Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

Shanahan, M. T.; Kanke, M.; Singh, A. P.; Villanueva, J. W.; McNairn, A. J.; Oyesola, O. O.; Bonfini, A.; Hung, Y.-H.; Sheahan, B.; Bloom, J. C.; Cubitt, R. L.; Curry, E. G.; Pitman, W. A.; Rinaldi, V. D.; Dekaney, C. M.; Ding, S.; Peck, B. C. E.; Schimenti, J. C.; Dow, L. E.; Buchon, N.; Tait-Wojno, E. D.; Sethupathy, P.

doi:10.1101/2020.10.01.321612

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bioRxiv · 10.1101/2020.10.01.321612

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

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Abstract

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

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Shanahan, M. T., Kanke, M., Singh, A. P., Villanueva, J. W., McNairn, A. J., Oyesola, O. O., Bonfini, A., Hung, Y.-H., Sheahan, B., Bloom, J. C., Cubitt, R. L., Curry, E. G., Pitman, W. A., Rinaldi, V. D., Dekaney, C. M., Ding, S., Peck, B. C. E., Schimenti, J. C., Dow, L. E., Buchon, N., Tait-Wojno, E. D., Sethupathy, P.. 2020-10-01. Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt. https://doi.org/10.1101/2020.10.01.321612

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