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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/PTEN f/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/PTEN f/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., (© 2023. The Author(s).)

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.

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., (Copyright © 2021. Published by Elsevier B.V.)

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;Kras G12D/+ 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 Kras G12D , Apc flox , Trp53 flox 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;Kras G12D/+ mice, PGC transcript-expressing cells with Kras G12D/+ 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 Kras G12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;Kras G12D/+ ;Apc flox/flox mice presented intramucosal dysplasia/carcinoma and Pgc-CreERT2;Kras G12D/+ ;Apc flox/flox ;Trp53 flox/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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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&#95;circ&#95;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&#95;circ&#95;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&#95;circ&#95;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&#95;circ&#95;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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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.

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 (P correction  = 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., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

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.

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 NH 2 -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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2015 Wiley Periodicals, Inc.)

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.

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.

Background: Pepsinogen C (PGC) plays an important role in sustaining the cellular differentiation during the process of gastric carcinogenesis. This study aimed to assess the role of PGC tagSNPs and their interactions with Helicobacter pylori (H. pylori) in the development of gastric cancer and its precursor, atrophic gastritis., Methods: Four PGC tagSNPs (rs6941539, rs6912200, rs3789210 and rs6939861) were genotyped by Sequenom MassARRAY platform in a total of 2311 subjects consisting of 642 gastric cancer, 774 atrophic gastritis, and 895 healthy control subjects. The mRNA and protein expression levels of PGC in gastric tissues and in serum were respectively measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunohistochemistry, and Eenzyme-linked immunoabsorbent assay (ELISA)., Results: We found associations between PGC rs3789210 CG/GG genotypes and reduced gastric cancer risk and between PGC rs6939861 A variant allele and increased risks of both gastric cancer and atrophic gastritis. As for the haplotypes of PGC rs6941539-rs6912200-rs3789210-rs6939861 loci, the TTCA and TTGG haplotypes were respectively associated with increased and reduced risks of both gastric cancer and atrophic gastritis; additionally, the CTCA haplotype was associated with increased atrophic gastritis risk. Very interestingly, rs6912200 CT/TT genotypes had a positive interaction with H. pylori, synergistically elevating the gastric cancer risk. Moreover, healthy subjects who carried rs6912200 CT, TT and CT/TT variant genotypes had lower histological and serum expression levels of PGC protein., Conclusions: Our findings highlight an important role of PGC rs3789210 and rs6939861 in altering susceptibility to atrophic gastritis and/or gastric cancer. Moreover, people who carry rs6912200 variant genotypes exhibit higher gastric cancer risk in case of getting H. pylori infection, which strongly suggest a necessity of preventing and/or eliminating H. pylori infection in those individuals.

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.

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.

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.

Purpose: The discovery of new diagnostic tools for the diagnosis of prostate cancer (PCa) has become an important field of research. In this study, we analyzed the diagnostic value of the expression of the pepsinogen C (PGC) and prostate-specific membrane antigen (PSMA) genes in tissue samples obtained from prostate biopsies., Materials and Methods: This study was comprised of 51 consecutive patients who underwent transrectal ultrasound (TRUS)-guided prostate biopsies between January 2010 and March 2010. The biopsies were performed with 12 cores, and an additional core was randomly retrieved from the peripheral zone from each patient for study purposes. The expression of the PGC and PSMA genes was analyzed from the cDNA from the samples via the qRT-PCR technology. The expression patterns of patients with PCa were compared with those of patients without a PCa diagnosis., Results: PSMA was overexpressed in only 43.4% of PCa cases, and PGC was overexpressed in 72.7% of cases. The median expression of PSMA was 1.5 times (0.1 to 43.9) and the median PGC expression was 8.7 times (0.1 to 50.0) the expression observed in prostatic tissue from TRUS-guided biopsies of normal patients. Analysis of patients with high-risk PCa indicated that PGC was overexpressed in 71.4% of cases (with a median expression of 10.6 times), and PSMA was overexpressed in only 35.7% of cases (with a median expression of 4.5 times). Among patients with low-risk PCa, PGC was also overexpressed in 71.4% of cases (with a median expression of 5.9 times), and PSMA was overexpressed in only 42.8% of cases (with a median expression of 2.5 times)., Conclusions: PGC gene expression is significantly higher in prostatic tissue in men affected by PCa when compared to normal prostates. Further analyses are necessary to confirm our results.

