Promoter methylation of RNF180 is associated with H.pylori infection and serves as a marker for gastric cancer and atrophic gastritis

Abstract

Promoter methylation (PM) of RING-finger protein (RNF) 180 affects gastric cancer (GC) prognosis, but its association with risk of GC or atrophic gastritis (AG) is unclear. We investigated relationships between RNF180 PM and GC or AG, and the effects of Helicobactor pylori (H.pylori) infection on RNF180 PM. This study included 513 subjects (159 with GC, 186 with AG, and 168 healthy controls [CON]) for RNF180 PM analysis, and another 55 GC patients for RNF180 gene expression analysis. Methylation was quantified using average methylation rates (AMR), methylated CpG site counts (MSC) and hypermethylated CpG site counts (HSC). RNF180 promoter AMR and MSC increased with disease severity. Optimal cut-offs were GC + AG: AMR > 0.153, MSC > 4 or HSC > 1; GC: AMR > 0.316, MSC > 15 and HSC > 6. Hypermethylation at 5 CpG sites differed significantly between GC/AG and CON groups, and was more common in GC patients than AG and CON groups for 2 other CpG sites. The expression of RNF180 mRNA levels in tumor were significantly lower than those in non-tumor, with the same as in hypermethylation than hypomethylation group. H.pylori infection increased methylation in normal tissue or mild gastritis, and increased hypermethylation risk at 3 CpG sites in AG. In conclusion, higher AMR, MSC and HSC levels could identify AG + GC or GC. Some RNF180 promoter CpG sites could identify precancerous or early-stage GC. H.pylori affects RNF180 PM in normal tissue or mild gastritis, and increases hypermethylation in 3 CpG sites in AG.

Keywords: CpG islands; atrophic gastritis; bisulfite genomic sequencing; gastric cancer; methylation.

Figure 1. The scatter plot of RNF180 gene promoter methylation

(A) The average methylation rate of RNF 180 gene in different gastric diseases; (B) The methylated CpG site counts of RNF 180 gene in different gastric diseases; (C) The hypemethylated CpG site counts RNF 180 gene in different gastric diseases.

Figure 2. The ROC curve of RNF180 gene promoter methylation

(A) GC + AG vs. CON., AUC = 0.802; (B) GC vs. AG + Con., AUC = 0.685; (C) Methylated CpG site counts, GC + AG vs. CON., AUC = 0.772; (D) Methylated CpG site counts, GC vs. AG + CON., AUC = 0.677; (E) Hypermethylated CpG site counts, GC + AG vs. CON., AUC = 0.813; (F) Hypermethylated CpG site counts, GC vs. AG + CON AUC = 0.654.