fragment regulated gene expression levels. Therefore, the methylation of this fragment may serve a role in regulating the TNFRSF10C gene expression levels. To determine the relationship between TNFRSF10C promoter methylation and its transcriptional expression levels in lung cancer cells, 5′-aza-deoxycytidine was used to treat lung cancer cells A549 and NCI-H1299. The results demonstrated that the mRNA expression levels of TNFRSF10C in the two lung cancer cell lines were significantly increased following demethylation treatment (A549, FC=8.000, P=0.0001; NCI-H1299, FC=3.163, P=1.143×10⁻⁵; Fig. 5B).