The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Stomach carcinogenesis progresses stepwise from normal mucosa/superficial gastritis, atrophic gastritis (GA) to gastric cancer (GC). Host factors independent of or combined with Helicobacter pylori infection may modulate the carcinogenesis process. In this two-stage study, we selected 24 putative functional tag single-nucleotide polymorphisms (tagSNPs) for six H.pylori-related host genes, MUC1, toll-like receptor 4 (TLR4), protein tyrosine phosphatase, non-receptor type 11 (PTPN11), IL-1B, PGC and PGA3-5, and analyzed their influence and interaction with H.pylori on the GA and GC risks. Using high-throughput genotyping, the 24 tagSNPs were preliminarily assessed in a screening population of 552 controls, 254 GA and 236 GC subjects; subsequently, five candidate tagSNPs for gastric diseases risk in the TLR4, PGC and PTPN11 genes were re-evaluated in a larger population of 1276 controls, 907GA and 714 GC subjects. We observed that PGC rs6458238, PGC rs4711690 and PTPN11 rs12229892 were associated with susceptibilities to GA and/or GC. Moreover, rs4711690 and rs12229892 and H.pylori demonstrated significant interaction effects on GA risk. In gastric cancerous specimens, we observed significantly higher messenger RNA level in the subjects carrying the PGC rs6458238 GA genotype than that in subjects with the common GG genotype. These findings indicated that genetic variations of two crucial H.pylori-related host genes, H.pylori's mucosal effecter PGC gene and H.pylori's cellular messenger PTPN11 gene, either dependent or independent of interaction with H.pylori, were associated with the risks of GC and/or GA that precede carcinoma. Functional studies and further independent large-scale studies especially in other ethnic populations are still needed to confirm our results.

We characterized the expression patterns of pepsinogen A (tPGA) and pepsinogen C (tPGC) in turbot (Scophthalmus maximus). Quantitative expression analysis showed that tPGC was preferentially expressed in early developmental stages, and that the tPGA mRNA expression level was higher in adult fish. Full-length cDNA constructs of tPGA and tPGC were 1307 bp (from which 377 amino acids were deduced); and 1430 bp (from which 385 amino acids were deduced), respectively. The deduced proteins of tPGA and tPGC possessed signal peptides of 17 amino acids and 20 amino acids respectively. The initial transcripts of tPGA and tPGC were detected at 22 days post hatching (dph), well after the formation of gastric glands (16 dph). This suggested that the morphologic development of gastric glands was not synchronous with their functional development. In addition, tPGA and tPGC mRNAs were also expressed in muscle and ovary at much lower levels than in stomach and esophagus. The distribution of tPGA and tPGC in the turbot was investigated using in-situ hybridization, and tPGA and tPGC were first detected in the esophagus and cardiac region of the stomach, and then throughout the stomach., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Aspartic proteases comprise a large group of enzymes involved in peptide proteolysis. This collection includes prominent enzymes globally categorized as pepsins, which are derived from pepsinogen precursors. Pepsins are involved in gastric digestion, a hallmark of vertebrate physiology. An important member among the pepsinogens is pepsinogen C (Pgc). A particular aspect of Pgc is its apparent single copy status, which contrasts with the numerous gene copies found for example in pepsinogen A (Pga). Although gene sequences with similarity to Pgc have been described in some vertebrate groups, no exhaustive evolutionary framework has been considered so far., Methodology/principal Findings: By combining phylogenetics and genomic analysis, we find an unexpected Pgc diversity in the vertebrate sub-phylum. We were able to reconstruct gene duplication timings relative to the divergence of major vertebrate clades. Before tetrapod divergence, a single Pgc gene tandemly expanded to produce two gene lineages (Pgbc and Pgc2). These have been differentially retained in various classes. Accordingly, we find Pgc2 in sauropsids, amphibians and marsupials, but not in eutherian mammals. Pgbc was retained in amphibians, but duplicated in the ancestor of amniotes giving rise to Pgb and Pgc1. The latter was retained in mammals and probably in reptiles and marsupials but not in birds. Pgb was kept in all of the amniote clade with independent episodes of loss in some mammalian species. Lineage specific expansions of Pgc2 and Pgbc have also occurred in marsupials and amphibians respectively. We find that teleost and tetrapod Pgc genes reside in distinct genomic regions hinting at a possible translocation., Conclusions: We conclude that the repertoire of Pgc genes is larger than previously reported, and that tandem duplications have modelled the history of Pgc genes. We hypothesize that gene expansion lead to functional divergence in tetrapods, coincident with the invasion of terrestrial habitats.

