BackgroundTo investigate the potential of serum miRNAs as biomarkers for early detection of gastric cancer (GC), a population-based study was conducted in Linqu, a high-risk area of GC in China.Methodology/Principal FindingsAll subjects were selected from two large cohort studies. Differential miRNAs were identified in serum pools of GC and control using TaqMan low density array, and validated in individual from 82 pairs of GC and control, and 46 pairs of dysplasia and control by real-time quantitative reverse transcription-polymerase chain reaction. The temporal trends of identified serum miRNA expression were further explored in a retrospective study on 58 GC patients who had at least one pre-GC diagnosis serum sample based on the long-term follow-up population. The miRNA profiling results demonstrated that 16 miRNAs were markedly upregulated in GC patients compared to controls. Further validation identified a panel of three serum miRNAs (miR-221, miR-744, and miR-376c) as potential biomarkers for GC detection, and receiver operating characteristic (ROC) curve-based risk assessment analysis revealed that this panel could distinguish GCs from controls with 82.4% sensitivity and 58.8% specificity. MiR-221 and miR-376c demonstrated significantly positive correlation with poor differentiation of GC, and miR-221 displayed higher level in dysplasia than in control. Furthermore, the retrospective study revealed an increasing trend of these three miRNA levels during GC development (P for trend<0.05), and this panel could classify serum samples collected up to 5 years ahead of clinical GC diagnosis with 79.3% overall accuracy.Conclusions/SignificanceThese data suggest that serum miR-221, miR-376c and miR-744 have strong potential as novel non-invasive biomarkers for early detection of GC.

Background: Genetic polymorphisms of Toll-like receptors (TLR) may influence the outcome of Helicobacter pylori infection and play important roles in gastric carcinogenesis. To screen the genetic variants of TLR2 and TLR5, and evaluate their associations with gastric cancer (GC) and its precursors, a population-based study was conducted in Linqu County, Shandong Province, China.Methods: Genetic variants were identified by PCR-based denaturing high-performance liquid chromatography and PCR-restriction fragment length polymorphism analysis in 248 GC cases, 846 subjects with advanced gastric lesions including 350 dysplasia and 496 intestinal metaplasia, and 496 superficial gastritis/mild chronic atrophic gastritis controls.Results: Nine allelic variants each were detected within the promoter and exons of TLR2 and TLR5. Among those, TLR2 c. À196 to À174 del carriers (ins/delþdel/del) showed a significantly decreased risk of GC (adjusted OR, 0.66; 95% CI: 0.48-0.90), whereas TLR5 rs5744174 C carriers (TCþCC) had an increased risk of GC (OR, 1.43; 95% CI: 1.03-1.97). Further analysis indicated an elevated risk of GC in subjects with the TLR5 rs5744174 TCþCC genotype and H. pylori infection (OR, 3.35; 95% CI: 2.13-5.26), and a significant interaction between rs5744174 and H. pylori infection was observed (OR, 2.15; 95% CI: 1.12-4.16).Conclusion: These findings suggest that TLR2 c. À196 to À174 ins>del, TLR5 rs5744174 and interaction between rs5744174 and H. pylori infection were associated with the development of GC.Impact: TLR2 and TLR5 polymorphisms may play important roles in the process of H. pylori-related gastric carcinogenesis. Cancer Epidemiol Biomarkers Prev; 20(12); 2594-602. Ó2011 AACR.

BackgroundMicroRNAs (miRNAs) have been implicated in various human diseases. Single nucleotide polymorphisms (SNPs) in inflammation-related miRNA may play an important role in Helicobacter pylori (H. pylori)-induced gastric lesions. To evaluate the associations between miRNA SNPs, H. pylori and gastric lesions, a population-based study was conducted in Linqu County, China.Methodology/Principal FindingsBased on serum miRNA array conducted in this population, two SNP loci (miR-146a rs2910164: G>C and miR-27a rs895819: T>C) were determined by polymerase chain reaction-restriction fragment length polymorphism in 2,380 participants with diverse gastric lesions. Using participants with superficial gastritis and mild chronic atrophic gastritis as the reference group, we found that rs2910164 CC carriers had a significantly increased risk of intestinal metaplasia [adjusted odds ratio (OR), 1.42; 95% confidence interval (CI), 1.03–1.97] and dysplasia (OR, 1.54; 95% CI, 1.05–2.25) compared to GG carriers, whereas no significant association was observed for rs895819. Stratified analysis by H. pylori infection indicated that rs2910164 C allele was associated with an increased risk of intestinal metaplasia and dysplasia only among individuals infected with H. pylori (CC vs. GG: OR, 1.53; 95% CI, 1.12–2.08, P for trend = 0.004). Participants who simultaneously carried variant alleles and H. pylori infection were more likely to develop intestinal metaplasia and dysplasia, although the interaction between genetic variants and H. pylori infection was not significant (P for interaction = 0.35 for rs2910164 and 0.92 for rs895819).Conclusions/SignificanceThese findings suggest that miR-146a rs2910164 polymorphism may contribute to the evolution of H. pylori-associated gastric lesions in this high-risk population.

