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

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.

Protocol for the long‐term culture of human primary keratinocytes from the normal colorectal mucosa

“…studies carried out on cell cultures obtained from tissue biopsies taken from experimental animals (Evans, Flint, & Potten, 1994), and human cancer specimens (Rousset, 1986). Experimental data obtained with human cell lines from colorectal carcinomas, such as Caco-2, HT29, and T84, which may spontaneously differentiate into intestinal-like cells (Moberg, Bell, Wahrer, Haber, & Hariharan, 2001;Peck et al, 2016), are limited by the cancerous nature of these cellular models (Whitehead, Vaneeden, Noble, Ataliotis, & Jat, 1993).…”

Evolutionary Dynamics of microRNA target sites across vertebrate evolution

“…The success of our method in enriching for deeply conserved miRNAs with known deeply conserved functions implies that miR-20 and miR-30 also have deeply conserved functions. miR-30 has been shown to play a role in thermogenesis, which may form part of a conserved pathway among mammals, and appears to play important roles in adipogenesis and intestinal epithelial cell homeostasis [58][59][60], while miR-20 has roles in suppressing angiogenesis and is a member of a miRNA family that is essential in mice [61][62][63]. Thus, both of these miRNAs may represent promising candidates for the discovery of conserved and essential miRNA-mediated regulatory pathways among mammals, and potentially other bilaterian species.…”

MicroRNAs at the epicenter of intestinal homeostasis

“…MiR‐30 was recently identified as an important regulator of intestinal proliferation and differentiation . The expression of miR‐30 was found to be higher in proliferating cells than in differentiated cells.…”