miR-30 Family Controls Proliferation and Differentiation of Intestinal Epithelial Cell Models by Directing a Broad Gene Expression Program That Includes SOX9 and the Ubiquitin Ligase Pathway.
Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3'-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis.
From the Curriculum in Genetics & Molecular Biology.
Bailey C E
0000-0002-4316-0966
BC
Peck
the Departments of Genetics.
John
J
Sincavage
the Departments of Genetics.
Sydney
S
Feinstein
Nutrition, and Cell Biology and Physiology, and.
Amanda T
AT
Mah
Cell Biology and Physiology, and.
James G
JG
Simmons
From the Curriculum in Genetics & Molecular Biology, Cell Biology and Physiology, and.
P Kay
PK
Lund
From the Curriculum in Genetics & Molecular Biology, the Departments of Genetics, the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 praveen_sethupathy@med.unc.edu.
Praveen
P
Sethupathy
D000818:Animals; D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
C546086:MIRN30b microRNA, human; D035683:MicroRNAs; C534715:Mirn30d microRNA, mouse; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D044767:Ubiquitin-Protein Ligases
differentiation; gene regulation; intestinal epithelium; microRNA (miRNA); proliferation