Purpose: Human pepsinogen C (PGC) is an aspartic protease produced specifically by the gastric mucosa, and is considered as a mature marker of gastric epithelium. This study examined the contributions of PGC polymorphisms and the Helicobacter pylori (H. pylori) infection to the risk of gastric cancer (GC), and its precancerous conditions in a Northeast Chinese population., Methods: The PGC insertion/deletion polymorphism was evaluated by polymerase chain reaction analysis, followed by direct DNA sequencing in 564 cases of GC, atrophic gastritis (AG), gastric ulcer (GU) and superficial gastritis (as control). All cases were frequency-matched 1:1 by gender and age (+/-5). H. pylori infection was identified by serum anti-H. pylori IgG measurement through enzyme-linked immunosorbent assay., Results: Patients with a homozygous PGC allele 1 genotype had a significant risk of AG [adjusted odds ratio (OR) 3.11; 95% confidence interval (CI) 1.44-6.71] or of GC (OR 3.00; 95% CI 1.38-6.51), and a significantly elevated risk of intestinal metaplasia (OR 1.90, 95% CI 1.11-3.27). PGC polymorphism with H. pylori infection increased risk of GU (OR 8.69; 95% CI 1.01-74.69), and AG (OR 11.12; 95% CI 1.37-90.84) or GC (OR 10.61; 95% CI 1.28-87.79) in a super-multiplicative manner. The S value was 5.40, 6.48 and 4.34; and the AP value was 72.09, 7.00 and 69.69%, respectively., Conclusions: The PGC gene polymorphism increases an individual's susceptibility to GC and its precancerous conditions. Moreover, the PGC gene polymorphism shows a positive link to H. pylori infection in the development of GC.

Background and Objective: Human pepsinogen C (PGC) is an aspartic protease synthesized in gastric mucosa. PGC gene insertion/deletion polymorphism, which is located between exon 7 and 8, has been found to associate with gastric cancer (GC) susceptibility. This study was to investigate the relationship between PGC polymorphism with protein expression of PGC in gastric mucosa and serum., Methods: PGC insertion/deletion polymorphism was evaluated by PCR, followed by direct DNA sequencing in 493 cases of GC, atrophic gastritis (AG), gastric erosion ulcer (GEU) and superficial gastritis (SG). PGC protein expression in gastric mucosa was measured by immunohistochemistry. The serum PGC level was determined by enzyme-linked immunosorbent assay (ELISA)., Results: In accordance with the following order SG-->GEU-->GA-->GC, the frequency of PGC homozygous allele 1 was gradually increased, which was higher in GC than in SG (P=0.018); while the protein expression of PGC in gastric mucosa was gradually decreased (P<0.01), along with a gradual decrease in the strong positive rate of PGC (P<0.05) except for SG vs. GEU. The serum level of PGC was significantly lower in SG than in GU(P=0.000) and GC(P=0.000). The frequency of PGC homozygous allele 1 was negatively correlated to PGC protein expression in gastric mucosa (r=-0.1085, P=0.023). From homozygous allele 1 to heterozygous allele 1, and to other genotypes, the PGC positive rate was gradually increased in gastric mucosa, with significant differences between homozygous allele 1 and other genotypes (P=0.009); while the strong-positive rate of PGC was gradually decreased only in SG group (P=0.047)., Conclusion: PGC gene insertion/deletion polymorphism is negatively related to PGC protein expression in gastric mucosa, but is not related to the serum PGC level.

