miRNome Profiling Analysis Reveals Novel Hepatocellular Carcinoma Diagnostic, Prognostic and Treatment-Related Candidate Biomarkers: Post hoc Analysis of SORAMIC Trial.

INTRODUCTION: Early diagnosis of hepatocellular carcinoma (HCC) as well as evaluation of prognosis and prediction of treatment efficacy remains challenging due to the missing specific non-invasive biomarkers. The aim of this study was to identify disease-specific microRNA (miRNA) patterns for diagnosis, prediction of prognosis, and treatment response in patients with HCC. METHODS: The study population included 42 HCC patients from SORAMIC clinical trial: 22 patients received sorafenib monotherapy, 20 patients underwent 90Y radioembolization in combination with sorafenib. 20 individuals were included in the control group. HCC patients underwent collection of plasma samples before and 7-9 weeks after the beginning of the treatment. Isolation of circulating miRNAs, preparation of small RNA sequencing libraries and next-generation sequencing were performed. Association analysis for novel diagnostic, prognostic, and treatment-related candidate biomarkers was performed. RESULTS: A total of 42 differentially expressed (16 up-regulated and 26 down-regulated) miRNAs were identified comparing baseline and control group plasma samples. hsa-miR-215-5p and hsa-miR-192-5p were down-regulated, while hsa-miR-483-5p and hsa-miR-23b-3p were up-regulated comparing baseline and 7-9 weeks post-sorafenib monotherapy samples. hsa-miR-215-5p was the sole down-regulated miRNA in the same combination therapy comparison. hsa-miR-183-5p, hsa-miR-28-3p, and hsa-miR-1246 were found to be significantly up-regulated comparing non-responders versus responders to sorafenib. High hsa-miR-215-5p expression was significantly associated with worse HCC patients' prognosis. CONCLUSIONS: Systematic miRNA profiling of highly characterized samples from SORAMIC study revealed a subset of potential miRNA biomarkers for HCC diagnosis and prognosis of sorafenib-treated patients' survival.

Constituents of stable commensal microbiota imply diverse colonic epithelial cell reactivity in patients with ulcerative colitis.

BACKGROUND: Despite extensive research on microbiome alterations in ulcerative colitis (UC), the role of the constituent stable microbiota remains unclear. RESULTS: This study, employing 16S rRNA-gene sequencing, uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis, the study highlights microbial constituents, featuring Phocaeicola, Collinsella, Roseburia, Holdemanella, and Bacteroides, that are not affected during the course of UC. Co-cultivation experiments, utilizing commensal Escherichia coli and Phocaeicola vulgatus, were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells, without inducing pathogen recognition and stress responses, offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients' response to the same bacteria, which indicate the need for more comprehensive, stratified analyses with an expanded sample size. CONCLUSION: This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota - Escherichia coli and Phocaeicola vulgatus - potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals.

5'-Isoforms of miR-1246 Have Distinct Targets and Stronger Functional Impact Compared with Canonical miR-1246 in Colorectal Cancer Cells In Vitro.

Colorectal cancer (CRC) is a multifactorial disease involving genetic and epigenetic factors, such as miRNAs. Sequencing-based studies have revealed that miRNAs have many isoforms (isomiRs) with modifications at the 3'- and 5'-ends or in the middle, resulting in distinct targetomes and, consequently, functions. In the present study, we aimed to evaluate the putative targets and functional role of miR-1246 and its two 5'-isoforms (ISO-miR-1246_a and ISO-miR-1246_G) in vitro. Commercial Caco-2 cells of CRC origin were analyzed for the expression of WT-miR-1246 and its 5'-isoforms using small RNA sequencing data, and the overabundance of the two miR-1246 isoforms was determined in cells. The transcriptome analysis of Caco-2 cells transfected with WT-miR-1246, ISO-miR-1246_G, and ISO-miR-1246_a indicated the minor overlap of the targetomes between the studied miRNA isoforms. Consequently, an enrichment analysis showed the involvement of the potential targets of the miR-1246 isoforms in distinct signaling pathways. Cancer-related pathways were predominantly more enriched in dysregulated genes in ISO-miR-1246_G and ISO-miR-1246_a, whereas cell cycle pathways were more enriched in WT-miR-1246. The functional analysis of WT-miR-1246 and its two 5'-isoforms revealed that the inhibition of any of these molecules had a tumor-suppressive role (reduced cell viability and migration and promotion of early cell apoptosis) in CRC cells. However, the 5'-isoforms had a stronger effect on viability compared with WT-miR-1246. To conclude, this research shows that WT-miR-1246 and its two 5'-isoforms have different targetomes and are involved in distinct signaling pathways but collectively play an important role in CRC pathogenesis.

