Prolonged culturing of colonic epithelial organoids derived from healthy individuals and ulcerative colitis patients results in the decrease of LINE-1 methylation level.

Patient-derived human intestinal organoids are becoming an indispensable tool for the research of digestive system in health and disease. However, very little is still known about the long-term culturing effect on global genomic methylation level in colonic epithelial organoids derived from healthy individuals as well as active and quiescent ulcerative colitis (UC) patients. In this study, we aimed to evaluate the epigenetic stability of these organoids by assessing the methylation level of LINE-1 during prolonged culturing. We found that LINE-1 region of both healthy control and UC patient colon tissues as well as corresponding epithelial organoids is highly methylated (exceeding 60%). We also showed that long-term culturing of colonic epithelial organoids generated from stem cells of healthy and diseased (both active and quiescent UC) individuals results in decrease of LINE-1 (up to 8%) methylation level, when compared to tissue of origin and short-term cultures. Moreover, we revealed that LINE-1 methylation level in sub-cultured organoids decreases at different pace depending on the patient diagnosis (healthy control, active or quiescent UC). Therefore, we propose LINE-1 as a potential and convenient biomarker for reliable assessment of global methylation status of patient-derived intestinal epithelial organoids in routine testing of ex vivo cultures.

The Translational Impact of Plant-Derived Xeno-miRNA miR-168 in Gastrointestinal Cancers and Preneoplastic Conditions.

INTRODUCTION: Diet is one of the most important factors contributing to the multistep process of carcinogenesis. The clinical relevance of exogenous food-derived xeno-microRNAs (miRNAs) in human diseases is poorly understood. In this study, we aimed to evaluate the potential clinical relevance of the xeno-miRNA miR-168 in the gastric mucosa along the preneoplastic conditions and gastric carcinogenesis. METHODS: For a systematic analysis, we included stomach tissues from patients with different pathologies, including normal mucosa (N), chronic non-atrophic (CNAG) and atrophic gastritis (CAG) and intestinal metaplasia (IM) (n = 72), matched non-tumorous (NT) and tumorous (T) gastric cancer (GC) tissues (n = 81), matched colorectal cancer (CRC) tissues (n = 40), and colon mucosa and faeces from controls and IBD patients. RESULTS: miR-168 was reproducibly detectable in all samples studied, with the highest levels in the proximal upper GI and in non-tumorous compared to tumorous tissues in both GC and CRC. There was no difference related to H. pylori positivity or inflammation grade, while higher miR-168 levels were observed in patients with moderate or severe AG/IM or OLGIM3/4. Survival analysis showed only a small, non-significant trend towards worse overall survival for patients with the highest to lowest miR-168 levels, while no differences were related to Lauren's classification. CONCLUSIONS: Food-derived xeno miRNAs are reproducibly detectable in the gastric and colonic mucosa. Although the clinically relevant function remains to be elucidated, higher levels of miR-168 in patients with moderate and severe IM merit further investigation.

Microbial composition of tumorous and adjacent gastric tissue is associated with prognosis of gastric cancer.

Helicobacter pylori (H. pylori) infection has been considered as the main causal factor in gastric carcinogenesis, but other bacterial species may also play an important role in pathophysiology of gastric cancer. The aim of the study was to explore the link between gastric cancer prognosis and the mucosal microbial community in tumorous and adjacent gastric tissue. The bacterial profile was analysed using 16S sequencing (V1-V2 region). Microbial differences were mostly characterized by lower relative abundances of H. pylori in tumorous gastric tissues. Bacterial community and outcome data analysis revealed the genus Fusobacterium and Prevotella significantly associated with worse overall survival in gastric cancer patients. In particular, Fusobacterium was associated with significant increase in hazard ratio in both univariable and multivariable analysis and independently validated using TCMA data. Phylogenetic biodiversity of Fusobacterium species in the stomach revealed F. periodonticum as the most prevalent in healthy subjects, while F. nucleatum was most abundant in patients with gastric cancer. Bacterial community network analysis in gastric cancer suggests substantial complexity and a strong interplay between F. nucleatum and Prevotella. In summary, mucosal microbial community in the stomach was associated with worse overall survival in gastric cancer patients. Strongest negative impact on prognosis was linked to the abundance of F. nucleatum in tumorous specimens, suggesting its translational relevance in management of gastric cancer patients.