Purpose: The diagnosis of prostate cancer in men with persistently increased prostate specific antigen after a negative prostate biopsy has become a great challenge for urologists and pathologists. We analyzed the diagnostic value of 6 genes in the tissue of patients with prostate cancer., Materials and Methods: The study was comprised of 50 patients with localized disease who underwent radical prostatectomy. Gene selection was based on a previous microarray analysis. Among 4,147 genes with different expressions between 2 pools of patients 6 genes (PSMA, TMEFF2, GREB1, TH1L, IgH3 and PGC) were selected. These genes were tested for diagnostic value using the quantitative reverse transcription polymerase chain reaction method. Initially malignant tissue samples from 33 patients were analyzed and in the second part of the study we analyzed benign tissue samples from the other 17 patients with prostate cancer. The control group was comprised of tissue samples of patients with benign prostatic hyperplasia., Results: Analysis of malignant prostatic tissue demonstrated that prostate specific membrane antigen was over expressed (mean 9 times) and pepsinogen C was under expressed (mean 1.3 x 10(-4) times) in all cases compared to benign prostatic hyperplasia. The other 4 tested genes showed a variable expression pattern not allowing for differentiation between benign and malignant cases. When we tested these results in the benign prostate tissues from patients with cancer, pepsinogen C maintained the expression pattern. In terms of prostate specific membrane antigen, despite over expression in most cases (mean 12 times), 2 cases (12%) presented with under expression., Conclusions: Pepsinogen C tissue expression may constitute a powerful adjunctive method to prostate biopsy in the diagnosis of prostate cancer cases.

Objective: This study was designed to investigate the interaction between pepsinogen C(PGC) insertion/deletion polymorphism and Helicobacter pylori(Hp) infection, together with its different subtype strains, in the development of gastric cancer (GC)., Methods: PGC Genotypes were determined by polymerase chain reaction (PCR) assay in 564 subjects with superficial gastritis (NOR), gastric ulcer (GU), atrophic gastritis (AG) and GC, who were frequency-matched as 1:1. Serum Hp-IgG antibodies were determined by an enzyme linked immunoadsorbent assay (ELISA). Hp genetic subtypes in 171 patients with Hp infection were determined by PCR methods., Results: In GU, AG and GC, the OR of interaction was 8.69 (P = 0.049), 11.16 (P = 0.02), and 10.61 (P = 0.03), respectively; the interaction index of PGC homozygous allele 1 genotype and Hp infection was 5.40, 6.48 or 4.34, respectively; the attributable proportions were 0.721, 0.770 and 0.697, respectively. In AG and GC, no significant interactions were observed between PGC polymorphism and Hp genetic subtypes., Conclusion: The findings of this study suggest that PGC insertion/deletion polymorphism and Hp infection seem to present a positive interaction in the development of gastric cancer. While no interactions may be present between PGC polymorphism and Hp genetic subtypes.

We aimed to study the role of an insertion/deletion polymorphism in the Pepsinogen C (PGC) gene in the clinical outcome of 172 breast cancer patients. The six polymorphic alleles were amplified using PCR. Our results indicate that patients carrying the allele 6 present a higher 5-year survival mean (83.4% of 6 allele carriers were alive at 5 years vs. only 68.6% of noncarriers, p=0.001), suggesting a role for this polymorphism in the outcome of breast cancer patients. We hypothesize that PGC polymorphism can be a predictive biomarker in breast cancer, contributing to an individual profile of great interest in clinical oncology.