Detailed Characterization of the Lung-Gut Microbiome Axis Reveals the Link between PD-L1 and the Microbiome in Non-Small-Cell Lung Cancer Patients.

Next-generation sequencing technologies have started a new era of respiratory tract research in recent years. Alterations in the respiratory microbiome between healthy and malignant conditions have been revealed. However, the composition of the microbiome varies among studies, even in similar medical conditions. Also, there is a lack of complete knowledge about lung-gut microbiome interactions in lung cancer patients. The aim of this study was to explore the lung-gut axis in non-small-cell lung cancer (NSCLC) patients and the associations between lung-gut axis microbiota and clinical parameters (CRP, NLR, LPS, CD8, and PD-L1). Lung tissue and fecal samples were used for bacterial 16S rRNA sequencing. The results revealed, for the first time, that the bacterial richness in lung tumor tissue gradually decreased with an increase in the level of PD-L1 expression (p < 0.05). An analysis of ß-diversity indicated a significant positive correlation between the genera Romboutsia and Alistipes in both the lung tumor biopsies and stool samples from NSCLC patients (p < 0.05). Survival analysis showed that NSCLC patients with higher bacterial richness in their stool samples had prolonged overall survival (HR: 2.06, 95% CI: 1.025-4.17, p = 0.0426).

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.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 µL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

Diagnostic and Prognostic Value of IL-10, FABP2 and LPS Levels in HCC Patients.

Hepatocellular carcinoma (HCC) still lacks valuable diagnostic and prognostic tools. This study aimed to investigate the potential diagnostic and prognostic value of baseline interleukin (IL)-10, fatty acid-binding protein 2 (FABP2) and lipopolysaccharide (LPS) levels in patients with HCC. Serum levels of IL-10, FABP2 and LPS in 47 newly diagnosed HCC patients and 50 healthy individuals were estimated and compared. The best cut-off points for baseline IL-10, FABP2 and LPS levels predicting overall survival (OS) were evaluated. Both levels of FABP2 and IL-10 were significantly higher in HCC patients vs. control group (median 2095 vs. 1772 pg/mL, p = 0.026; 9.94 vs. 4.89 pg/mL, p < 0.001) and may serve as potential biomarkers in complex HCC diagnostic tools. The cut-off value of 2479 pg/mL for FABP2 was determined to have the highest sensitivity (66.7%) and specificity (55.6%) to distinguish patients with a median OS longer than 17 months. However, the median OS of patients with high and low levels of FABP2 were not significantly different (p = 0.896). The prognostic value of LPS as well as FABP2 and IL-10 for HCC patients appears to be limited.

A scan of all coding region variants of the human genome, identifies 13q12.2-rs9579139 and 15q24.1-rs2277598 as novel risk loci for pancreatic ductal adenocarcinoma.