Differential Expression of Long Noncoding RNA HOTAIR in Intestinal Metaplasia and Gastric Cancer.

INTRODUCTION: High expression of HOTAIR promotes tumor growth and carries a dismal prognosis for the patient. We investigated the prognostic value of HOTAIR expression in gastric cancer (GC) and systematically delineate the expression in relation to Helicobacter pylori infection and preneoplastic changes. METHODS: HOTAIR expression was analyzed in surgical paired tissue samples of patients with GC and biopsy samples from patients with atrophic gastritis and/or intestinal metaplasia (AG ± -IM), chronic nonatrophic gastritis, and controls. The cancer genome atlas (TCGA) data were used for validation. HOTAIR expression was evaluated in sera and ascites of patients with GC. Quantitative HOTAIR expression analysis was performed using quantitative polymerase chain reaction, and LINE-1 methylation was assessed by bisulfite pyrosequencing. RESULTS: HOTAIR was more frequently detected in tumor tissues compared with adjacent gastric mucosa (65.4% vs 8.6%). HOTAIR expression was associated with depth of tumor invasion and tumor location and with shorter overall survival in patients with diffuse-type GC as confirmed in the TCGA cohort. HOTAIR was not detectable in controls but was found in 2.2% of patients with chronic nonatrophic gastritis and 18.3% of patients with AG ± IM, which was further associated with IM, grade of IM, and H. pylori positivity. DISCUSSION: HOTAIR expression was associated with GC and preneoplastic changes of stomach mucosa. Although HOTAIR expression was strongly linked to IM, HOTAIR expression was only associated with worse prognosis in Lauren diffuse and not intestinal type of GC. Further studies are needed to evaluate the value of HOTAIR as diagnostic and predictive biomarker in IM and translational therapeutic relevance of HOTAIR in diffuse-type GC.

Fusobacterium nucleatum is associated with worse prognosis in Lauren's diffuse type gastric cancer patients.

Fusobacterium nucleatum (F. nucleatum) is frequently detected in primary colorectal cancer (CRC) and matching metastasis, and has been linked to a worse prognosis. We investigated the presence of F. nucleatum in gastric cancer (GC) and gastric preneoplastic conditions of the stomach, and its potential prognostic value in GC patients. Fusobacterium spp. and F. nucleatum were quantified in various specimens from gastrointestinal tract including paired CRC and GC tissues using probe-based qPCR. Fusobacterium spp. and F. nucleatum were more frequently found in tumorous tissue of CRC and GC compared to non-tumorous tissues. The frequency and bacterial load were higher in CRC compared to GC patients. F. nucleatum positivity showed no association to chronic gastritis or preneoplastic conditions such as intestinal metaplasia. F. nucleatum-positivity was associated with significantly worse overall survival in patients with Lauren's diffuse type, but not with intestinal type GC. There was no association with gender, Helicobacter pylori-status, tumor stage or tumor localization. However, F. nucleatum was positively associated with patient's age and a trend for a lower global long interspersed element-1 DNA methylation. In conclusion, our work provides novel evidence for clinical relevance of F. nucleatum in GC by showing an association between F. nucleatum positivity with worse prognosis of patients with Laurens's diffuse type gastric cancer. Further studies are necessary to explore related mechanistic insights and potential therapeutic benefit of targeted antibiotic treatment in GC patients.

miR-20b and miR-451a Are Involved in Gastric Carcinogenesis through the PI3K/AKT/mTOR Signaling Pathway: Data from Gastric Cancer Patients, Cell Lines and Ins-Gas Mouse Model.

Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP, and miR-451a: CAV1 and TSC1. Loss-of-function experiments revealed that down-regulation of miR-20b and up-regulation of miR-451a expression exhibits an anti-tumor effect in vitro (miR-20b: reduced viability, colony formation, increased apoptosis rate, and miR-451a: reduced colony forming ability). To summarize, the present study identified that expression of miR-20b and miR-451a are deregulated in vitro and in vivo and have a tumor suppressive role in GC through regulation of the PI3K/AKT/mTOR signaling pathway.

