In Vitro Identification of miR-718 targets and miR-718-induced epigenetic modifications

Abstract Epigenetic modifications are now recognized as contributing factors in many diseases. Modifications in gene expression can be induced by DNA methylation/hydroxymethylation, histone changes, and non-coding RNAs rather than changes in gene sequence. Of particular interest are microRNAs (miRNAs), a class of non-coding

RNAs that target messenger RNA molecules with complementary sequences for degradation. MiR-718 is a little-studied miRNA that may play a role in cancers, cardiovascular disease, and neurological disorders. According to miRbase.org, miR-718's predicted targets include genes that affect calcium signaling,

mitochondrial health, autophagy, and apoptosis. Current literature indicates miR-718 upregulation leads to a decrease in PTEN (Phosphatase and Tensin homolog) mRNA and indicates the role of PTEN in apoptosis and angiogenesis in cancer cells and the cardiovascular system, respectively, from small samples of human

subjects. The current state of literature regarding miR-718 is limited, yet miR-718 has the potential to impact various cell pathways involved in different diseases and disorders. My laboratory upregulated miR-718 in SH- SY5Y cells (a human neuronal model), using a mimic siRNA to miR-718. Our preliminary RT-qPCR data show

miR-718 upregulation downregulates expression of PTEN, DNA methyltransferase 3A (DNMT3A), Methyl- CpG-Binding protein 2 (MECP2), and miR-132. We chose these genes because PTEN, DNMT3A, and MECP2 are predicted targets of miR-718, and because several calcium channel genes are predicted targets (calcium

signaling affects CREB phosphorylation and hence transcription of genes), miR-132 would be an indirect target of miR-718. We also stained miR-718 mimic-treated SH-SY5Y cells with Tetramethylindocarbocyanine Perchlorate (“DiI”) to determine if miR-718 upregulation affects dendritic spines. Our generated preliminary

data show miR-718 upregulation leads to increased dendritic spines. Because of the potential to impact gene expression and epigenetics genome-wide, we propose to (1) determine the effects of miR-718 upregulation on gene expression of miR-718 predicted targets using RNA sequencing (RNAseq), and (2) investigate the effects

of miR-718 upregulation on DNA methylation via DNA methylation sequencing and histone modifications using Histone Modification Arrays, Western Blotting, and flow cytometry. We expect that if we upregulate miR-718, targets of miR-718 will be downregulated in RNAseq data, validating those genes as targets of miR-718. We

also expect that because our preliminary data shows miR-718 upregulation leads to a significant decrease in DNMT3A and MECP2, our epigenetic studies will reveal loss of DNA methylation and histone modifications.