Predictive single nucleotide polymorphism markers for acute oral mucositis in patients with nasopharyngeal carcinoma treated with radiotherapy

Abstract

The aim of this study was to investigate the association between the susceptibility of severe oral mucositis (OM) in Chinese nasopharyngeal carcinoma (NPC) patients treated with radiotherapy and single nucleotide polymorphisms (SNPs) across the whole genome. SNPs were screened in a total of 24 patients with NPC and an additional 6 were subjected to mRNA expression analysis. Patients were subdivided into CTC 0-2 (CTC toxicity grade 0, 1, and 2) and CTC 3+ (CTC toxicity grade 3 and above) groups according to their CTC (common toxicity criteria) scores. The GTEx dataset was used to performed eQTL analyses and in-vitro functional assays were performed for eQTL-associated genes. Our data identified 7 functional SNPs associated with the development of OM. We observed that rs11081899-A, located in the 5'-UTR of the ZNF24 gene, was significantly correlated with a higher risk of severe mucositis (OR = 14.631, 95% CI = 2.61-105.46, p = 1.2 × 10^-4), and positively associated with ZNF24 mRNA expression (p = 4.1 × 10^-6) from GTEx dataset. In addition, high ZNF24 mRNA expression was associated with severe OM in patients with NPC (p = 0.02). Further functional assays revealed that ZNF24 knockdown reduced p65 expression and suppressed TNF-α-induced NF-κB activation and pro-inflammatory cytokines release. These findings suggested that rs11081899-A may be a genetic susceptibility factor for radiation-induced OM in patients with NPC, although its value in clinical application needs to be further verified in a large cohort. Also, we suggested that downregulation of ZNF24 may attenuate the development of mucositis by suppressing NF-κB activation.

Keywords: ZNF24; genetic polymorphism; nasopharyngeal carcinoma; oral mucositis; radiotherapy.

Figure 1. eQTL analysis of rs11081899

(A) Boxplots from GTEx data showing the effect of rs11081899 genotypes on ZNF24 expression in skin-sun-exposed tissue. Numbers below each boxplot indicate the sample size of each genotype. (B) Gene eQTL Visualizer plot showing significant eQTLs for ZNF24 in skin-sun-exposed tissue. An eQTL appears as a circle and a rectangle box on the heat map. The color and size of the circle represent the effect size and p-value of the eQTL results, respectively. Additionally, the closer a SNP is to the transcription start site (TSS), the darker the box is. Numbers in black ovals indicate sample sizes. The TSS is drawn as a flag and the direction of the flag indicates the transcription direction.

Figure 2. Association between ZNF24 mRNA expression and severe oral mucositis

The relative ZNF24 mRNA level of peripheral blood lymphocytes in 6 patients with NPC (3 from the CTC 0-2 group, 3 from the CTC 3+ group) was examined by RT-qPCR. GAPDH was selected as the internal control. *p < 0.05.

Figure 3. Effect of ZNF24 on NF-κB

(A) RT-qPCR analysis of ZNF24 mRNA expression level after siRNA transfection treatment in GM0639 cells for 24 and 48 h. Nontargeting siRNA (si-NC) was used as a control. Data is represented as mean ± SD. (B) ZNF24 knockdown reduced p65 expression as determined by western blot analysis. Nontargeting siRNA treatment was used as a control. Protein loading was evaluated by β-actin. (C) Downregulation of ZNF24 markedly inhibited TNF-α-induced NF-κB activation. GM0639 cells were treated with siRNA transfection for 48 h and then with TNF-α (1 nM) for the indicated times. Nuclear extracts were prepared and analyzed by western blot using antibodies against p65 and phospho-specific p65 (Ser 536). Nontargeting siRNA treatment was used as a control. Histon-H3 was detected as the loading control. Each assay was performed in triplicate. *p < 0.001.

Figure 4. Effect of ZNF24 on TNF-α-induced IL-6 and IL-1β expression

GM0639 cells were treated with TNF-α (0, 1 nM) for the indicated times after siRNA treatment for 48 h. (A, B) After TNF-α treatment for 1 h, cells were collected and total RNA was extracted. IL6 and IL1B mRNA expression was detected by RT-qPCR. (C, D) Cell supernatants were collected at 0.5, 1, and 6 h after TNF-α treatment and the levels of IL-6 and IL-1β secretion were detected by ELISA. Each assay was performed in triplicate. Data was represented as mean ± SD. *p < 0.05, **p < 0.01.