Comparison of genomic and transcriptional microbiome analysis in gastric cancer patients and healthy individuals

BACKGROUND Helicobacter pylori and the stomach microbiome play a crucial role in gastric carcinogenesis,and detailed characterization of the microbiome is necessary for a better understanding of the pathophysiology of the disease.There are two common modalities for microbiome analysis:DNA(16S rRNA gene)and RNA(16S rRNA transcript)sequencing.The implications from the use of one or another sequencing approach on the characterization and comparability of the mucosal microbiome in gastric cancer(GC)are poorly studied.AIM To characterize the microbiota of GC using 16S rRNA gene and its transcript and determine difference in the bacterial composition.METHODS In this study,316 DNA and RNA samples extracted from 105 individual stomach biopsies were included.The study cohort consisted of 29 healthy control individuals and 76 patients with GC.Gastric tissue biopsy samples were collected from damaged mucosa and healthy mucosa at least 5 cm from the tumor tissue.From the controls,healthy stomach mucosa biopsies were collected.From all biopsies RNA and DNA were extracted.RNA was reverse transcribed into cDNA.V1-V2 region of bacterial 16S rRNA gene from all samples were amplified and sequenced on an Illumina MiSeq platform.Bray-Curtis algorithm was used to construct sample-similarity matrices abundances of taxonomic ranks in each sample type.For significant differences between groups permutational multivariate analysis of variance and Mann-Whitney test followed by false-discovery rate test were used.RESULTS Microbial analysis revealed that only a portion of phylotypes(18%-30%)overlapped between microbial profiles obtained from DNA and RNA samples.Detailed analysis revealed differences between GC and controls depending on the chosen modality,identifying 17 genera at the DNA level and 27 genera at the RNA level.Ten of those bacteria were found to be different from the control group at both levels.The key taxa showed congruent results in various tests used;however,differences in 7 bacteria taxa were found uniquely only at the DNA level,and 17 uniquely only at the RNA level.Furthermore,RNA sequencing was more sensitive for detecting differences in bacterial richness,as well as differences in the relative abundance of Reyranella and Sediminibacterium according to the type of GC.In each study group(control,tumor,and tumor adjacent)were found differences between DNA and RNA bacterial profiles.CONCLUSION Comprehensive microbial study provides evidence for the effect of choice of sequencing modality on the microbiota profile,as well as on the identified differences between case and control.

Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

AIM:To investigate the role of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium(DSS)-induced colitis.METHODS:Balb/c mice were divided into three groups:8 mice with acute DSS-induced colitis(3.5% DSS solution;7 d),8 mice with chronic DSS-induced colitis(3.5% DSS solution for 5 d + water for 6 d;4 cycles;total:44 d) and 12 mice without DSS supplementation as a control group.Primary colonic epithelial cells were isolated using chelation method.The cells were cultivated in the presence of mediators(lipopolysaccharide(LPS),apocynin or diphenyleneiodonium).Viability of cells was assessed by fluorescent microscopy.Production of reactive oxygen species(ROS) by the cells was measured fluorometrically using Amplex Red.Production of tumour necrosis factor-alpha(TNF-α) by the colonic epithelial cells was analysed by ELISA.Nox1 gene expression was assessed by real-time PCR.RESULTS:Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups,whereas decreased viability,increased ROS production,and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls.The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis.Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.CONCLUSION:Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.

Atrophic gastritis and gastric cancer tissue miRNome analysis reveals hsa-miR-129-1 and hsa-miR-196a as potential early diagnostic biomarkers

BACKGROUND Gastric cancer(GC)is one of the most frequently diagnosed tumor globally.In most cases,GC develops in a stepwise manner from chronic gastritis or atrophic gastritis(AG)to cancer.One of the major issues in clinical settings of GC is diagnosis at advanced disease stages resulting in poor prognosis.Micro RNAs(mi RNAs)are small noncoding molecules that play an essential role in a variety of fundamental biological processes.However,clinical potential of mi RNA profiling in the gastric cancerogenesis,especially in premalignant GC cases,remains unclear.AIM To evaluate the AG and GC tissue mi RNomes and identify specific mi RNAs’potential for clinical applications(e.g.,non-invasive diagnostics).METHODS Study included a total of 125 subjects:Controls(CON),AG,and GC patients.All study subjects were recruited at the Departments of Surgery or Gastroenterology,Hospital of Lithuanian University of Health Sciences and divided into the profiling(n=60)and validation(n=65)cohorts.Total RNA isolated from tissue samples was used for preparation of small RNA sequencing libraries and profiled using next-generation sequencing(NGS).Based on NGS data,deregulated mi RNAs hsa-mi R-129-1-3 p and hsa-mi R-196 a-5 p were analyzed in plasma samples of independent cohort consisting of CON,AG,and GC patients.Expression level of hsa-mi R-129-1-3 p and hsa-mi R-196 a-5 p was determined using the quantitative real-time polymerase chain reaction and 2-ΔΔCt method.RESULTS Results of tissue analysis revealed 20 differentially expressed mi RNAs in AG group compared to CON group,129 deregulated mi RNAs in GC compared to CON,and 99 altered mi RNAs comparing GC and AG groups.Only 2 mi RNAs(hsa-mi R-129-1-3 p and hsa-mi R-196 a-5 p)were identified to be step-wise deregulated in healthy-premalignant-malignant sequence.Area under the curve(AUC)-receiver operating characteristic analysis revealed that expression level of hsa-mi R-196 a-5 p is significant for discrimination of CON vs AG,CON vs GC and AG vs GC and resulted in AUCs:88.0%,93.1%and 66.3%,respectively.Comparing results in tissue and plasma samples,hsa-mi R-129-1-3 p was significantly down-regulated in GC compared to AG(P=0.0021 and P=0.024,tissue and plasma,respectively).Moreover,analysis revealed that hsa-mi R-215-3 p/5 p and hsa-mi R-934 were significantly deregulated in GC based on Helicobacter pylori(H.pylori)infection status[log2 fold change(FC)=-4.52,P-adjusted=0.02;log2 FC=-4.00,P-adjusted=0.02;log2 FC=6.09,P-adjusted=0.02,respectively].CONCLUSION Comprehensive mi RNome study provides evidence for gradual deregulation of hsa-mi R-196 a-5 p and hsa-mi R-129-1-3 p in gastric carcinogenesis and found hsami R-215-3 p/5 p and hsa-mi R-934 to be significantly deregulated in H.pylori carrying GC patients.

