Integrated transcriptomic analysis reveals miRNA-hub mRNA-TF interactions and key regulatory targets in STEC infected...

Shiga toxin-producing Escherichia coli (STEC) is a leading foodborne pathogen responsible for hemolytic uremic syndrome (HUS). This pathogen poses a severe threat to global public health. MicroRNAs (miRNAs) are increasingly recognized as essential post-transcriptional regulators and therapeutic targets. Despite this, their exact regulatory networks and roles in STEC pathogenesis remain largely unknown.

An in vitro infection model was established using human intestinal epithelial cells (HIECs), with optimal infection time and bacterial load determined via CCK-8 assays and DAPI staining. High-throughput RNA sequencing (RNA-seq) was performed to profile miRNA and mRNA expression, followed by RT-qPCR validation. Differentially expressed miRNAs and their target mRNAs were identified by integrating miRanda predictions with transcriptomic data. Target functions were annotated using GO and KEGG enrichment analyses. A protein-protein interaction (PPI) network was constructed to identify core hub genes, and upstream transcription factors (TFs) were predicted using the TRRUST and hTFtarget databases, culminating in the construction of an integrated miRNA-hub mRNA-TF regulatory network.

Infection of HIECs with STEC (10⁶ CFU/mL) for 1 hour induced profound cellular structural damage, accompanied by the differential expression of 652 target mRNAs (301 upregulated and 351 downregulated). Functional enrichment revealed that these targets are predominantly involved in inflammatory responses, apoptosis, and cell proliferation. Through the PPI network, 10 core hub genes (including TNF, CXCL8, CCN2, and TGFB2) were identified, along with 11 highly correlated regulatory TFs. Based on the integrated network analysis, has-miR-3121-3p, hsa-miR-219b-5p, and hsa-miR-543 were pinpointed as master regulatory miRNAs, suggesting they orchestrate critical host signaling pathways during infection.

STEC infection drastically reprograms the transcriptomic landscape of HIECs, triggering the dysregulation of inflammation and apoptosis-related pathways. This study is the first to delineate a comprehensive miRNA-mRNA-TF regulatory network for STEC infection, highlighting miR-3121-3p, miR-219b-5p, and miR-543 as key molecular mediators. These findings provide novel insights into the molecular pathogenesis of STEC and lay a crucial foundation for exploring potential regulatory and host-directed therapeutic targets.