One pepsinogen C gene was cloned from the gastric mucosa of orange-spotted grouper (Epinephelus coioides) for the first time. This gene consisted of nine exons interrupted by eight introns. The nucleotide sequence with the coding region and 3'-flanking region was also determined. The deduced polypeptide sequence was composed of a prosegment of 55 residues and a pepsin moiety of 332 residues. The putative substrate-binding subsites were well conserved among fish with the exception of the residue Thr292 in the S(2) subsite and displayed a significantly low value of hydropathy and high flexibility. These differences may affect the catalytic function and substrate specificity. RT-PCR assay followed by Southern blot revealed that pepsinogen C was primarily distributed in the stomach, but also expressed in various tissues, including gill, intestine, pyloric ceca, esophagus and ovary. This is the first observation of pepsinogen C expression in various tissues of one species of fish. Pepsinogen C transcript was first detected at 41 dph and continuously expressed through to adult fish, coinciding with the pepsin-like enzymes activity during developmental stages. Pepsin-like enzymes activity was present in the early larva stage, increased significantly at the end of juvenile stage and remained at similar levels in young fish and adult. Northern blot analysis suggested that three forms of transcripts were expressed differently during experimental periods. Our results suggest that pepsin C possesses any other functions besides digestion.

In this study the sea bass (Dicentrarchus labrax) pepsinogen C gene was isolated. The nucleotide sequences of all exons are presented. The organization of the gene is compatible with that of other aspartic proteinases. The predicted 388-residue amino acid (aa) sequence of sea bass pepsinogen C consists of a signal sequence of 16 amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two characteristic active-site aspartic acids. We also analyzed fasting-induced changes in the expression of progastricsin mRNA, using real-time RT-PCR absolute quantification. Progastricsin mRNA copy number was downregulated under conditions of negative energy balance, such as starvation, and upregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass progastricsin gene and support a role of this gastric digestive enzyme in the regulation of food intake in sea bass.

Purpose: Transcription factor SOX2 is expressed in normal gastric mucosae but not in the normal colon. We aimed to clarify the role of SOX2 with reference to pepsinogen expression in the gastrointestinal epithelium., Methods: We analyzed expression of SOX2 and pepsinogens, differentiation markers of the stomach, in ten gastric cancer (GC) and ten colorectal cancer (CRC) cell lines. The effects of over-expression and down-regulation of SOX2 on pepsinogen expression were also examined., Results: Six GC and five CRC cell lines showed SOX2 expression on RT-PCR. Expression of pepsinogen A was detectable in eight GC and seven CRC cell lines, whereas the majority of the cell lines expressed pepsinogen C. Over-expression of SOX2 up-regulated expression of pepsinogen A but not that of pepsinogen C in 293T human embryonic kidney cells, and some GC and CRC cell lines. Moreover, pepsinogen A expression was significantly reduced by SOX2 RNA interference in two GC cell lines., Conclusion: These data suggest that SOX2 plays an important role in regulation of pepsinogen A, and ectopic expression of SOX2 may be associated with abnormal differentiation of colorectal cancer cells.

l-glutamate not only confers cognitive discrimination for umami taste in the oral cavity, but also conveys sensory information to vagal afferent fibers in the gastric mucosa. We used RT-PCR, western blotting, and immunohistochemistry to demonstrate that mGluR1 is located in glandular stomach. Double staining revealed that mGluR1 is found at the apical membrane of chief cells and possibly in parietal cells. Moreover, a diet with 1% l-glutamate induced changes in the expression of pepsinogen C mRNA in stomach mucosa. These data suggest that mGluR1 is involved in the gastric phase regulation of protein digestion.

