The relationship between pepsinogen C and gastric carcinogenesis: a transgene and population study.

BACKGROUND: Pepsinogen C (PGC) is expressed in chief cells, fundic mucous neck cells, and pyloric gland cells of gastric epithelium and also in breast, prostate, lung, and seminal vesicles. METHODS: We explored the clinicopathological and prognostic significances of PGC mRNA using pathological and bioinformatics analyses. We generated PGC knockout and PGC-cre transgenic mice to observe the effects of PGC deletion and PTEN abrogation in PGC-positive cells on gastric carcinogenesis. Finally, we observed the effects of altered PGC expression on aggressive phenotypes by CCK8, Annexin V staining, wound healing and transwell assays and analyzed the partner proteins of PGC using co-IP (co-immunoprecipitation) and double fluorescence staining. RESULTS: PGC mRNA level was inversely correlated with the T and G stage and a short survival of gastric cancer (p < 0.05). PGC protein expression was negatively linked to lymph node metastasis, dedifferentiation, and low Her-2 expression of gastric cancer (p < 0.05). No difference in body weight or length was evident between wild-type (WT) and PGC knockout (KO) mice (p > 0.05), but PGC KO mice had a shorter survival than WT mice (p < 0.05). No gastric lesions were observed in the mucosa of the granular stomach in PGC KO mice, which displayed lower frequency and severity of gastric lesion than in WT mice after treated with MNU. Transgenic PGC-cre mice showed high cre expression and activity in the lung, stomach, kidney, and breast. Gastric cancer and triple-negative lobular breast adenocarcinoma were found in PGC-cre/PTENf/f mice with two previous pregnancies and breast feeding, but breast cancer was not seen in transgenic mice exposed to either estrogen or progesterone, or those with two previous pregnancies and no breast feeding. PGC suppressed proliferation, migration, invasion, and induced apoptosis, and interacted with CCNT1, CNDP2 and CTSB. CONCLUSION: PGC downregulation was seen in gastric cancer, but PGC deletion resulted in resistance to chemically-induced gastric carcinogenesis. PGC expression suppressed the proliferation and invasion of gastric cancer cells possibly by interacting with CCNT1, CNDP2 and CTSB. Spontaneous triple-negative lobular adenocarcinoma and gastric cancer were seen in PGC-cre/PTENf/f mice, and the breast carcinogenesis was closely linked to pregnancy and breast feeding, but not to single exposure to estrogen or progesterone, or pregnancy. Limiting either pregnancy or breast feeding might help to prevent hereditary breast cancer.

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

BACKGROUND: A 100-bp insertion/deletion polymorphism in the pepsinogen C gene has been associated with the risk of gastric cancer (GC). OBJECTIVE: We analyzed the relationships of the 100-bp insertion/deletion polymorphism with GC, atrophic gastritis (AG), and intestinal metaplasia (IM) in the Mexican general population (MGP). METHODS: We studied the genomic DNA of subjects with GC n = 80, AG and IM n = 60, controls n = 110, and the MGP n = 97. PGC gene insertion/deletion polymorphism was identified by means of PCR, capillary electrophoresis and GeneScan software. RESULTS: Different allele sizes of PGC polymorphism were observed in the studied groups, from 266 bp to 499 bp, which were grouped for the analysis as short alleles of 266-399 bp, medium alleles of 400-433 bp and large alleles of 434-499 bp. Carriers of one or two medium alleles, had an increased risk of GC, with OR of 1.99 (CI95% 1.08-3.67 p = 0.026) compared to homozygotes (no medium/no medium). CONCLUSIONS: Previous studies have related PGC short alleles to risk for or protection against GC depending on the ethnic origin of the population. In our study, medium alleles were related to risk for GC. Further studies are required to establish the importance of this polymorphism in the origin of gastric neoplasia.

