microRNA-mediated response to pathophysiological stress in the intestine

Abstract

microRNAs (miRNAs) are short, 22-nt non-coding RNAs that modulate gene expressions post-transcriptionally. miRNAs are predicted to potentially target hundreds of genes simultaneously, rendering them crucial regulatory hubs of cellular signaling. Gene suppressions mediated by miRNAs are implicated in various biological processes such as embryogenesis and tumorigenesis. Here we present a transgenic mouse strain that allows for the study of inducible and reversible inhibition of the global miRNA activity in vivo via the expression of T6B, an inhibitor of the miRNA-induced silencing complex (miRISC) assembly. Using this model, we characterized the role of global miRNA function in the intestine, which served as a unique system for the study of miRNA as its physiology involved diverse cellular processes such as stem cell renewal, proliferation, and differentiation. While being expendable during homeostasis, proper miRNA function proved to be essential for intestinal regeneration after DSS-induced colitis, supporting the notion that miRNAs served as a buffer of homeostasis against pathophysiological stress such as inflammation. In addition to the inflammatory stress, the intestine is frequently subjected to oncogene-induced stress. Colorectal cancer (CRC) represents the third most common malignancy worldwide, accounting for ~10% of cancer-related mortality. Mutation of the oncogene K-RAS was detected in 44% of CRC patients, prompting it as a main driver of tumorigenesis. To investigate the interplay between miRNA and oncogenic K-RAS, we profiled the physiological miRNA target landscape in the mouse colonic epithelium and tumor expressing K-RasG12D using Halo-Enhanced Argonaute Pulldown (HEAP). Integrating that with matching chromatin accessibility, transcriptomic, proteomic, and phospho-proteomic data, we uncovered a surprising global suppression of miRNA function by oncogenic K-Ras. Further mechanistic investigations revealed that oncogenic K-Ras suppressed Csnk1a1/Csnk2a1 expression and activity, potentially hypo-phosphorylating Ago2 (Ser825-S835). This subsequently triggered enhanced Ago2:mRNA binding, expanded detection of the miRNA target repertoire, and global de-repression of miRNA targets that we observed in colonic tissues expressing oncogenic K-Ras. Our findings connected a potent regulatory mechanism of miRNA to oncogenic K-Ras and provided a mechanistic link between hyperactive K-Ras signaling and the up-regulation of hundreds of miRNA target genes. To complement our findings regarding the intestinal response to pathophysiological stress, we performed functional analysis using the transcriptomic profiles of intestines under inflammatory or K-Ras-induced oncogenic stress to further understand their context-specific nature. Using different mouse models of inflammatory bowel disease (IBD), we characterized varying degrees of responsiveness (resolution of inflammation) in the intestine to MK2 inhibition. Transcriptomic profiling of the intestines with/without treatment in these models established an MK2 responder signature that was enriched in the inflammatory monocytes and neutrophils. Furthermore, analysis of transcriptomic perturbations from endogenous expressions of various oncogenic Gly12 mutants of K-Ras (K-RasG12D, K-RasG12C, K-RasG12V) in colonic tumors established both an allele-agnostic and allele-specific signatures that could facilitate the functional stratification of patients and developments of allele-specific therapies in the era of precision medicine. Collectively, the work presented in this dissertation underscored the essential role of miRNAs in the intestinal response to stress, the global suppression of miRNA function by oncogenic K-Ras, and the context-specific facet of both inflammatory and oncogene-induced stress in the intestine.