Objective: To assess the relationship between the insert-deletion polymorphism in the Human pepsinogen (PGC) gene and susceptibility to gastric cancer, together with its precursors, and to investigate the interaction between PGC polymorphism and H. pylori infection in the development of gastric cancer., Methods: PGC gene polymorphism in 141 patients with gastric cancer and 564 matched non-cancer controls were detected by polymerase chain reaction (PCR), and the relation between the PGC polymorphism and gastric cancer was examined. Serum H. pylori-IgG was determined with ELISA method. The odds ratios and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression model. The effect-modified model was used to evaluate the PGC-H. pylori interaction., Results: No significant association between the frequency of homogenous allele 1 of PGC gene and the sex and age of the subjects was observed. The subjects with the homogenous allele 1 had an increased risk of developing atrophic gastritis (OR: 3.103, 95% CI: 1.440-6.686), and gastric cancer (OR: 2.962, 95% CI: 1.370-6.404), and Intestinal metaplasia (OR: 1.659, 95% CI: 0.998-2.757) comparing with those with the non-homogenous allele 1. For subjects carrying both homogenous allele 1 and H. pylori-IgG positive, there was an significant increased risk of developing atrophic gastritis and gastric cancer (likelihood test ratio test: P = 0.023, P = 0.005)., Conclusion: PGC gene polymorphism may be associated with susceptibility to gastric cancer and atrophic gastritis. The PGC gene polymorphism and H. pylori infection seem to display an interaction in the development of gastric cancer.

Aim: To study the role of an insertion/deletion polymorphism in the pepsinogen C (PGC) gene, an effective marker for terminal differentiation of the stomach mucosa, in the susceptibility to the development of gastric lesions., Methods: The study was performed with 99 samples of known gastric lesions and 127 samples without evidence of neoplastic disease. PCR was employed and the 6 polymorphic alleles were amplified: Allele 1 (510 bp), Allele 2 (480 bp), Allele 3/4 (450/460 bp), Allele 5 (400 bp) and Allele 6 (310 bp)., Results: Our results revealed that Allele 6 carriers seemed to have protection against the development of any gastric lesion (OR = 0.34; P<0.001), non-dysplastic lesions associated with gastric adenocarcinoma such as atrophy or intestinal metaplasia (OR = 0.28; P<0.001) or invasive GC (OR = 0.39; P = 0.004)., Conclusion: Our study reveals that the Allele 6 carrier status has a protective role in the development of gastric lesions, probably due to its association with higher expression of PGC. Moreover, the frequency of Allele 6 carriers in the control group is far higher than that obtained in Asian populations, which might represent a genetic gap between Caucasian and Asian populations.

Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and real-time reverse transcription-PCR, revealing similar up-regulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkhead 6 (Foxl1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.

The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S- and 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.

Aim: To identify a molecular marker for gastric cancer, and to investigate the relationship between the polymorphism of pepsinogen C (PGC) gene and the genetic predisposition to gastric cancer., Methods: A total of 289 cases were involved in this study. 115 cases came from Shenyang area, a low risk area of gastric cancer, including 42 unrelated controls and 73 patients with gastric cancer. 174 cases came from Zhuanghe area, a high-risk area of gastric cancer, including 113 unrelated controls, and 61 cases from gastric cancer kindred families. The polymorphism of PGC gene was detected by polymerase chain reaction (PCR) and the relation between the genetic polymorphism of PGC and gastric cancer was examined., Results: Four alleles, 31 0bp (allele 1), 400 bp (allele 2), 450 bp (allele 3), and 480 bp (allele 4) were detected by PCR. The frequency of allele 1 was higher in patients with gastric cancer than that in controls. Genotypes containing homogenous allele 1 were significantly more frequent in patients with gastric cancer than that in controls (0.33, 0.14, chi(2)=3.86, P<0.05). There was no significant difference between the control group of Zhuanghe and the group of gastric cancer kindred. But the frequency of allele 1 was higher in control group of Zhuanghe area than that in control group of Shenyang area and genotypes containing homogenous allele 1 were significantly more frequent in the control group of Zhuanghe area than those in control group of Shenyang area (0.33, 0.14, chi(2)=4.32, P<0.05). In the group of gastric cancer kindred the frequency of allele 1 was significantly higher than that in control group of Shenyang area (0.5164, 0.3571, chi(2)=4.47, P<0.05). Genotypes containing homogenous allele 1 were significantly more frequent in the group of gastric cancer kindred than those in control group of Shenyang area (0.36, 0.14, chi(2)=4.91, P<0.05)., Conclusion: These results suggest that there is some relation between pepsinogen C gene polymorphism and gastric cancer, and the person with homogenous allele 1 predisposes to gastric cancer than those with other genotypes. Pepsinogen C gene polymorphism may be used as a genetic marker for a genetic predisposition to gastric cancer. The distribution of pepsinogen C gene polymorphism in Zhuanghe, a high-risk area of gastric cancer, is different from that in Shenyang, a low risk area of gastric cancer.

