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.

Reply

“…2 In De Re et al, 1 the histology positivity ranged from 13% among control subjects to 37% among first-degree relatives. In De Re et al 3 the antibody serologic positivity (using a similar cutoff to our study) ranged from 14% among control subjects to 55% among patients with gastric cancer. By comparison, in our North American cohort the histologic positivity was 8%, and serologic positivity was 14%.…”

“…They note studies from low gastric cancer-incidence regions of Europe (the Netherlands, Norway, and Italy) demonstrating higher discrimination of serologic biomarkers using receiver operating curve analysis. [1][2][3] However, there exist several important differences in these European populations compared with our multiethnic North American population. Most notably, the population prevalence of Helicobacter pylori (Hp) carriage is higher in the European samples.…”

“…Another important difference between the referenced studies is that the Italian study specifically recruited patients with autoimmune gastritis, 1,3 whereas most patients with atrophic gastritis/intestinal metaplasia in our cohort were not diagnosed with autoimmunemediated disease. Autoimmune gastritis seems to be a distinct clinical entity characterized by corpuspredominant inflammation, although there seems to be some overlap with prior Hp infection.…”

Medium and large alleles of the PGC gene are risk factors for gastric cancer

“…The PGC gene coding for the pepsinogen II protein consists of nine exons, and different lengths of the TATA box are represented by short (308-378 bp), medium (400-413 bp) and large (434-479 bp) repeats, which are located between exons 7 and 8 [13]. This polymorphic length of the TATA box is better known as the 100-bp insertion/deletion polymorphism and has been related to peptic ulcers and gastric cancer.…”

“…DNA was extracted from leucocytes of peripheral blood by the Miller method [12]. The PGC gene insertion/deletion polymorphism was identified by means of PCR and capillary electrophoresis using the following primers: forward 5'-FAM/GGCCAGATCTGCGTGTTTTA-3' and reverse 5'-AGCCCTAAGCCTGTTTTTGG-3' [13]. PCR was performed in a 15-L reaction mixture comprising 200 ng genomic DNA, each primer at 5 pM, 0.5 U Taq polymerase (Invitrogen, USA), 1X PCR buffer, 1.5 mM MgCl2, 2.0 mM deoxynucleotide triphosphate mix (dNTP Set; Vivantis, Malaysia), and 5% DMSO.…”