BACKGROUND: Early detection of colorectal cancer (CRC) significantly improves its management and patients' survival. Circular RNAs (circRNAs) are peculiar covalently closed transcripts involved in gene expression modulation whose dysregulation has been extensively reported in CRC cells. However, little is known about their alterations in the early phases of colorectal carcinogenesis. METHODS: In this study, we performed an integrative analysis of circRNA profiles in RNA-sequencing (RNA-Seq) data of 96 colorectal cancers, 27 adenomas, and matched adjacent mucosa tissues. We also investigated the levels of cognate linear transcripts and those of regulating RNA-binding proteins (RBPs). Levels of circRNA-interacting microRNAs (miRNAs) were explored by integrating data of small RNA-Seq performed on the same samples. RESULTS: Our results revealed a significant dysregulation of 34 circRNAs (paired adj. p < 0.05), almost exclusively downregulated in tumor tissues and, prevalently, in early disease stages. This downregulation was associated with decreased expression of circRNA host genes and those encoding for RBPs involved in circRNA biogenesis, including NOVA1, RBMS3, and MBNL1. Guilt-by-association analysis showed that dysregulated circRNAs correlated with increased predicted activity of cell proliferation, DNA repair, and c-Myc signaling pathways. Functional analysis showed interactions among dysregulated circRNAs, RBPs, and miRNAs, which were supported by significant correlations among their expression levels. Findings were validated in independent cohorts and public datasets, and the downregulation of circLPAR1(2,3) and circLINC00632(5) was validated by ddPCR. CONCLUSIONS: These results support that multiple altered regulatory mechanisms may contribute to the reduction of circRNA levels that characterize early colorectal carcinogenesis.
- Publication type
- Journal Article MeSH
Long-term dysbiosis of the gut microbiome has a significant impact on colorectal cancer (CRC) progression and explains part of the observed heterogeneity of the disease. Even though the shifts in gut microbiome in the normal-adenoma-carcinoma sequence were described, the landscape of the microbiome within CRC and its associations with clinical variables remain under-explored. We performed 16S rRNA gene sequencing of paired tumour tissue, adjacent visually normal mucosa and stool swabs of 178 patients with stage 0-IV CRC to describe the tumour microbiome and its association with clinical variables. We identified new genera associated either with CRC tumour mucosa or CRC in general. The tumour mucosa was dominated by genera belonging to oral pathogens. Based on the tumour microbiome, we stratified CRC patients into three subtypes, significantly associated with prognostic factors such as tumour grade, sidedness and TNM staging, BRAF mutation and MSI status. We found that the CRC microbiome is strongly correlated with the grade, location and stage, but these associations are dependent on the microbial environment. Our study opens new research avenues in the microbiome CRC biomarker detection of disease progression while identifying its limitations, suggesting the need for combining several sampling sites (e.g., stool and tumour swabs).
- Publication type
- Journal Article MeSH
Actinotignum schaalii is an emerging, opportunistic pathogen and its connection to non-infectious diseases and conditions, such as prostate or bladder cancer, or chronic inflammation has been proposed. Here, we analyzed 297 urine, ureteral and urinary catheter samples from 128 patients by Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis and Sequencing (PCR-DGGE-S), and culture, and 29 of these samples also by 16S rRNA Illumina sequencing, to establish A. schaalii's prevalence in urinary tract-related samples, its relation to other bacteria, and its potential association with patients' conditions and samples' characteristics. A. schaalii-positive samples were significantly more diverse than A. schaalii negative and between-group diversity was higher than intra-group. Propionimicrobium lymphophilum, Fusobacterium nucleatum, Veillonella sp., Morganella sp., and Aerococcus sp. were significantly more often present in A. schaalii-positive samples; thus, we suggest these species are A. schaalii's concomitants, while Enterobacter and Staphylococcaceae were more often identified in A. schaalii-negative samples; therefore, we propose A. schaalii and these species are mutually exclusive. Additionally, a significantly higher A. schaalii prevalence in patients with ureter stricture associated hydronephrosis (p = 0.020) was noted. We suggest that A. schaalii could be an early polybacterial biofilm colonizer, together with concomitant species, known for pro-inflammatory features.
- Publication type
- Journal Article MeSH
Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.
- MeSH
- DNA, Bacterial genetics MeSH
- Adult MeSH
- Feces microbiology MeSH
- Phylogeny MeSH
- Gram-Negative Bacteria classification genetics isolation & purification MeSH
- Gram-Positive Bacteria classification genetics isolation & purification MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Reagent Kits, Diagnostic MeSH
- Reproducibility of Results MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Sequence Analysis, DNA MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Healthy Volunteers MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
