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

Bailey C.E. Peck, John Sincavage, Sydney Feinstein, Amanda T. Mah, James G. Simmons, P. Kay Lund, Praveen Sethupathy

Research output: Contribution to journalArticle

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Abstract

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.

LanguageEnglish (US)
Pages15975-15984
Number of pages10
JournalJournal of Biological Chemistry
Volume291
Issue number31
DOIs
StatePublished - Jul 29 2016

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Ligases
Ubiquitin
Gene expression
Epithelial Cells
Gene Expression
MicroRNAs
Proteasome Endopeptidase Complex
Genes
Homeostasis
Transcription Factors
Tuners
RNA Sequence Analysis
Proteins
Caco-2 Cells
Enterocytes
Differentiation Antigens
Cell proliferation
3' Untranslated Regions
Bioinformatics
Computational Biology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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. / Peck, Bailey C.E.; Sincavage, John; Feinstein, Sydney; Mah, Amanda T.; Simmons, James G.; Lund, P. Kay; Sethupathy, Praveen.

In: Journal of Biological Chemistry, Vol. 291, No. 31, 29.07.2016, p. 15975-15984.

Research output: Contribution to journalArticle

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