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.

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.

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.

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.

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.

The Role of miR-375-3p and miR-200b-3p in Gastrointestinal Stromal Tumors.

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST-miR-375-3p and miR-200b-3p-in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA-target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3'UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively affected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST.

Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.