Protective effect of the combination of essential oil from patchouli and tangerine peel against gastric ulcer in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oil (EO) is the main extract of patchouli and tangerine peel with antiinflammatory, antiulcer, and other functions. However, the efficacy and mechanism of the combination of EO from patchouli and tangerine peel against gastric ulcer (GU) are unclear. AIM OF THE STUDY: This study aims to reveal the protective effect of the combination of EO from patchouli and tangerine peel against GU in rats, as well as explore the optimal ratio and possible mechanism of EO in GU treatment. MATERIALS AND METHODS: The GU model is executed via water immersion and restraint stress. The repair effect of EO in different proportions on gastric mucosa injury and the effects on serum gastrin (GAS), pepsinogen C (PGC), prostaglandin E2 (PGE2), and 5-hydroxytryptamine in GU rats were observed. The optimal ratio obtained was used in the second part to set different dose groups for further experiment. The effects of the different EO doses on gastric mucosal ulcer formation and gastric acid secretion were evaluated. The morphology of chief and parietal cells were observed via transmission electron microscopy. The contents of GAS, PGC, substance P (SP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cholecystokinin (CCK), PGE2, and motilin (MTL) in serum in different groups were detected via enzyme-linked immunosorbent assay. Expressions of epidermal growth factor (EGF) and trefoil factor 2 (TFF2) protein in gastric tissues were detected via immunohistochemistry, and expressions of c-Jun N-terminal kinase (JNK), P53, Bcl-2-associated X protein (Bax), and Caspase-3 protein in gastric tissues were detected via western blotting. RESULTS: The EO from patchouli and tangerine peel at 1:2 ratio of compatibility significantly improved gastric mucosal injury, decreased serum GAS and PGC contents, and increased the PGE2 level in serum (p < 0.05). The mixture of EO from patchouli and tangerine peel (Mix-EO) can reduce the formation of gastric mucosal ulcers, reduce gastric mucosal injury, improve the expansion of the endoplasmic reticulum of the chief cells, repair mitochondrial damage, and inhibit the secretion of gastric acid by parietal cells. Mix-EO at 300 mg/kg can reduce the expression of serum GAS, PGC, SP, CCK, and cAMP/cGMP (p < 0.05 or 0.01); increase the expression of EGF and TFF2 protein in gastric tissues (p < 0.01); and inhibit the expression of JNK, p53, Bax, and Caspase-3 proteins (p < 0.01). CONCLUSION: The combination of EO from patchouli and tangerine peel can repair the gastric mucosal damage in GU rats and prevent the occurrence of ulcers by inhibiting the secretion of gastric acid, enhancing the defensive ability of gastric mucosa, and suppressing the apoptosis of gastric epithelial cells. Moreover, the optimal compatible ratio of patchouli and tangerine peel is 1:2.

Induction of Gastric Cancer by Successive Oncogenic Activation in the Corpus.

BACKGROUND & AIMS: Metaplasia and dysplasia in the corpus are reportedly derived from de-differentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C (PGC) transcript-expressing cells represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model. METHODS: We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments and histologic and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC transcript-expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC transcript-expressing cells with activated Kras, inactivated Apc, and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice. RESULTS: Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC transcript-expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma and Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma. CONCLUSIONS: The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.

Pepsinogen C expression-related lncRNA/circRNA/mRNA profile and its co-mediated ceRNA network in gastric cancer.

The expression of pepsinogen C (PGC) is considered an ideal negative biomarker of gastric cancer, but its pathological mechanisms remain unclear. This study aims to analyze competing endogenous RNA (ceRNA) networks related to PGC expression at a post-transcriptional level and build an experimental basis for studying the role of PGC in the progression of gastric cancer. RNA sequencing technology was used to detect the differential expression (DE) profiles of PGC-related long non-coding (lnc)RNAs, circular (circ)RNAs, and mRNAs. Ggcorrplot R package and online database were used to construct DElncRNAs/DEcircRNAs co-mediated PGC expression-related ceRNA networks. In vivo and in vitro validations were performed using quantitative reverse transcription-PCR (qRT-PCR). RNA sequencing found 637 DEmRNAs, 698 DElncRNAs, and 38 DEcircRNAs. The PPI network of PGC expression-related mRNAs consisted of 503 nodes and 1179 edges. CFH, PPARG, and MUC6 directly interacted with PGC. Enrichment analysis suggested that DEmRNAs were mainly enriched in cancer-related pathways. Eleven DElncRNAs, 13 circRNAs, and 35 miRNA-mRNA pairs were used to construct ceRNA networks co-mediated by DElncRNAs and DEcircRNAs that were PGC expression-related. The network directly related to PGC was as follows: SNHG16/hsa_circ_0008197-hsa-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p-PGC. qRT-PCR validation results showed that PGC, PPARG, SNHG16, and hsa_circ_0008197 were differentially expressed in gastric cancer cells and tissues: PGC positively correlated with PPARG (r = 0.276, P = 0.009), SNHG16 (r = 0.35, P = 0.002), and hsa_circ_0008197 (r = 0.346, P = 0.005). PGC-related DElncRNAs and DEcircRNAs co-mediated complicated ceRNA networks to regulate PGC expression, thus affecting the occurrence and development of gastric cancer at a post-transcriptional level. Of these, the network directly associated with PGC expression was a SNHG16/hsa_circ_0008197-mir-98-5p/hsa-let-7f-5p/hsa-let-7c-5p - PGC axis. This study may form a foundation for the subsequent exploration of the possible regulatory mechanisms of PGC in gastric cancer.

