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.
Highlights
MicroRNAs are a class of small non-coding RNAs of approximately 22 nucleotides in length that are endogenously expressed in mammalian cells
Using dermal wound healing and oral squamous cell carcinoma as independent model systems, two of our recent studies showed that the miR-99 family members play an important role in regulating proliferation and migration of skin and oral mucosa epithelial cells, respectively [4,5]
Our dermal wound healing study showed that the miR-99 family members are co-regulated during the wound healing process, and the downregulation of miR-99 family contributes to re-epithelialization of the wound by regulating cell proliferation, apoptosis and cell migration [4]
Summary
MicroRNAs are a class of small non-coding RNAs of approximately 22 nucleotides in length that are endogenously expressed in mammalian cells. We functionally assessed the role of selected target genes, including Homeobox A1 (HOXA1), in miR-99 family-mediated regulation of proliferation and cell migration in epithelial cells. Hsa-miR-99a, hsa-miR-99b, hsa-miR-100 and non-targeting microRNA mimic (Dharmacon), and gene specific siRNAs for mTOR, HOXA1, CTDSPL, NMT1, TMEM30A, SMARCA5 and Bcl-2 (Santa Cruz Biotechnology) were transfected into the cells using DharmaFECT Transfection Reagent 1 as described previously [20,21].
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