The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid β-Oxidation-Related Genes in Intestinal Stem Cells

Abstract

Cell division, growth, and differentiation are energetically costly and dependent processes. In adult stem cell-based epithelia, cellular identity seems to be coupled with a cell’s metabolic profile and vice versa. It is thus tempting to speculate that resident stem cells have a distinct metabolism, different from more committed progenitors and differentiated cells. Although investigated for many stem cell types in vitro, in vivo data of niche-residing stem cell metabolism is scarce. In adult epithelial tissues, stem cells, progenitor cells, and their progeny have very distinct functions and characteristics. In our study, we hypothesized and tested whether stem and progenitor cell types might have a distinctive metabolic profile in the intestinal lineage. Here, taking advantage of the genetically accessible adult Drosophila melanogaster intestine and the availability of ex vivo single cell sequencing data, we tested that hypothesis and investigated the metabolism of the intestinal lineage from stem cell (ISC) to differentiated epithelial cell in their native context under homeostatic conditions. Our initial in silico analysis of single cell RNAseq data and functional experiments identify the microRNA miR-277 as a posttranscriptional regulator of fatty acid β-oxidation (FAO) in the intestinal lineage. Low levels of miR-277 are detected in ISC and progressively rising miR-277 levels are found in progenitors during their growth and differentiation. Supporting this, miR-277-regulated fatty acid β-oxidation enzymes progressively declined from ISC towards more differentiated cells in our pseudotime single-cell RNAseq analysis and in functional assays on RNA and protein level. In addition, in silico clustering of single-cell RNAseq data based on metabolic genes validates that stem cells and progenitors belong to two independent clusters with well-defined metabolic characteristics. Furthermore, studying FAO genes in silico indicates that two populations of ISC exist that can be categorized in mitotically active and quiescent ISC, of which the latter relies on FAO genes. In line with an FAO dependency of ISC, forced expression of miR-277 phenocopies RNAi knockdown of FAO genes by reducing ISC size and subsequently resulting in stem cell death. We also investigated miR-277 effects on ISC in a benign and our newly developed CRISPR-Cas9-based colorectal cancer model and found effects on ISC survival, which as a consequence affects tumor growth, further underlining the importance of FAO in a pathological context. Taken together, our study provides new insights into the basal metabolic requirements of intestinal stem cell on β-oxidation of fatty acids evolutionarily implemented by a sole microRNA. Gaining knowledge about the metabolic differences and dependencies affecting the survival of two central and cancer-relevant cell populations in the fly and human intestine might reveal starting points for targeted combinatorial therapy in the hope for better treatment of colorectal cancer in the future.

Inflammation- and Metastasis-Related Proteins Expression Changes in Early Stages in Tumor and Non-Tumor Adjacent Tissues of Colorectal Cancer Samples

“…Unfortunately, there are no animal models for the different CRC stages (I, II, III, IV) since there are only animal models for early or late CRC. This situation makes it difficult to investigate the different markers in vivo and across the different stages, despite the existence of some advanced studies about the use of zebrafish xenografts and Drosophila melanogaster [ 42 , 43 ].…”

Noncoding RNA Regulation of Hormonal and Metabolic Systems in the Fruit Fly Drosophila

“…Similarly, miR-277 regulates fatty acid β-oxidation (FAO) enzymes in intestinal stem cells. The differentially expressed genes are identified in miR-277 mutants [ 52 ]. Validations suggested disrupting homeostasis and progenitor cell survival with miR-277 mutants [ 52 ].…”

“…The differentially expressed genes are identified in miR-277 mutants [ 52 ]. Validations suggested disrupting homeostasis and progenitor cell survival with miR-277 mutants [ 52 ]. The alteration of fat metabolism by miR-14 suggested that the triacylglycerol and diacylglycerol levels differ in miR-14 mutants [ 53 ].…”

Steroid hormone-induced wingless ligands tune female intestinal size in Drosophila

“…Even though Notch-tumours recapitulate important steps of CRC tumorigenesis, N is not frequently mutated in CRC. Therefore, we also investigated an autonomous function for Crol/ZNF267 in a CRISPR-Cas9 based model of sporadic CRC 48 targeting the most frequently mutated genes with a multiplex guideRNA array (Fig. 6K) [48][49][50] .…”

“…Therefore, we also investigated an autonomous function for Crol/ZNF267 in a CRISPR-Cas9 based model of sporadic CRC 48 targeting the most frequently mutated genes with a multiplex guideRNA array (Fig. 6K) [48][49][50] . CRC in ISC has severe and pleiotropic cellular phenotypes and results in early fly demise 48 .…”