Helicobacter pylori (Hp) IgG ELISA of the New-Generation GastroPanel® Is Highly Accurate in Diagnosis of Hp-Infection in Gastroscopy Referral Patients.

BACKGROUND/AIM: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients. PATIENTS AND METHODS: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C). RESULTS: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000). CONCLUSION: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings.

LncRNAs adjacent to pepsinogen C in gastric diseases and diagnostic efficiencies of joint detection in gastric diseases.

Aims: We aimed to explore diagnostic efficiencies of long noncoding RNAs (lncRNAs) adjacent to PGC combining with sPGC and anti-Helicobacter pylori IgG in identifying GC (gastric cancer) and precancerous disease. Patients & methods: A total of 265 patients with different gastric diseases were collected. ELISA was to detect sPGC and anti-H. pylori IgG. LncRNAs was determined by qRT-PCR. Results: The area under receiver operating characteristic curve of lncRNAs in discriminating GC+AG (atrophic gastritis) and superficial gastritis (SG) were 79.0, 68.1 and 75.9%. The diagnostic performance of lncRNAs with sPGC had increasing trends in distinguishing GC from non-GC, SG from GC+AG comparing with lncRNAs, with no statistic difference. Diagnosis efficacies of lncRNAs with anti-H. pylori IgG improved dramatically. Conclusions: Serum lncRNAs could distinguish GC, AG and SG. Diagnosis efficiencies of lncRNAs with sPGC and anti-H. pylori-IgG could be improved.

SNP interactions of PGC with its neighbor lncRNAs enhance the susceptibility to gastric cancer/atrophic gastritis and influence the expression of involved molecules.

Multidimensional interactions of multiple factors are more important in promoting cancer initiation. Gene-gene interactions between protein-coding genes have been paid great attention, while rare studies refer to the interactions between encoding and noncoding genes. Our research group previously found encoding gene PGC polymorphisms could affect the susceptibility to atrophic gastritis (AG) and gastric cancer (GC). Interestingly, several SNPs in long noncoding RNA (lncRNA) genes, just adjacent to PGC, were found to be associated with AG risk and GC prognosis afterward. This study aims to explore the SNP interactions between PGC and its neighbor lncRNAs on the risk of AG and GC. Genotyping for seven PGC SNPs and seven lncRNA SNPs was conducted using Sequenom MassARRAY platform in a total of 2228 northern Chinese subjects, including 536 GC cases, 810 AG cases, and 882 controls. We found 15 pairwise PGC-lncRNAs SNPs had interactions: Five pairs were associated with AG risk, and ten pairs were associated with GC risk. Moreover, two GC-related interactions PGC rs6939861 with lnc-C6orf-132-1 rs7749023 and rs7747696 survived the Bonferroni correction (Pcorrection  = 0.049 and 0.007, respectively). Several combinations showed obvious epistasis and cumulative effects on disease risk. Some three-way interactions of SNPs with smoking and drinking could also be observed. Besides, a few interacting SNPs showed correlations with the expression levels of PGC protein and related lncRNAs in serum. Our study would provide research clues for further screening combination biomarkers uniting both protein-coding and noncoding genes with the potential in prediction of the susceptibility to GC and its precursor.

Correlation between negative expression of pepsinogen C and a series of phenotypic markers of gastric cancer in different gastric diseases.