Pepsinogen C (PGC) is the inactive precursor of pepsin C, which is a member of the aspartic proteinase family of proteolytic enzymes, and is found within the vertebrate stomach. The precursor molecule is synthesised within the gastric epithelial cells and secreted into the gastric lumen where it undergoes an autocatalytic activation under acidic conditions to the proteolytic molecule. However, the synthesis of PGC has also been reported in numerous non-gastric tissues including the prostate gland and the Brunner's gland of the small intestine. The physiological significance of PGC in these tissues is not known and studies are limited by the lack of appropriate in vitro cell models. We report here the use of the rat intestinal cell line, IEC-6, as an in vitro model to study the role of pepsinogen C in the intestine. PGC expression was detected in the IEC-6 cells by RT-PCR and immunocytochemistry, using a PGC specific antibody, localised the protein to cytoplasmic secretory granules. Enzymatic assay confirmed the presence of a functional protein exhibiting pepsin activity. Using semi-quantitative RT-PCR we showed that PGC gene expression was up-regulated by the gastro-intestinal hormones gastrin and secretin, and forskolin which stimulates adenylate cyclase activity. This study demonstrates that the IEC-6 cells provide a unique in vitro model for studying pepsinogen C and its potential role(s) in the small intestine., (Copyright 2003 S. Karger AG, Basel)

Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudo-pepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S4 through S'3 subsites. Hydrophobic and aromatic amino acids are preferred at the P1 and P'1 positions. Interactions at additional subsites are important in some cases, for example with cleavage of kappa-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones.

Although to date the phylogenetic position of the provisional order Eulipotyphla has been assessed by various molecular markers, it has not been conclusively clarified due to low statistical supporting values and inconsistent results. To clarify the phylogenetic position of Eulipotyphla, we cloned cDNAs for pepsinogens A and C from five mammalian species belonging to four different orders and determined their nucleotide sequences. Molecular phylogenetic analysis based on the 1st and 2nd codon positions of the protein-coding region of cDNA sequences strongly supported the close relationship between Eulipotyphla and Chiroptera. Carnivora was found to be a sister group to these two orders. The monophyly of the order Rodentia and that of the cohort Glires (Rodentia and Lagomorpha) was also shown by the present phylogenetic trees of pepsinogens., (Copyright 2001 Academic Press.)

An up-regulated cDNA fragment was isolated using a differential display polymerase chain reaction between ovulatory and postovulatory brook trout ovarian tissues. Using this fragment as a probe, a full-length cDNA of 1783 base pairs was obtained from an ovarian cDNA library. The cDNA presumably codes for a 383-amino acid protein with strong sequence similarity to an aspartic protease, progastricsin (EC 3.4.23.3), also known as pepsinogen C. On Northern blots of ovarian tissue, the trout progastricsin cDNA hybridized with a 1.8-kilobase transcript that was strongly up-regulated 4-6 days after ovulation. Of all other tissues tested, a transcript was only detected in the stomach. A recombinant trout progastricsin protein was produced and used to raise an antibody. On Western blots of ovarian tissue, the progastricsin antibody recognized a single 39-kDa protein that was present in the ovary only following ovulation. On Western blots of coelomic fluid, the 39-kDa protein was strongly detected 4-10 days after ovulation. The trout progastricsin was immunocytochemically localized to the granulosa cells of postovulatory follicles, suggesting that it is released from this tissue into the coelomic fluid following ovulation. Progastricsin has been found in the stomach, prostate, seminal vesicle, seminal fluid, and pancreas of vertebrates; however, this is the first report of a progastricsin in an animal ovary.