Coding sequence variants comprise a small fraction of the germline genetic variability of the human genome. However, they often cause deleterious change in protein function and are therefore associated with pathogenic phenotypes. To identify novel pancreatic ductal adenocarcinoma (PDAC) risk loci, we carried out a complete scan of all common missense and synonymous SNPs and analysed them in a case-control study comprising four different populations, for a total of 14 538 PDAC cases and 190 657 controls. We observed a statistically significant association between 13q12.2-rs9581957-T and PDAC risk (P = 2.46 × 10-9), that is in linkage disequilibrium (LD) with a deleterious missense variant (rs9579139) of the URAD gene. Recent findings suggest that this gene is active in peroxisomes. Considering that peroxisomes have a key role as molecular scavengers, especially in eliminating reactive oxygen species, a malfunctioning URAD protein might expose the cell to a higher load of potentially DNA damaging molecules and therefore increase PDAC risk. The association was observed in individuals of European and Asian ethnicity. We also observed the association of the missense variant 15q24.1-rs2277598-T, that belongs to BBS4 gene, with increased PDAC risk (P = 1.53 × 10-6). rs2277598 is associated with body mass index and is in LD with diabetes susceptibility loci. In conclusion, we identified two missense variants associated with the risk of developing PDAC independently from the ethnicity highlighting the importance of conducting reanalysis of genome-wide association studies (GWASs) in light of functional data.

Sterile Fecal Filtrate From A Healthy Donor Improves Microbial Diversity In Patients With Hepatic Encephalopathy.

BACKGROUND AND AIMS: Hepatic encephalopathy (HE) remains one of the most debilitating complications of liver cirrhosis. Changes in gut microbiome composition have been linked to liver diseases and its complications including HE. Recent randomized controlled trials showed fecal microbiota transplantation to be safe and effective in HE treatment, however transferring unidentified live bacteria could cause various complications, including infections, especially in immunocompromised patients. This study aimed to evaluate the safety and efficacy of sterile fecal filtrate transfer (SFFT) for the modulation of the intestinal microbiome of patients with cirrhosis and HE. METHODS: A custom-made air pressure filtration device was used for the sterile fecal filtrate preparation. Seven patients received SFFT from the same healthy donor. Patients were monitored at least 30 days after the procedure. Cognition tests, blood and stool sampling were performed to assess the safety and efficacy of SFFT on HE, liver function, and stool microbiome composition on follow-up days 7 and 30. RESULTS: SFFT was well tolerated and resulted in fluctuations in the microbial composition of study participants: α-diversity increased in 4/7 of the patients, without robust engraftment of donors' microbial composition as assessed by ß-diversity analysis. No significant effect on cognition tests or liver function was noted after the procedure. One death occurred three months after the procedure, however, it was not related to the SFFT. CONCLUSIONS: Despite the effect on the gut microbiome, we did not observe robust improvement in patients' liver function or HE cognition tests after the procedure.

Mucin-microbiome signatures shape the tumor microenvironment in gastric cancer.

BACKGROUND AND AIMS: We aimed to identify mucin-microbiome signatures shaping the tumor microenvironment in gastric adenocarcinomas and clinical outcomes. METHODS: We performed high-throughput profiling of the mucin phenotypes present in 108 gastric adenocarcinomas and 20 functional dyspepsia cases using validated mucin-based RT-qPCRs with subsequent immunohistochemistry validation and correlated the data with clinical outcome parameters. The gastric microbiota was assessed by 16S rRNA gene sequencing, taxonomy, and community composition determined, microbial networks analyzed, and the metagenome inferred in association with mucin phenotypes and expression. RESULTS: Gastric adenocarcinomas with an intestinal mucin environment or high-level MUC13 expression are associated with poor survival. On the contrary, gastric MUC5AC or MUC6 abundance was associated with a more favorable outcome. The oral taxa Neisseria, Prevotella, and Veillonella had centralities in tumors with intestinal and mixed phenotypes and were associated with MUC13 overexpression, highlighting their role as potential drivers in MUC13 signaling in GC. Furthermore, dense bacterial networks were observed in intestinal and mixed mucin phenotype tumors whereas the lowest community complexity was shown in null mucin phenotype tumors due to higher Helicobacter abundance resulting in a more decreased diversity. Enrichment of oral or intestinal microbes was mucin phenotype dependent. More specifically, intestinal mucin phenotype tumors favored the establishment of pro-inflammatory oral taxa forming strong co-occurrence networks. CONCLUSIONS: Our results emphasize key roles for mucins in gastric cancer prognosis and shaping microbial networks in the tumor microenvironment. Specifically, the enriched oral taxa associated with aberrant MUC13 expression can be potential biomarkers in predicting disease outcomes. Video Abstract.

