The proposed exploratory research will examine whether the effects of estrogen (E2) on cell proliferation and cholesterol metabolism are exerted, in part, through control of specific microRNA expression. Cellular cholesterol levels are maintained through several dynamic and complex mechanisms, which involve cholesterol synthesis, storage, efflux, uptake and intracellular transport. Cellular hypertrophy and proliferation increases the demand for cholesterol, which is required for new membranogenesis. E2 is a potent mitogenic steroid that induces hypertrophy and proliferation, and promotes tumor progression of several organs (e.g., breast, uterus). E2 exerts broad pleiotropic control on cellular processes, including increasing cholesterol synthesis and uptake. MicroRNAs (miRNAs) represent a large class of recently-discovered regulatory molecules. MicroRNAs are hormonally regulated in Drosophila, and show developmental and cell-specific expression in mammals. Human breast cancer MCF-7 cells and liver HepG2 cells will be treated with E2 or the antiestrogen, ICI 182780.
Specific Aim 1 will screen for E2-regulated expression of miRNAs by microarray, and confirm results by RNase protection assay. The messenger RNAs (mRNAs) of several proteins that are involved in cholesterol metabolism and are regulated by E2 are predicted targets of miRNAs.
Specific Aim 2 is to examine the effects of decreased miRNA on cell proliferation and aspects of cellular cholesterol homeostasis, and the ability of E2 to regulate these processes. Specific miRNAs, as selected by being regulated by E2 (Aim 1) or by being predicted to target mRNAs encoding cholesterol-related proteins, will be inhibited using 2' O-methyl-modified miRNA decoys.
Specific Aim 3 involves the search for targets of miRNAs from Aims 1 & 2 using databases.
Aim 3 also includes the development of an experimental approach to the discovery of miRNA targets by utilizing strepavidin pull-down of biotinylated miRNA-nucleoprotein complexes formed in vitro, followed by cDNA amplification, cloning and sequencing. This research will broaden our understanding of how E2 exerts its effects on normal cell function, as well as on the progression of estrogen-responsive cancers. This increased insight will improve our ability to design estrogen-related compounds for the use in treating symptoms associated with menopause, ovarian dysfunction, and estrogen-responsive cancers. ? ?
