Altered fecal small RNA profiles in colorectal cancer reflect gut microbiome composition in stool samples

Dysbiotic configurations of the human gut microbiota have been linked with colorectal cancer (CRC). Human small non-coding RNAs are also implicated in CRC and recent findings suggest that their release in the gut lumen contributes to shape the gut microbiota. Bacterial small RNAs (bsRNAs) may also play a role in carcinogenesis but their role is less explored. Here, we performed small RNA and shotgun sequencing on 80 stool specimens of patients with CRC, or adenomas, and healthy subjects collected in a cross-sectional study to evaluate their combined use as a predictive tool for disease detection. We reported a considerable overlap and correlation between metagenomic and bsRNA quantitative taxonomic profiles obtained from the two approaches. Furthermore, we identified a combined predictive signature composed by 32 features from human and microbial small RNAs and DNA-based microbiome able to accurately classify CRC from healthy and adenoma samples (AUC= 0.87). In summary we reported evidence that host-microbiome dysbiosis in CRC can be observed also by altered small RNA stool profiles. Integrated analyses of the microbiome and small RNAs in the human stool may provide insights for designing more accurate tools for diagnostic purposes. Small RNA-Seq was performed on small RNA extracted from stool samples from a cohort of 80 subjects (29 CRC patients, 27 adenomas and 24 controls). CRC patients were recruited at the first CRC diagnosis and had not received any treatment prior to fecal sample collection. Naturally evacuated fecal samples were obtained from all patients previously instructed to self-collect the specimen at home before any bowel preparation for the colonoscopy. The stool was collected in Stool Nucleic Acid Collection and Transport Tubes with RNA stabilizing solution (Norgen Biotek Corp) and returned at the time of performing a colonoscopy in the endoscopy unit or at the time of blood sampling. RNA was extracted using the Stool Total RNA Purification Kit (Norgen Biotek Corp). Small RNA (sRNA) transcripts were converted into barcoded cDNA libraries. Library preparation was performed with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Protocol E7330, New England BioLabs Inc., USA). The obtained libraries were subjected to the Illumina sequencing pipeline, passing through clonal cluster generation on a single-read flow cell (Illumina Inc., USA) by bridge amplification on the cBot (TruSeq SR Cluster Kit v3-cBOT-HS, Illumina Inc., USA) and 50 cycles sequencing-by-synthesis on the HiSeq 2000 (Illumina Inc., USA).