Large-scale integrative analysis of fecal samples sequencing data from healthy and CRC patients supports novel miRNA-mediated host-microbiota interactions

Dysregulated microRNA (miRNA) profiles were repeatedly reported in studies on Colorectal Cancer (CRC) tissues. More recently, analyses of stool from CRC patients have clearly shown that altered miRNA levels reflect the presence of neoplastic lesions. Then, studies in model organisms reported that miRNAs released into the gut lumen, and detectable in stool, can modulate microbial gene expression. However, a comprehensive investigation of miRNA-microbiota interactions in human is lacking. Combined profiling of fecal miRNAs and microbial species in a large population of patients and healthy subjects could provide novel insights into altered host-microbiota interactions in CRC. An integrative analysis of fecal shotgun metagenomic sequencing and small RNA-Seq data of 1,333 subjects from ten European cohorts was performed. The study included 338 CRC patients, 173 subjects with precancerous lesions, 199 with other gastrointestinal diseases, and 623 controls. Microbial profiling was performed using MetaPhlAn 4.1 while a Docker4Seq pipeline was applied for fecal miRNA quantification. Analysis of miRNA and microbial profiles was performed using differential analysis methods (DESeq2, SIAMCAT), and meta-analysis strategies to investigate significant and coherent miRNA-microbial associations across cohorts. From the omics analysis, a significant decrease (p<0.05) of miRNA heterogeneity was observed from healthy to late-stage CRC, while increased microbial diversity in CRC was driven by the introgression of oral species. Detectable fecal miRNAs across cohorts were confirmed to be transcriptionally active in the normal intestinal tissue and, some of them, dysregulated in tumor tissue. Analyzing miRNA levels with respect to abundances of 461 commonly detected microbial species, 2,223 consistent miRNA-microbial associations across cohorts were identified. Despite the observed associations were predominantly positive and detected in CRC (n=916, 47%), 780 associations (35%) were uniquely detected in controls. and only 3% of them shared among disease classes. These associations involved oral species, prevalently in late-stage CRC. Our findings indicate that gut microbial composition is associated with fecal miRNA profiles. Most of the associations are specific to CRC, may reflect alterations in both the fecal miRNome and microbiome, and support the role of miRNAs as potential mediators of cross-kingdom interactions.