Polymorphisms in Pepsinogen C and miRNA Genes Associate with High Serum Pepsinogen II in Gastric Cancer Patients

Abstract

Background: Pepsinogen (PG) II (PGII) is a serological marker used to estimate the risk of gastric cancer but how PGII expression is regulated is largely unknown. It has been suggested that PGII expression, from the PGC (Progastricsin) gene, is regulated by microRNAs (miRNA), but how PGII levels vary with Helicobacter pylori (H. pylori) infection and miRNAs genotype remains unclear.

Methods: Serum levels of PGI and PGII were determined in 80 patients with gastric cancer and persons at risk for gastric cancer (74 first-degree relatives of patients, 62 patients with autoimmune chronic atrophic gastritis, and 2 patients with dysplasia), with and without H. pylori infection. As control from the general population, 52 blood donors were added to the analyses. Associations between PGII levels and genetic variants in PGC and miRNA genes in these groups were explored based on H. pylori seropositivity and the risk for gastric cancer. The two-dimensional difference in gel electrophoresis (2D-DIGE) and the NanoString analysis of messenger RNA (mRNAs) from gastric cancer tissue were used to determine the pathways associated with increased PGII levels.

Results: PGII levels were significantly higher in patients with gastric cancer, and in those with H. pylori infection, than in other patients or controls. A PGI/PGII ratio ≤ 3 was found better than PGI < 25 ng/mL to identify patients with gastric cancer (15.0% vs. 8.8%). For two genetic variants, namely rs8111742 in miR-Let-7e and rs121224 in miR-365b, there were significant differences in PGII levels between genotype groups among patients with gastric cancer (p = 0.02 and p = 0.01, respectively), but not among other study subjects. Moreover, a strict relation between rs9471643 C-allele with H. pylori infection and gastric cancer was underlined. Fold change in gene expression of mRNA isolated from gastric cancer tissue correlated well with polymorphism, H. pylori infection, increased PGII level, and pathway for bacteria cell entry into the host.

Conclusions: Serum PGII levels depend in part on an interaction between H. pylori and host miRNA genotypes, which may interfere with the cut-off of PGI/PGII ratio used to identify persons at risk of gastric cancer. Results reported new findings regarding the relation among H. pylori, PGII-related host polymorphism, and genes involved in this interaction in the gastric cancer setting.

Keywords: Helicobacter pylori; PGC; RNAs; gastric cancer; polymorphisms; serum pepsinogen II.

Figure 1

PGC (Progastricsin) gene, coding for the Pepsinogen II protein, structure and interactions with endogenous miRNAs tested in the study. The nine exons are indicated with black rectangles. * Potential methylation sites are indicated as M1 to M12. Different lengths of TATA box repeats (Group 1, short; Group 2, medium; Group 3, long) are present in the intronic region between exons 7 and 8, which includes two methylation sites (M11, M12) [29].

Figure 2

Serum levels of pepsinogen I and II, by study group. The boxes indicate the median and 25th and 75th percentiles, and the outside whiskers indicate the range. (A) Pepsinogen I. Kruskal-Wallis test, p < 0.0001, Jonckheere-Terpstra test for trend not significant. (B) Pepsinogen II. Kruskal-Wallis test, p < 0.0001, Jonckheere-Terpstra test for trend p < 0.0001, significantly different (p < 0.05) average rank (Conover analysis). CTRL, controls; FDR-GC, first-degree relatives of gastric cancer patients; ACAG, autoimmune chronic atrophic gastritis; DIS, dysplasia; GC, gastric cancer.

Figure 3

Receiver operating characteristic curve for predicting the presence of anti-H. pylori IgG from serum Pepsinogen (PG) II levels.

Figure 4

Positive relationship between serum PGII levels and abundance of PGII from the paired patient samples.

Figure 5

Box plot showing median increased of PGII standard abundance in H. pylori-related GC tissue specimens.

Figure 6

PG II-related protein-protein interaction network generated using STRING. The network is made up of the 21 significant genes (Benjamin corrected p < 0.05) reported in Table 5. Colored nodes indicated the first choice of interactors.

Figure 7

Polymorphism-related protein-protein interaction networks generated using STRING. Colored nodes indicated the first choice of interactors. (A) The network is made up of the 11 genes (p < 0.05) reported in Table 8 for the rs9471643 SNPs. (B) The network is made up of the 4 genes reported in Table 7 for the pre-miRNA rs8111742 and rs121224 SNPs.