Two pepsinogens (Pg C and Pg A) were isolated from the stomach of adult Xenopus laevis by Q-Sepharose, Sephadex G-75, and Mono-Q column chromatographies. Autolytic conversion and activation of the purified Pgs into the pepsins were examined by acid treatment. We determined the amino acid sequences from the NH2-termini of Pg C, pepsin C, Pg A, and pepsin A. Based on the sequences, the cDNAs for Pg C and Pg A were cloned from adult stomach RNA, and the complete amino acid sequences of the Pg C and Pg A were predicted. In addition, a Pg A cDNA was cloned from the stomach of adult bullfrog Rana catesbeiana, and the primary structure of the Pg A was predicted. Molecular phylogenetic analysis showed that such anuran Pg C and Pg A belong to the Pg C group and the Pg A group in vertebrates, respectively. The molecular properties of Pg C and Pg A, such as size, sequences of the activation peptide and active site, profile of autolytic activation, and pH dependency of proteolytic activity of the activated forms, pepsin C and pepsin A, resemble those of Pgs found in other vertebrates. However, the hemoglobin-hydrolyzing activity of Xenopus pepsin C is completely inhibited in the presence of equimolar pepstatin, an inhibitor of aspartic proteinases. Thus, the Xenopus pepsin C differs significantly from other vertebrate pepsins C in its high susceptibility to pepstatin, and closely resembles A-type pepsins.

Pepsinogens A and C, and prochymosin were purified from four species of adult New World monkeys, namely, common marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), squirrel monkey (Saimiri sciureus), and capuchin monkey (Cebus apella). The occurrence of prochymosin was quite unique since this zymogen is known to be neonate-specific and, in primates, it has been thought that the prochymosin gene is not functional. No multiple form has been detected for any type of pepsinogen except that two pepsinogen-A isozymogens were identified in capuchin monkey. Pepsins A and C, and chymosin hydrolyzed hemoglobin optimally at pH 2-2.5 with maximal activities of about 20, 30, and 15 units/mg protein. Pepsins A were inhibited in the presence of an equimolar amount of pepstatin, and chymosins and pepsins C needed 5- and 100-fold molar excesses of pepstatin for complete inhibition, respectively. Hydrolysis of insulin B chain occurred first at the Leu15-Tyr16 bond in the case of pepsins A and chymosins, and at either the Leu15-Tyr16 or Tyr16-Leu17 bond in the case of pepsins C. The presence of different types of pepsins might be advantageous to New World monkeys for the efficient digestion of a variety of foods. Molecular cloning of cDNAs for three types of pepsinogens from common marmoset was achieved. A phylogenetic tree of pepsinogens based on the nucleotide sequence showed that common marmoset diverged from the ancestral primate about 40 million years ago.

Background: Peptic ulcer has multifactorial aetiology, including genetic factors. We have identified a family with pepsinogen Group A levels higher than normal, with a high prevalence of ulcer disease and a low prevalence of Helicobacter pylori infection., Aims: Performing linkage analysis in the identified family, Patients and Methods: We examined the segregation of pepsinogens with microsatellite dinucleotide repeat DNA markers along chromosome 11 (D11S480, PYGM) for pepsinogen Group A and along chromosome 6 [D6S105, D6S 1610, TRMI) for pepsinogen Group C., Results: In markers examined along chromosome 11, linkage analysis provided no evidence for significant causal mutation but, controlling for some risk factors we observed that the probability of falling ill, increases. The linkage analysis along chromosome 6 for pepsinogen Group C did not show a uniform genetic profile., Conclusions: This study evaluates the hypothesis of peptic ulcer inheritance at least in a small group of patients without the common risk factors.