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.

Genetic and non-genetic risk factors for early-onset pancreatic cancer.

BACKGROUND: Early-onset pancreatic cancer (EOPC) represents 5-10% of all pancreatic ductal adenocarcinoma (PDAC) cases, and the etiology of this form is poorly understood. It is not clear if established PDAC risk factors have the same relevance for younger patients. This study aims to identify genetic and non-genetic risk factors specific to EOPC. METHODS: A genome-wide association study was performed, analysing 912 EOPC cases and 10 222 controls, divided into discovery and replication phases. Furthermore, the associations between a polygenic risk score (PRS), smoking, alcohol consumption, type 2 diabetes and PDAC risk were also assessed. RESULTS: Six novel SNPs were associated with EOPC risk in the discovery phase, but not in the replication phase. The PRS, smoking, and diabetes affected EOPC risk. The OR comparing current smokers to never-smokers was 2.92 (95% CI 1.69-5.04, P = 1.44 × 10-4). For diabetes, the corresponding OR was 14.95 (95% CI 3.41-65.50, P = 3.58 × 10-4). CONCLUSION: In conclusion, we did not identify novel genetic variants associated specifically with EOPC, and we found that established PDAC risk variants do not have a strong age-dependent effect. Furthermore, we add to the evidence pointing to the role of smoking and diabetes in EOPC.

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.

Common variability in oestrogen-related genes and pancreatic ductal adenocarcinoma risk in women.

The incidence of pancreatic ductal adenocarcinoma (PDAC) is different among males and females. This disparity cannot be fully explained by the difference in terms of exposure to known risk factors; therefore, the lower incidence in women could be attributed to sex-specific hormones. A two-phase association study was conducted in 12,387 female subjects (5436 PDAC cases and 6951 controls) to assess the effect on risk of developing PDAC of single nucleotide polymorphisms (SNPs) in 208 genes involved in oestrogen and pregnenolone biosynthesis and oestrogen-mediated signalling. In the discovery phase 14 polymorphisms showed a statistically significant association (P < 0.05). In the replication none of the findings were validated. In addition, a gene-based analysis was performed on the 208 selected genes. Four genes (NR5A2, MED1, NCOA2 and RUNX1) were associated with PDAC risk, but only NR5A2 showed an association (P = 4.08 × 10-5) below the Bonferroni-corrected threshold of statistical significance. In conclusion, despite differences in incidence between males and females, our study did not identify an effect of common polymorphisms in the oestrogen and pregnenolone pathways in relation to PDAC susceptibility. However, we validated the previously reported association between NR5A2 gene variants and PDAC risk.

Diurnal variation of clock genes expression and other sleep-wake rhythm biomarkers among acute ischemic stroke patients.

There is accumulating evidence about sleep-wake rhythm disturbances as potential modifiable risk factors of both incident and recurrent stroke and less favorable outcomes after stroke. To our best knowledge this is the first study designed to investigate clock genes expression profiles in ischemic stroke patients and their relations to other biological and behavioral sleep-wake rhythm biomarkers, sleep structural and clinical stroke features. Altogether, 27 ischemic stroke patients (20 males) with the median age of 56 years and 25 gender and age matched controls were investigated with neurological and objective examination, scales, polysomnography, actigraphy and 24-h blood sampling for melatonin and clock genes profiles. Median melatonin plasma concentrations at four time points at 7, 11 p.m., 3 a.m. and 12 p.m. did not differ significantly between patients and controls, only early morning melatonin concentration at 7 a.m. was significantly lower and cortisol plasma concentration - significantly higher among stroke patients. All four clock genes (ARNTL (BMAL1), NR1D1 (Rev-erbα/ß), PER1, and PER3) showed significant time-of-day variation in both patients' and controls' groups, except expression of NR1D1 (Rev-erbα/ß) at 7 a.m. and PER1 at 12 p.m. differed significantly. In conclusion, acute ischemic stroke patients tended to preserve most of diurnal variation of sleep-wake rhythm molecular patterns. Nevertheless, early morning time point showing higher cortisol and lower melatonin concentrations and lower NR1D1 (Rev-erbα/ß) expression, as well as lower PER1 midday expression reflect specific circadian desynchrony features in different loops of the molecular circadian clock system.

