MicroRNA-99 Family Members Suppress Homeobox A1 Expression in Epithelial Cells

Abstract

The miR-99 family is one of the evolutionarily most ancient microRNA families, and it plays a critical role in developmental timing and the maintenance of tissue identity. Recent studies, including reports from our group, suggested that the miR-99 family regulates various physiological processes in adult tissues, such as dermal wound healing, and a number of disease processes, including cancer. By combining 5 independent genome-wide expression profiling experiments, we identified a panel of 266 unique transcripts that were down-regulated in epithelial cells transfected with miR-99 family members. A comprehensive bioinformatics analysis using 12 different sequence-based microRNA target prediction algorithms revealed that 81 out of these 266 down-regulated transcripts are potential direct targets for the miR-99 family. Confirmation experiments and functional analyses were performed to further assess 6 selected miR-99 target genes, including mammalian Target of rapamycin (mTOR), Homeobox A1 (HOXA1), CTD small phosphatase-like (CTDSPL), N-myristoyltransferase 1 (NMT1), Transmembrane protein 30A (TMEM30A), and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5). HOXA1 is a known proto-oncogene, and it also plays an important role in embryonic development. The direct targeting of the miR-99 family to two candidate binding sequences located in the HOXA1 mRNA was confirmed using a luciferase reporter gene assay and a ribonucleoprotein-immunoprecipitation (RIP-IP) assay. Ectopic transfection of miR-99 family reduced the expression of HOXA1, which, in consequence, down-regulated the expression of its downstream gene (i.e., Bcl-2) and led to reduced proliferation and cell migration, as well as enhanced apoptosis. In summary, we identified a number of high-confidence miR-99 family target genes, including proto-oncogene HOXA1, which may play an important role in regulating epithelial cell proliferation and migration during physiological disease processes, such as dermal wound healing and tumorigenesis.

In Vivo Activation of a Conserved MicroRNA Program Induces Mammalian Heart Regeneration

“…Bioinformatic predictions allow for the pre-screening of potential miRNA target sequences, however they do not unequivocally identify target genes and potential targets need to be validated by further biochemical approaches. As reported in other species, luciferase reporter assays confirmed microRNA binding predictions to both the human and zebrafish fntb and smarca5 3′ UTRs (Mueller et al, 2012; Brenner et al, 2012; Chen et al, 2013; Liang et al, 2014; Sun et al, 2011) (Figures S1D and S1E). Together, these results demonstrate that both fntb and smarca5 mRNAs are direct targets of miR-99/100.…”

Laser Capture Microdissection of Epithelium from a Wound Healing Model for MicroRNA Analysis

“…3, demonstrating a decrease in the miR-100 level in epithelial cells harvested from a wounded area as compared to epithelial cells from an unwounded area (control sample). This is in agreement with our previously published findings [11, 12]. …”

Identification of differentially expressed miRNAs through high-throughput sequencing in the chicken lung in response to Mycoplasma gallisepticum HS

“…Li et al, 2014). miR-99 family members may play an important role in regulating epithelial cell proliferation and migration during physiological disease processes (Chen et al, 2013), and miR-126 could be a promising biomarker for cancer prognosis prediction, especially in patients with digestive or respiratory system cancers (Dong et al, 2016). Our study suggests that miR-146, miR-101, miR-99, miR-122 and miR-126 might be very useful for further investigating the underlying mechanisms of miRNAs in MG infection.…”

“…Li et al, 2014;Stik et al, 2013), avian leucosis (H. Li et al, 2014;Q. Wang et al, 2013), infectious bursal disease (Y.-s. Wang et al, 2013), and ovarian carcinoma (Lee et al, 2012). All of these studies suggest that miRNAs may play important roles in MG infection in chicken.…”