Gastric tumorigenesis is a multistep process initiated by chronic superficial gastritis (SG), followed by atrophic gastritis (AG), then intestinal metaplasia (IM), and finally by dysplasia and adenocarcinoma according to the Correa model. Pepsinogen C (PGC) decreases gradually during progression of cancer, which makes PGC an ideal negative marker for GC. To explore the correlation between PGC and other positive tumor markers in different gastric diseases, we observed the expression of PGC, MG7-Ag, MMP9, NM23, Ki-67, and E-cadherin by immunohistochemistry, quantitative RT-PCR, and immunoblot analysis. Our results showed that in SG, PGC was highly expressed while malignant phenotype markers were rarely expressed. In contrast with SG, malignant phenotype markers were highly expressed while the positive rate of PGC reached only 1.44% in GC. So there was no coexpression of PGC and malignant phenotype markers in SG or GC tissues. Only in the AG group, which is well-known to be gastric precancerous disease, coexpression of PGC and malignant phenotype markers was detected. Our results suggested that the expression of PGC in AG was negatively correlated with that of MG7-Ag and MMP9. Of all AG, those with low expression of PGC and high expression of MG7-Ag and MMP9 may possess a greater potential of malignant transformation. Combined detection of negative marker PGC and positive markers MG7-Ag and MMP9 could be used as a potential follow-up panel for monitoring dynamical progression of AG and improving the detection efficiency of high-risk individuals of gastric cancer, and then taking necessary interventions on the target population.

[Molecular Cloning,Sequence Analysis,and Expression of Pepsinogens C Gene from Adult Alligator Sinensis Stomach].

Objective To investigate the molecular clone and structural features of pepsinogen C(PGC) gene in the stomach of Alligator sinensis,explore the phylogenetic relationships and tissue distribution,and analyze the variation of PGC expression in the stomachs of adult Alligator sinensis at different life stages. Methods The full-length cDNA of PGC gene of Alligator sinensis was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends and then sequenced.The physical and chemical parameters and advanced structures of the PGC protein were predicted by bioinformatics methods and tools.The PGC amino acid sequences of the Alligator sinensis and other vertebrates were compared by Clustal X software.The neighbor-joining phylogenetic tree was built by MEGA 6 software.Immunohistochemistry was used to locate PGC in the gastric mucosa of Alligator sinensis.The variation of the PGC mRNA levels in the stomach at different life stages was detected by quantitative real-time polymerase chain reaction.Results Reverse transcription polymerase chain reaction and rapid amplification of cDNA ends revealed a 1568 bp cDNA full-length sequence containing 1167 bp open reading frame,which encoded 388 amino acids.The PGC gene of Alligator sinensis had been deposited in the GenBank Data Libraries under the accession number of KY799383.Bioinformatics analysis predicted that the Alligator sinensis PGC had a theoretical relative molecular mass of 41 998 with a theoretical isoelectric point of 4.16.In addition,the three-dimensional structure of the PGC was constructed by homology modeling to predict its active site with two essential aspartyl residues and six essential cysteine residues involved in forming three disulphide bonds.The neighbor-joining phylogenetic tree of vertebrates from the amino acids sequences of PGC showed all crocodiles were clustered as a group,and the PGC of Alligator sinensis was the closest to Alligator mississippiensis.Alligator sinensis PGC was specifically expressed in the gastric mucosa,and its expressions significantly differed during reproduction and hibernation significantly(P<0.05).Conclusions Alligator sinensis PGC gene is highly conserved in evolution.Its protein is a gastric specific digestive proteinase that belongs to a aspartic proteinase family.

Comparative proteomic analysis of human malignant ascitic fluids for the development of gastric cancer biomarkers.