Food-Derived Xeno-microRNAs: Influence of Diet and Detectability in Gastrointestinal Tract-Proof-of-Principle Study.

SCOPE: Diet is amongst the most crucial factors contributing to the multistep process of carcinogenesis. The role of exogenous microRNAs (miRNAs) is still debatable. In this proof-of-principle work, the presence of miRNAs in a variety of foods, its stability to processing, and detectability in GI mucosa and feces are studied and the effect of short-term diet on human- or plant-derived miRNAs in feces and blood is examined. METHODS AND RESULTS: Animal and plant miRNAs are detected in all foods irrespective of processing. Animal-derived foods showed the highest miRNA level and the lowest is found in cheese and milk. The impact of the short-term vegetarian or meat-rich diet on blood and feces miRNA is evaluated in healthy subjects using qPCR and Affymetrix profiling. Diet is not associated with changes in ultraconserved miRNAs. However, a vegetarian diet is associated with an increase of miR-168 in feces but not in blood. Overall, plant miR-168 is detectable in normal GI mucosa and in colorectal cancer. CONCLUSIONS: Food provides a great source of miRNAs and diet may be associated with changes in xenomiRs. Plant-derived miR-168 is ubiquitously present in feces, normal mucosa, and cancer. Further studies are needed to evaluate the functional interaction between diet-derived miRNAs and GI tract.

LINE-1 hypomethylation is not a common event in preneoplastic stages of gastric carcinogenesis.

LINE-1 hypomethylation is widely accepted as marker for global genomic DNA hypomethylation, which is a frequent event in cancer. The aim of the study was to evaluate LINE-1 methylation status at different stages of gastric carcinogenesis and evaluate its prognostic potential in clinical settings. LINE-1 methylation was analyzed in 267 tissue samples by bisulfite pyrosequencing including primary colorectal cancer tissues (T-CRC) with corresponding adjacent colon mucosa (N-CRC), gastric cancer tissues (T-GC) with corresponding gastric mucosa (N-GC), normal gastric tissues (N), chronic non-atrophic and atrophic gastritis (CG). LINE-1 methylation level was lower in both T-GC and T-CRC when compared to paired adjacent tissues. No difference was observed for LINE-1 methylation status between patients with normal gastric mucosa, CG and N-GC. LINE-1 methylation in T-GC but not N-GC tended to correlate with age. Subgroup stratification analysis did not reveal significant differences in LINE-1 methylation status according to tumor stage, anatomical location, histological subtype, differentiation grade. We observed similar overall survival data between patients with high or low LINE-1 levels. In summary, LINE-1 hypomethylation is a characteristic feature in GC but not very common in early preneoplastic stages of gastric carcinogenesis. Prognostic role of LINE-1 hypomethylation in GC patients could not be confirmed in this cohort.

Polymorphisms of microRNA target genes IL12B, INSR, CCND1 and IL10 in gastric cancer.

AIM: To evaluate associations between miRNA target genes IL12B, INSR, CCND1 and IL10 polymorphisms and gastric cancer (GC) in European population. METHODS: Gene polymorphisms were analyzed in 508 controls and 474 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 IL10 genes are not linked with the presence of GC.

MiRNA profiling of gastrointestinal stromal tumors by next-generation sequencing.

Deregulation of miRNAs has been observed virtually in all major types of cancer, whereas the miRNA signature in GIST is not well characterized yet. In this study the first high-throughput miRNA profiling of 15 paired GIST and adjacent normal tissue samples was performed using small RNA-seq approach and differentially expressed miRNAs as well as isomiRNAs were defined. Highly significantly deregulated miRNAs were selected for validation by Taq-Man low-density array in replication group of 40 paired samples. Validated miRNAs were further subjected to enrichment analysis, which revealed significantly enriched KEGG pathways in the main GIST associated pathways. Further, we used an integrated analysis of miRNA-mRNA correlations for KIT and PDGFRA target genes and found a significant correlation between all of the enriched miRNAs and their target gene KIT. Results of the phenotype analysis showed miR-509-3p to be up-regulated in epithelioid and mixed cell types compared to spindle type, whereas miR-215-5p showed negative correlation with risk grade of GIST. These data reveal a detailed miRNA profile of GIST and highlight new candidates that may be important in the development of malignant disease.