ObjectiveTo identify serum biomarkers that may predict the short or long term outcomes of anti-Helicobacter pylori (H. pylori) treatment, a follow-up study was performed based on an intervention trial in Linqu County, China. MethodsA total of 529 subjects were selected randomly from 1,803 participants to evaluate total anti-H. pylori immunoglobulin G (IgG) and 10 specific antibody levels before and after treatment at 1-, 2- and 7.3-year. The outcomes of anti-H. pylori treatment were also parallelly assessed by13C-urea breath test at 45-d after treatment and 7.3-year at the end of follow-up. ResultsWe found the medians of anti-H. pylori IgG titers were consistently below cut-off value through 7.3 years in eradicated group, however, the medians declined in recurrence group to 1.2 at 1-year after treatment and slightly increased to 2.0 at 7.3-year. While the medians were significantly higher (>3.0 at 2- and 7.3-year) among subjects who failed the eradication or received placebo. For specific antibody responses, baseline seropositivities of FliD and HpaA were reversely associated with eradication failure [for FliD, odds ratio (OR)=0.44, 95% confidence interval (95% CI): 0.27–0.73; for HpaA, OR=0.32, 95% CI: 0.17–0.60]. The subjects with multiple positive specific antibodies at baseline were more likely to be successfully eradicated in a linear fashion (Ptrend=0.006). ConclusionsOur study suggested that total anti-H. pylori IgG level may serve as a potential monitor of long-term impact on anti-H. pylori treatment, and priority forH. pylori treatment may be endowed to the subjects with multiple seropositive antibodies at baseline, especially for FliD and HapA.

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.

Background: To evaluate the relationship between methylation status of blood leukocyte DNA and risk of gastric cancer, a population-based study was conducted in Linqu County.Methods: Methylation levels of IGFII and N33 were determined by quantitative methylation-specific PCR. The temporal trend of methylation levels during gastric cancer development was investigated in 133 gastric cancer cases from two cohorts with pre-and/or post-gastric cancer samples. As the references of pre-GCs, 204 intestinal metaplasia (IM) or dysplasia (DYS) subjects who did not progress to gastric cancer during the followup period were selected. Meanwhile, 285 subjects with superficial gastritis/chronic atrophic gastritis (SG/ CAG) were also selected as controls.Results: IGFII median methylation level was significantly higher in gastric cancer cases than those with SG/CAG (61.47% vs. 49.73%; P < 0.001). IGFII and N33 methylation levels were elevated at least 5 years ahead of clinical gastric cancer diagnosis comparing with SG/CAG (63.38% vs. 49.73% for IGFII, 9.12% vs. 5.70% for N33; all P < 0.001). Furthermore, the frequency of hypermethylated IGFII was markedly increased in IM or DYS subjects who progressed to gastric cancer in contrast to those who remained with IM and DYS, and adjusted ORs were 12.52 [95% confidence interval (CI), 3.81-41.15] for IM and 10.12 (95% CI, 2.68-38.22) for DYS. Similar results were also found for N33 in subjects with IM (OR, 3.77; 95% CI,.Conclusions: Our findings suggested that hypermethylated IGFII and N33 in blood leukocyte DNA were associated with risk of gastric cancer in a Chinese population.Impact: IGFII and N33 methylation status may be related to gastric carcinogenesis. Cancer Epidemiol Biomarkers Prev; 23(10); 2019-26. Ó2014 AACR.

BackgroundMethylation is a common epigenetic modification which may play a crucial role in cancer development. To investigate the association between methylation of COX-2 in blood leukocyte DNA and risk of gastric cancer (GC), a nested case–control study was conducted in Linqu County, Shandong Province, a high risk area of GC in China.MethodsAssociation between blood leukocyte DNA methylation of COX-2 and risk of GC was investigated in 133 GCs and 285 superficial gastritis (SG)/ chronic atrophic gastritis (CAG). The temporal trend of COX-2 methylation level during GC development was further explored in 74 pre-GC and 95 post-GC samples (including 31 cases with both pre- and post-GC samples). In addition, the association of DNA methylation and risk of progression to GC was evaluated in 74 pre-GC samples and their relevant intestinal metaplasia (IM)/dysplasia (DYS) controls. Methylation level was determined by quantitative methylation-specific PCR (QMSP). Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated by unconditional logistic regression analysis.ResultsThe medians of COX-2 methylation levels were 2.3 % and 2.2 % in GC cases and controls, respectively. No significant association was found between COX-2 methylation and risk of GC (OR, 1.15; 95 % CI: 0.70-1.88). However, the temporal trend analysis showed that COX-2 methylation levels were elevated at 1–4 years ahead of clinical GC diagnosis compared with the year of GC diagnosis (3.0 % vs. 2.2 %, p = 0.01). Further validation in 31 GCs with both pre- and post-GC samples indicated that COX-2 methylation levels were significantly decreased at the year of GC diagnosis compared with pre-GC samples (1.5 % vs. 2.5 %, p = 0.02). No significant association between COX-2 methylation and risk of progression to GC was found in subjects with IM (OR, 0.50; 95 % CI: 0.18–1.42) or DYS (OR, 0.70; 95 % CI: 0.23–2.18). Additionally, we found that elder people had increased risk of COX-2 hypermethylation (OR, 1.55; 95 % CI: 1.02–2.36) and subjects who ever infected with H. pylori had decreased risk of COX-2 hypermethylation (OR, 0.54; 95 % CI: 0.34–0.88).ConclusionsCOX-2 methylation exists in blood leukocyte DNA but at a low level. COX-2 methylation levels in blood leukocyte DNA may change during GC development.

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