OBJECTIVES: Malignant ascites is a sign of peritoneal seeding, which is one of the most frequent forms of incurable distant metastasis. Because the development of malignant ascites is associated with an extremely poor prognosis, determining whether it resulted from peritoneal seeding has critical clinical implications in diagnosis, choice of treatment, and active surveillance. At present, the molecular characterizations of malignant ascites are especially limited in case of gastric cancer. We aimed to identify malignant ascites-specific proteins that may contribute to the development of alternative methods for diagnosis and therapeutic monitoring and also increase our understanding of the pathophysiology of peritoneal seeding. DESIGN & METHODS: First, comprehensive proteomic strategies were employed to construct an in-depth proteome of ascitic fluids. Label-free quantitative proteomic analysis was subsequently performed to identify candidates that can differentiate between malignant ascitic fluilds of gastric cancer patients from benign ascitic fluids. Finally, two candidate proteins were verified by ELISA in 84 samples with gastric cancer or liver cirrhosis. RESULTS: Comprehensive proteome profiling resulted in the identification of 5347 ascites proteins. Using label-free quantification, we identified 299 proteins that were differentially expressed in ascitic fluids between liver cirrhosis and stage IV gastric cancer patients. In addition, we identified 645 proteins that were significantly expressed in ascitic fluids between liver cirrhosis and gastric cancer patients with peritoneal seeding. Finally, Gastriscin and Periostin that can distinguish malignant ascites from benign ascites were verified by ELISA. CONCLUSIONS: This study identified and verified protein markers that can distinguish malignant ascites with or without peritoneal seeding from benign ascites. Consequently, our results could be a significant resource for gastric cancer research and biomarker discovery in the diagnosis of malignant ascites.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Association of Polymorphisms in three pri-miRNAs that Target Pepsinogen C with the Risk and Prognosis of Gastric Cancer.

We aimed to explore the associations of polymorphisms in three microRNAs (miRNAs) (let-7e rs8111742, miR-365b rs121224 and miR-4795 rs1002765) that target PGC with the risk and prognosis of gastric cancer/atrophic gastritis. Sequenom's MassArray was used to genotype the miRNA polymorphisms in 724 gastric cancer cases, 862 atrophic gastritis cases and 862 controls in a Chinese population. We found that let-7e rs8111742 and miR-4795 rs1002765 were associated with the risk of gastric cancer in the H. pylori-positive subgroup. MiR-365b rs121224 was associated with the risk of intestinal-type gastric cancer in the alcohol consumption subgroup. Intestinal-type gastric cancer patients at Borrmann stages III-IV who carry the miR-365b rs121224 GG genotype had better prognosis compared with those who carry the CG or CC genotypes. MiR-365b rs121224 was associated with Lauren typing and TNM staging, in which the distribution of GG genotype carriers in intestinal-type gastric cancer and the TNM stage I-II subgroup was higher than that of CG or CC genotypes, which contrasted with the distribution in diffuse-type gastric cancer or TNM III-IV groups. These findings suggested that the polymorphisms in these miRNAs might be biomarkers for gastric cancer risk and prognosis, especially for populations infected with Helicobacter pylori or who consume alcohol.

Polymorphic rs9471643 and rs6458238 upregulate PGC transcription and protein expression in overdominant or dominant models.

The pepsinogen C (PGC) gene encodes a major differentiation biomarker for gastric mucosa and has two single nucleotide polymorphisms, rs9471643 G>C and rs6458238 G>A, within its 5' upstream region that are involved in gastric carcinogenesis. However, in what genetic models the two polymorphisms modulate disease risk and how they relate to gastric carcinogenesis needs further study. We fitted the most appropriate genetic models to the PGC polymorphisms and validated their robustness; then with knowledge of the genetic model, we investigated the influence of functional variant alleles or genotypes on gene expression in vitro and in vivo. We confirmed that rs9471643 CG genotype was stably associated with reduced gastric cancer risk in complete overdominant model. This favorable CG genotype was also associated with reduced atrophic gastritis risk in subjects carrying rs6458238 AG/AA genotype. The G>C transition at rs9471643 enhanced promoter activity and transcription factor binding ability, and the CG genotype was consistently associated with elevated levels of PGC mRNA, in situ protein and serum protein in complete overdominant model based-analyses. Additionally, rs6458238 AG/AA genotype was associated with reduced atrophic gastritis risk in dominant model. Its favorable A allele was related to higher promoter activity and lower transcription factor binding ability, and the AG/AA genotype showed association with elevated levels of serum PGC protein in dominant model based-analyses. Our results suggest that rs9471643 CG and rs6458238 AG/AA genotypes have important roles in up-regulating PGC expression, which may partially explain why individuals with these favorable genotypes have decreased risks of getting gastric cancer.

Pepsinogen-II 100 bp ins/del gene polymorphism and its elevated circulating levels are associated with gastric cancer, particularly with Helicobacter pylori infection and intestinal metaplasia.