Polymorphisms of micro RNA target genes IL12B, INSR, CCND1 and IL10 in gastric cancer

AIM To evaluate associations between mi RNA target genes IL12B,INSR,CCND1 and IL10 polymorphisms and gastric cancer(GC)in European population.METHODS Gene polymorphisms were analyzed in 508 controls and474 GC patients from 3 tertiary centers in Germany,Lithuania and Latvia.Controls were patients from the out-patient departments,who were referred for upper endoscopy because of dyspeptic symptoms and had no history of previous malignancy.Gastric cancer(GC)patients had histopathological verification of gastric adenocarcinoma.Genomic DNA was extracted using salting out method from peripheral blood mononuclear cells.IL12B T>G(rs1368439),INSR T>C(rs1051690),CCND1 A>C(rs7177)and IL10 T>C(rs3024498)SNPs were genotyped by the real-time polymerase chain reaction.Associations between gene polymorphism and GC were evaluated using multiple logistic regression analysis with adjustment for sex,age and country of birth.RESULTS We observed similar distribution of genotypes and allelic frequencies of all polymorphisms between GC patients and controls except of INSR rs1051690.The frequency of the T allele of INSR gene was significantly higher in GC patients than in controls(23.26%and 19.19%respectively,P=0.028).CT genotype was also more prevalent in patients compared to control group(38.48%and 30.12%respectively,P<0.021).Logistic regression analysis revealed that only one polymorphism(rs1051690 in INSR gene)was associated with increased risk of GC.Carriers of CT genotype had higher odds of GC when compared to CC genotype(OR=1.45,95%PI:1.08-1.95,P=0.01).Similar association was observed in a dominant model for INSR gene,where comparison of TT+CT vs CC genotypes showed an increased risk of GC(OR=1.44,95%PI:1.08-1.90,P=0.01).Other analyzed SNPs were not associated with the presence of GC.CONCLUSION INSR rs1051690 SNP is associated with increased risk of GC,while polymorphisms in IL12B,CCND1 and IL10genes are not linked with the presence of GC.

Plasma Nogo-A and placental growth factor levels are associated with portal hypertension in patients with liver cirrhosis

BACKGROUND Clinically significant portal hypertension(CSPH) and severe portal hypertension(SPH) increase the risk for decompensation and life-threatening complications in liver cirrhosis. Pathologic angiogenesis might contribute to the formation of these conditions. Placental growth factor(PlGF) and Nogo-A protein are biomarkers of pathological angiogenesis, but data on their role in liver cirrhosis and portal hypertension is scarce.AIM To determine plasma levels of PlGF and Nogo-A in patients with liver cirrhosis,CSPH, SPH and potential to predict portal hypertension.METHODS A cohort of 122 patients with hepatitis C virus and/or alcohol-induced liver cirrhosis with characterized hepatic venous pressure gradient(HVPG) were included in the study. Demographic data, medical history, Child-Turcotte-Pugh and Model of End Stage liver disease score, clinical chemistry, liver stiffnessvalues were recorded on the day of the procedure prior HVPG measurement. The degree of portal hypertension was determined by the invasive HVPG measurement. Nogo-A and PlGF plasma levels were evaluated using enzyme linked immunosorbent assay. The control group consisted of 30 healthy age-and sex-matched individuals.RESULTS Peripheral PlGF levels were higher and Nogo-A levels were lower in patients with liver cirrhosis(23.20 vs 9.85;P < 0.0001 and 2.19 vs 3.12;P = 0.004 respectively). There was a positive linear correlation between peripheral levels of PlGF and HVPG(r = 0.338, P = 0.001) and negative linear correlation between the peripheral Nogo-A levels and HVPG(r =-0.267, P = 0.007). PlGF levels were higher in CSPH and SPH(P = 0.006;P < 0.0001) whereas Nogo-A levels were lower(P = 0.01;P < 0.033). Area under the curve for the diagnosis of CSPH for PlGF was 0.68(P = 0.003) and for Nogo-A-0.67(P = 0.01);for SPH 0.714(P <0.0001) and 0.65(P = 0.014) respectively. PlGF levels were higher and Nogo-A levels were lower in patients with esophageal varices(P < 0.05). PlGF cut-off value of 25 pg/mL distinguished patients with CSPH at 55.7% sensitivity and76.7% specificity;whereas Nogo-A cut-off value of 1.12 ng/mL was highly specific(93.1%) for the diagnosis of CSPH.CONCLUSION Plasma PlGF levels were higher while Nogo-A levels were lower in patients with liver cirrhosis and portal hypertension. Biomarkers showed moderate predictive value in determining CSPH and SPH.