Gut microbial similarity in twins is driven by shared environment and aging.

BACKGROUND: Human gut microbiome composition is influenced by genetics, diet and environmental factors. We investigated the microbial composition in several gastrointestinal (GI) compartments to evaluate the impact of genetics, delivery mode, diet, household sharing and aging on microbial similarity in monozygotic and dizygotic twins. METHODS: Fecal, biopsy and saliva samples were obtained from total 108 twins. DNA and/or RNA was extracted and the region V1-V2 of the 16S rRNA gene was amplified and sequenced. Bray-Curtis similarity was used for further microbiome comparisons, Mann-Whitney test was applied to evaluate the significant differences between groups and Spearman test was applied to reveal potential correlations between data. FINDINGS: The global bacterial profiles were grouped into two clusters separating the upper and lower GI. The upper GI microbiome composition was strictly dependent on the Helicobacter pylori status. With a positivity rate of 55%, H. pylori completely colonized the stomach and separated infected twins from non-infected twins irrespective of zygosity status. Lower GI microbiome similarity between the twins was defined mainly by household-sharing and aging; whereas delivery mode and host genetics had no influence. There was a progredient decrease in the bacterial similarity with aging. Shared vs. non-shared phylotypes analysis showed that in both siblings the shared phylotypes progressively diminished with aging, while the non-shared phylotypes increased. INTERPRETATION: Our findings strongly highlight the aging and shared household as they key determinants in gut microbial similarity and drift in twins irrespective of their zygotic state. FUNDING: This work was supported by the grant of the Research Council of Lithuania (Project no. APP-2/2016) and also partially supported by the funds of European Commission through the "European funds for regional development" (EFRE) as well as by the regional Ministry of Economy, Science and Digitalization as part of the "LiLife" Project as part of the "Autonomy in old Age" research group (Project ID: ZS/2018/11/95324).

Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using antimicrobial protein KvarIa.

BACKGROUND: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. METHODS: Mouse infection model was established based on infection of antibiotic-treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit®, a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. RESULTS: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit® S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. CONCLUSIONS: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is suppressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit®-protected klebicin KvarIa.

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.

miRNome Profiling and Functional Analysis Reveal Involvement of hsa-miR-1246 in Colon Adenoma-Carcinoma Transition by Targeting AXIN2 and CFTR.

Regulatory changes occurring early in colorectal cancer development remain poorly investigated. Since the majority of cases develop from polyps in the adenoma-carcinoma transition, a search of early molecular features, such as aberrations in miRNA expression occurring prior to cancer development, would enable identification of potentially causal, rather than consequential, candidates in the progression of polyp to cancer. In the current study, by employing small RNA-seq profiling of colon biopsy samples, we described differentially expressed miRNAs and their isoforms in the adenoma-carcinoma transition. Analysis of healthy-adenoma-carcinoma sequence in an independent validation group enabled us to identify early deregulated miRNAs including hsa-miR-1246 and hsa-miR-215-5p, the expressions of which are, respectively, gradually increasing and decreasing. Loss-of-function experiments revealed that inhibition of hsa-miR-1246 lead to reduced cell viability, colony formation, and migration rate, thereby indicating an oncogenic effect of this miRNA in vitro. Subsequent western blot and luciferase reporter assay provided evidence of hsa-miR-1246 being involved in the regulation of target AXIN2 and CFTR genes' expression. To conclude, the present study revealed possible involvement of hsa-miR-1246 in early colorectal cancer development and regulation of tumor suppressors AXIN2 and CFTR.