BACKGROUND: Polymorphism in the gene of pepsinogen-II (PG-II) and its serum level are effective biomarkers for terminal differentiation of gastric mucosa into gastritis, intestinal metaplasia (IM), and gastric cancer (GC) in relationship to Helicobacter pylori infection. METHODS: Genotyping of the PG-II 100 bp insertion/deletion (ins/del) polymorphism was performed in patients with GC (n = 192) and age- and gender-matched H. pylori-associated dyspepsia (n = 180) and healthy subjects (HS, n = 240) by PCR. IgG anti-H. pylori (in all subjects) and serum PG-II levels were estimated in 145 patients each with GC and dyspepsia and in 65 healthy controls (HC) using ELISA (Biohit Oyj, Finland). RESULTS: Five alleles were amplified by PCR: allele 5 (510 bp), allele 4 (480 bp), allele 3 (450 bp), allele 2 (400 bp), and allele 1 (shorter allele, 310 bp). Allele 1 carriage was infrequent, and serum PG-II level was higher among patients with GC than in HC [OR 0.43 (95 % CI, 0.29-0.85), p < 0.001 and mean ± SD; 17.53 ± 12.60 vs. 12.77 ± 7.53 µg/l, p = 0.005, respectively], particularly in the presence of H. pylori [OR 0.42 (0.25-0.71), p = 0.001 and 18.78 ± 12.63 vs. 13.97 ± 8.14, p = 0.034]. However, allele 1 carriage and PG-II levels were comparable among patients with GC and dyspepsia. Patients with IM also carried allele 1 infrequently and had higher levels of PG-II than those without [OR 0.5 (0.29-0.85), p = 0.011 and 20.07 ± 14.22 vs. 16.61 ± 12.08, p = 0.048]. CONCLUSIONS: Carriage of the shorter allele of the PG-II 100 bp ins/del polymorphism and elevated levels of PG-II are associated with GC, particularly with H. pylori infection and IM.

SNP interactions of Helicobacter pylori-related host genes PGC, PTPN11, IL1B, and TLR4 in susceptibility to gastric carcinogenesis.

A series of host genes that respond to Helicobacter pylori (H. pylori) infection are involved in the process of gastric carcinogenesis. This study sought to examine interactions among polymorphisms of H. pylori-related genes PGC, PTPN11, TLR4, and IL1B and assess whether their interaction effects were modified by H. pylori infection. Thirteen polymorphisms of the aforementioned genes were genotyped by the Sequenom MassARRAY platform in 714 gastric cancer patients, 907 atrophic gastritis cases and 1276 healthy control subjects. When we considered the host genetic effects alone, gene-gene interactions consistently decreased the risks of gastric cancer and/or atrophic gastritis, including three two-way interactions: PGC rs6912200-PTPN11 rs12229892, PGC rs4711690-IL1B rs1143623 and PTPN11 rs12229892-IL1B rs1143623 and a three-way interaction: PGC rs4711690-PGC rs6912200-PTPN11 rs12229892. When the effect modification of H. pylori infection was evaluated, the cumulative effects of the aforementioned three-way interaction on atrophic gastritis susceptibility switched from being beneficial to being risky by the status of H. pylori infection. These data showed that SNP interactions among H. pylori-related genes PGC, PTPN11, and IL1B, are associated with susceptibility to gastric carcinogenesis. Moreover, we provided important hints of an effect modification by H. pylori infection on the cumulative effect of PGC and PTPN11 polymorphisms. Functional experiments and further independent large-scale studies especially in other ethnic populations are still needed to confirm our results.

Expression of serum let-7c, let-7i, and let-7f microRNA with its target gene, pepsinogen C, in gastric cancer and precancerous disease.