PNPLA3 and RNF7 Gene Variants are Associated with the Risk of Developing Liver Fibrosis and Cirrhosis in an Eastern European Population.

BACKGROUND AND AIMS: Genome-wide association studies have revealed an association between the risk of developing liver fibrosis or cirrhosis and the single nucleotide polymorphisms (SNPs) of the PNPLA3, RNF7, MERTK and PCSK7 genes. We aimed to validate these results in an Eastern European population. METHODS: We evaluated the associations between the PNPLA3 (rs738409), RNF7 (rs16851720), MERTK (rs4374383) and PCSK7 (rs236918) variants and liver fibrosis and cirrhosis in a series of consecutive patients recruited at the Department of Gastroenterology, Lithuanian University of Health Sciences Hospital, during the period 2012-2015. The study included 317 individuals with liver cirrhosis, 154 individuals with liver fibrosis, and 498 controls. The studied SNPs were determined using RT-PCR TaqMan assays. RESULTS: MERTK and PCSK7 SNPs were not associated with liver fibrosis or cirrhosis. The PNPLA3 SNP rs738409 was associated with a higher risk of developing liver fibrosis (aOR: 1.65, P=0.001) and cirrhosis (aOR: 1.92, P=5.57*10-7). PNPLA3 genotypes were also associated with higher risk of developing liver fibrosis and cirrhosis in dominant (aOR: 1.98, P=2.20*10-5; aOR: 1.67, P=0.008, respectively) and recessive (aOR: 3.94, P=5.16*10-5; aOR: 3.02, P=0.003, respectively) models. RNF7 rs16851720 was associated with liver cirrhosis comparing CC vs. AA + CA genotypes (aOR: 0.26, P=0.020). CONCLUSION: Our study showed that PNPLA3 rs738409 and RNF7 rs16851720 confer an increased risk of developing liver fibrosis and cirrhosis in this Eastern European population, while the MERTK and PCSK7 SNPs are not associated with these conditions.

Associations between NOD2, IRGM and ORMDL3 polymorphisms and pediatric-onset inflammatory bowel disease in the Lithuanian population.

BACKGROUND AND OBJECTIVE: Recent GWAS and meta-analyses have revealed about 200 susceptibility genes/loci for inflammatory bowel diseases (IBD). However, only a small number of studies were performed in early-onset IBD. The aim of this study was to assess the association between NOD2, IL23R, ATG16L1, IRGM, IL10, NKX2-3 and ORMDL3 variants and early-onset IBD. MATERIALS AND METHODS: A total of 76 affected individuals (30 with Crohn's disease [CD] and 46 with ulcerative colitis [UC]) at the age of ≤17 years and 158 matched controls recruited in Lithuania were genotyped for the known genetic susceptibility variants in NOD2 (Arg702Trp (rs2066844), Gly908Arg (rs2066845) and Leu1007insC (rs2066847)), IL23R (rs11209026), ATG16L1 (rs2241880), IRGM (rs4958847), IL10 (rs3024505), NKX2-3 (rs11190140) and ORMDL3 (rs2872507) genes. RESULTS: Variants in NOD2 (Leu1007insC) and IRGM genes increased risk for CD (OR=6.56, 95% CI: 2.54-16.91, P=1.21×10-5 and OR=2.32, 95% CI: 1.05-5.14, P=0.033; respectively); whereas a variant in ORMDL3 gene was strongly associated with UC (OR=1.99, 95% CI: 1.23-3.20, P=4.15×10-3). CONCLUSIONS: The results confirmed that polymorphisms in NOD2 (Leu1007insC) and IRGM genes are associated with increased risk of CD; whereas the ORMDL3 variant is associated with susceptibility to UC in the Lithuanian early-onset IBD population.

