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.

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.

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. MicroRNAs (miRNAs) are small noncoding molecules that play an essential role in a variety of fundamental biological processes. However, clinical potential of miRNA profiling in the gastric cancerogenesis, especially in premalignant GC cases, remains unclear. AIM: To evaluate the AG and GC tissue miRNomes and identify specific miRNAs' 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 miRNAs hsa-miR-129-1-3p and hsa-miR-196a-5p were analyzed in plasma samples of independent cohort consisting of CON, AG, and GC patients. Expression level of hsa-miR-129-1-3p and hsa-miR-196a-5p was determined using the quantitative real-time polymerase chain reaction and 2-ΔΔCt method. RESULTS: Results of tissue analysis revealed 20 differentially expressed miRNAs in AG group compared to CON group, 129 deregulated miRNAs in GC compared to CON, and 99 altered miRNAs comparing GC and AG groups. Only 2 miRNAs (hsa-miR-129-1-3p and hsa-miR-196a-5p) 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-miR-196a-5p 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. Compar-ing results in tissue and plasma samples, hsa-miR-129-1-3p 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-miR-215-3p/5p and hsa-miR-934 were significantly deregulated in GC based on Helicobacter pylori (H. pylori) infection status [log2 fold change (FC) = -4.52, P-adjusted = 0.02; log2FC = -4.00, P-adjusted = 0.02; log2FC = 6.09, P-adjusted = 0.02, respectively]. CONCLUSION: Comprehensive miRNome study provides evidence for gradual deregulation of hsa-miR-196a-5p and hsa-miR-129-1-3p in gastric carcinogenesis and found hsa-miR-215-3p/5p and hsa-miR-934 to be significantly deregulated in H. pylori carrying GC patients.

Fecal Microbiome Transplantation for Recurrent Clostridioides difficile Infection: Treatment Efficacy, Short and Long-term Follow-up Results from Consecutive Case Series.

BACKGROUND AND AIMS: Many studies have shown a high effectiveness of fecal microbiota transplantation (FMT) in treatment of recurrent or refractory Clostridioides difficile infection (CDI). Nevertheless, data on long term outcomes and complications after FMT are still lacking. We aimed to evaluate the efficacy, the peri- procedural safety profile and the long-term efficacy and safety of FMT for recurrent CDI during a median follow up period of 24 months. METHODS: Our study included 60 consecutive patients that were treated from 2015 to 2019 for recurrent CDI. In all patients FMT was performed through the nasoenteric tube placed during gastroscopy. Fresh donor feces were used for FMT from unrelated donors. Pre-FMT preparation included CDI treatment with oral vancomycin 500 mg q.i.d. for at least five days and proton pump inhibitor (PPI) administration before FMT. Follow up data included information about recurrent CDI episodes, early and late complications, health status at 3, 12 and 24 months after FMT. RESULTS: FMT was performed for 60 patients (median age 72.5 years) with recurrent CDI. Clinical improvement after the first FMT procedure was observed in 48 patients (80%). Ten of 12 initially non-responding patients had a clinical resolution after a second FMT leading to an increased overall cure rate of 96.7 %. The remaining two patients needed a third FMT with a final overall cure rate of 100%. Nine of 60 patients were under immunosuppressive therapy. Six immunosuppressed patients were in the group of initial responders and the remaining three in the initially non-responder group. We observed a very low rate of adverse events in the short and long-term after FMT. During the first eight weeks after the FMT procedure, the death of three patients occurred, but they were not related to the FMT procedure. Patients were followed up for a median of 20 months, with the range from 12 to 55 months. During the follow-up period no long-term serious adverse events (SAE) were documented. CONCLUSIONS: Our study confirms excellent efficacy rates of FMT in the treatment of recurrent CDI. In addition, this study shows that it is possible to avoid short term SAE when FMT is administered via a nasoenteric tube by following a very stringent peri-procedural patient follow-up protocol. Our study also demonstrates good safety with a low rate of long-term adverse events after FMT.

Association of Long Non-Coding RNA Polymorphisms with Gastric Cancer and Atrophic Gastritis.

Long non-coding RNAs (lncRNA) play an important role in the carcinogenesis of various tumours, including gastric cancer. This study aimed to assess the associations of lncRNA ANRIL, H19, MALAT1, MEG3, HOTAIR single-nucleotide polymorphisms (SNPs) with gastric cancer and atrophic gastritis. SNPs were analyzed in 613 gastric cancer patients, 118 patients with atrophic gastritis and 476 controls from three tertiary centers in Germany, Lithuania and Latvia. Genomic DNA was extracted from peripheral blood leukocytes. SNPs were genotyped by the real-time polymerase chain reaction. Results showed that carriers of MALAT1 rs3200401 CT genotype had the significantly higher odds of atrophic gastritis than those with CC genotype (OR-1.81; 95% CI 1.17-2.80, p = 0.0066). Higher odds of AG were found in a recessive model (CC vs. TT + CT) for ANRIL rs1333045 (OR-1.88; 95% CI 1.19-2.95, p = 0.0066). Carriers of ANRIL (rs17694493) GG genotype had higher odds of gastric cancer (OR-4.93; 95% CI 1.28-19.00) and atrophic gastritis (OR-5.11; 95% CI 1.10-23.80) compared with the CC genotype, and carriers of HOTAIR rs17840857 TG genotype had higher odds of atrophic gastritis (OR-1.61 95% CI 1.04-2.50) compared with the TT genotype; however, the ORs did not reach the adjusted significance threshold (p < 0.007). In summary, our data provide novel evidence for a possible link between lncRNA SNPs and premalignant condition of gastric cancer, suggesting the involvement of lncRNAs in gastric cancer development.

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.