This study examined the expression patterns of serum let-7 microRNA (miRNA) and its target gene, pepsinogen C (PGC), in gastric cancer (GC) and precancerous disease patients to evaluate their diagnostic efficiency for GC and its precursor and to investigate any correlation between the two. Serum samples were taken from 638 patients, including 214 GC patients, 222 atrophic gastritis (AG) patients, and 202 controls (CON). The expression of serum let-7 miRNA was detected in control-AG (precancerous disease) through to GC patients using quantitative reverse-transcription polymerase chain reaction. Serum PGC was determined by enzyme-linked immuno-sorbent assay. PGC expression in situ was detected by immunohistochemistry staining. The luciferase reporter gene system was used to verify correlation between let-7 miRNA and its predicted target gene. The results showed that serum let-7c, let-7i, and let-7f demonstrated significant differences in the CON-AG-GC sequence (P = 0.017, P < 0.001, P = 0.003, respectively); let-7c was significantly lower in the AG group, and let-7i and let-7f were significantly higher in the GC group. Significantly different expressions of serum PGC were found among the three diseases, and also between AG vs. CON, and GC vs. CON (P = 0.027, P = 0.001, respectively). Linear-regression analysis suggested that serum let-7c was negatively correlated to the expression of PGC (r = -0.096, P = 0.047), and serum let-7c, let-7i, and let-7f showed no association with PGC expression in tissue. In addition, serum let-7c, let-7f, and let-7i showed significant correlations with environment factors. Serum let-7c, let-7i, and let-7f demonstrated significant differences in the CON-AG-GC disease sequence indicating that let-7 miRNA might have value by serving as potential biomarker in the diagnosis of GC or its precancerous diseases. There were significant negative correlations between serum let-7c and its target gene PGC expression.

Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach.

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

The interaction effects of pri-let-7a-1 rs10739971 with PGC and ERCC6 gene polymorphisms in gastric cancer and atrophic gastritis.

BACKGROUND: The aim of this study was to investigate the interaction effects of pri-let-7a-1 rs10739971 with pepsinogen C (PGC) and excision repair cross complementing group 6 (ERCC6) gene polymorphisms and its association with the risks of gastric cancer and atrophic gastritis. We hoped to identify miRNA polymorphism or a combination of several polymorphisms that could serve as biomarkers for predicting the risk of gastric cancer and its precancerous diseases. METHODS: Sequenom MassARRAY platform method was used to detect polymorphisms of pri-let-7a-1 rs10739971 G → A, PGC rs4711690 C → G, PGC rs6458238 G → A, PGC rs9471643 G → C, and ERCC6 rs1917799 in 471 gastric cancer patients, 645 atrophic gastritis patients and 717 controls. RESULTS: An interaction effect of pri-let-7a-1 rs10739971 polymorphism with ERCC6 rs1917799 polymorphism was observed for the risk of gastric cancer (P interaction = 0.026); and interaction effects of pri-let-7a-1 rs10739971 polymorphism with PGC rs6458238 polymorphism (P interaction = 0.012) and PGC rs9471643 polymorphism (P interaction = 0.039) were observed for the risk of atrophic gastritis. CONCLUSION: The combination of pri-let-7a-1 rs10739971 polymorphism and ERCC6 and PGC polymorphisms could provide a greater prediction potential than a single polymorphism on its own. Large-scale studies and molecular mechanism research are needed to confirm our findings.

Amplification of steroid-mediated SP-B expression by physiological levels of caffeine.

Factors positively influencing surfactant homeostasis in general and surfactant protein B (SP-B) expression in particular are considered of clinical importance regarding an improvement of lung function in preterm infants. The objective of this study was to identify effects of physiological levels of caffeine on glucocorticoid-mediated SP-B expression in vitro and in vivo. Levels of SP-B and pepsinogen C were quantified by quantitative real-time RT-PCR or immunoblotting in NCI-H441 cells daily exposed to caffeine and/or dexamethasone (DEX). In vivo, SP-B expression was analyzed in bronchoalveolar lavage (BAL) of preterm sheep exposed to antenatal DEX and/or postnatal caffeine. If DEX and caffeine were continuously present, SP-B mRNA and protein levels were increased for up to 6 days after induction (P < 0.05). Additionally, caffeine enhanced SP-B mRNA expression in DEX-pretreated cells (P < 0.05). Moreover, caffeine amplified DEX-induced pepsinogen C mRNA expression (P < 0.05). After short-term treatment with caffeine in vivo, only slightly higher SP-B levels could be detected in BAL of preterm sheep following antenatal DEX, combined with an increase of arterial oxygen partial pressure (P < 0.01). Our data demonstrated that the continuous presence of caffeine in vitro is able to amplify DEX-mediated SP-B expression. In contrast, short-term improvement of lung function in vivo is likely to be independent of altered SP-B transcription and translation. An impact of caffeine on release of surfactant reservoirs from lamellar bodies could, however, quickly affect SP-B content in BAL, which has to be further investigated. Our findings indicate that caffeine is able to amplify main effects of glucocorticoids that result from changes in surfactant production, maturation, and release.