TPMT and ITPA genetic variants in Lithuanian inflammatory bowel disease patients: Prevalence and azathioprine-related side effects.

PURPOSE: Inter-individual thiopurine metabolism variability can influence treatment outcomes in inflammatory bowel disease (IBD) patients. Genetic polymorphisms in thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) were linked with toxicity of azathioprine (AZA). The aim of the study was to investigate frequencies of TPMT and ITPA polymorphisms in Lithuanian IBD patients and analyze their association with AZA-related adverse events. MATERIALS/METHODS: Polymorphisms in TPMT (TPMT*2,*3B,*3C,*3A) and ITPA (rs1127354, rs7270101) genes were determined using PCR-RFLP and TaqMan(®) genotyping assays. 551 consecutive Lithuanian IBD patients were genotyped. The use of AZA and its side effects were assessed retrospectively according to hospital medical records. RESULTS: Frequencies of TPMT*3A, TPMT*3B and TPMT*3C alleles were 3.1%, 0.5% and 0.1%, respectively. TPMT*2 genetic variant was not detected in the study group. The distribution of minor alleles for ITPA rs1127354 and rs7270101 polymorphisms was 9.9% and 10.5%, respectively. AZA was prescribed in 82 patients and it provoked myelotoxicity in 11%, hepatotoxicity in 6.1%, dyspepsia in 6.1%, and pancreatitis in 3.6% of cases. Among patients who had AZA-related myelotoxicity, 11.1% were TPMT compound heterozygous, 44.4% had heterozygous genotype (P<0.01). Frequencies of ITPA minor alleles were similar among the patients with and without AZA-related side effects. CONCLUSION: Frequencies of TPMT and ITPA variant alleles in Lithuanian IBD group were similar to those observed in the Northern-Eastern Europe Caucasian populations. Polymorphisms in TPMT might be associated with myelotoxicity and leukopenia in AZA treated patients, while ITPA variant alleles appear not to be linked with treatment-related side effects.

Epigenetic silencing of miR-137 is a frequent event in gastric carcinogenesis.

MicroRNAs (miRNA) are involved in posttranscriptional regulation of gene expression and are dysregulated during carcinogenesis. CpG island methylation of miR-137 is a common event in different cancers; however, the role of miR-137 in gastric cancer (GC) remains largely unexplored. In this study we aimed to characterize the epigenetic alterations of miR-137 in gastric carcinogenesis. We analyzed total 295 tissues including paired primary gastric cancer (T-GC) with corresponding adjacent gastric mucosa (N-GC), paired primary colorectal cancer (CRC) tissues with corresponding non-tumorous mucosa, gastric tissues from controls (N), and patients with chronic/atrophic gastritis (CG) with and without Helicobacter pylori infection. Bisulfite pyrosequencing and TaqMan RT-PCR were used to analyze miR-137 methylation and expression, respectively. Survival differences were evaluated using Kaplan-Meier analyses. miR-137 CpG island methylation was more frequent in tumorous compared to non-tumorous conditions and higher in CRC than in GC. In comparison to N-GC, miR 137 methylation level was lower in N and CG tissues, which correlates with Correas cascade. MiR-137 methylation inversely correlates with global LINE-1 methylation and miR-137 expression. miR-137 methylation was higher in intestinal type GC compared to diffuse one, and higher in antrum compared to cardia and corpus, however, miR-137 methylation was associated with worse prognosis in diffuse, but not in intestinal type of GC. The expression in colon was significantly higher compared to any gastric tissues suggesting functional difference. In summary, miR-137 methylation is a frequent event in gastrointestinal cancers which occurs early in stepwise manner during gastric carcinogenesis and inversely correlates with global methylation. © 2015 Wiley Periodicals, Inc.

Gene polymorphisms of micrornas in Helicobacter pylori-induced high risk atrophic gastritis and gastric cancer.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population. METHODS: Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR. RESULTS: Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR -2.34, 95% CI 1.08-5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14-5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC. CONCLUSIONS: Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.