Estrogen Regulation of MicroRNA Expression

Women outlive men, but life expectancy is not influenced by hormone replacement (estrogen + progestin) therapy. Estrogens appear to protect brain, cardiovascular tissues, and bone from aging. Estrogens regulate genes directly through binding to estrogen receptors alpha and beta (ERα and ERβ) that are ligand-activated transcription factors and indirectly by activating plasma membrane-associated ER which, in turns, activates intracellular signaling cascades leading to altered gene expression. MicroRNAs (miRNAs) are short (19-25 nucleotides), naturally-occurring, non-coding RNA molecules that base-pair with the 3’ untranslated region of target mRNAs. This interaction either blocks translation of the mRNA or targets the mRNA transcript to be degraded. The human genome contains ~ 700-1,200 miRNAs. Aberrant patterns of miRNA expression are implicated in human diseases including breast cancer. Recent studies have identified miRNAs regulated by estrogens in human breast cancer cells, human endometrial stromal and myometrial smooth muscle cells, rat mammary gland, and mouse uterus. The decline of estradiol levels in postmenopausal women has been implicated in various age-associated disorders. The role of estrogen-regulated miRNA expression, the target genes of these miRNAs, and the role of miRNAs in aging has yet to be explored.


INTRODUCTION
Women live longer than men. Estrogens (estradiol, estrone, and estriol) are steroid hormones that regulate development and homeostasis in a wide variety of tissues including the brain, reproductive tract, vasculature, and breast. Estradiol (E 2 ) is synthesized in the ovary and is the primary estrogen in premenopausal women. Animal studies have shown that higher estrogen levels in females protect against aging by upregulating the expression of antioxidant, longevity-related genes, e.g., selenium-dependent glutathione peroxidase (GPx) and Mn-superoxide dismutase (Mn-SOD) [1], by protecting against stroke-related injury [2], by vasorelaxing effects [3], by direct myocardial protection [4], and by activating the insulin receptor substrate (IRS)-1 signaling pathway [5]. Women's life expectancy seems not to be influenced by hormone replacement therapy (HRT = estrogens plus a progestin, usually conjugated equine estrogens and medoxyprogesterone acetate (MPA)) in postmenopausal women, but atherosclerosis and bone loss are considerably delayed. In addition, HRT protects against Alzheimer's disease (AD) [6], perhaps by suppressing elevated gonadotropin levels, i.e., luteinizing hormone (LH), in postmenopausal women since elevated LH is thought to play a key role in AD pathogenesis [7]. Other age-associated impairments are also reduced by estrogen. For example, premenopausal women have a reduced risk for cataracts compared with men of the same age group and women in the Farmingham study who used estrogen replacement therapy (ERT) showed reduced *Address correspondence to this author at the Department of Biochemistry & Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292, USA; Tel: 502-852-3668; Fax: 502-852-6222; E-mail: carolyn.klinge@louisville.edu risk for cataracts [8]. miRNAs are a class of naturallyoccurring, small, non-coding RNA molecules that are related to, but distinct from, small interfering RNAs (siRNAs) which regulate mRNA translation or stability [9][10][11]. There are very few studies on the hormonal regulation of miRNAs expression. Select changes in microRNA (miRNA) expression correlate with diagnostic markers used in breast cancer therapies, e.g., estrogen receptor (ER ) and tumor grade [12][13][14][15][16][17][18][19][20][21][22]. However, there are only 5 reports that E 2 regulates miRNA expression that will be reviewed here. It is highly likely that hormones play a major role in regulating miRNAs by both genomic (transcriptional) and non-genomic mechanisms of action. Identification and characterization of estrogen-regulated miRNAs may provide new biomarkers and therapeutic targets in aging as well as in diseases including breast cancer.

Genomic ER Activities
Initiation of transcription is a complex event occurring through the cooperative interaction of multiple factors at the target gene promoter. I will use the term ER to refer to either ER or ER or to both subtypes. I will refer to each subtype individually as pertinent to the known functions of these two proteins. Estrogen action is primarily mediated through binding to ER. ER and ER are members of the steroid/nuclear receptor superfamily of proteins of which there are 48 members in mammals [23]. ER and ER are highly conserved within the DNA binding domain (DBD, C domain), but differ in their N-and C-termini [24]. Structurally, ER has 6 domains lettered A-F from N-to C-terminus. ER is believed to be the ancestral steroid receptor originating 600-1200 million years ago, presumably because of the role of estrogens in reproduction and maturation [25,26]. In the simplest model, the binding of estradiol (E 2 ) via hydrogen bonding to residues within the ligand binding pocket of the ligand binding domain (LBD, E domain) results in conformational changes termed activation [27]. These conformational changes induced by E 2 binding result in loosening of contact between the N-terminus and the Cterminus of ER and exposes nuclear localization signals within the DNA binding domain (DBD, C domain) and hinge region (D domain) as well as altering structure of the LBD [28]. Crystal structure studies of the LBD of ER excluding the F domain shows that the LBD has 12 alpha helices and E 2 binding repositions helix 12 such that activation function-2 (AF-2) is exposed [29]. Helix 12 acts as a "switch" controlling accessibility of the coregulator interaction site. Ligand binding also facilitates ER dimerization (homodimerization or heterodimerization of ER and ER ). E 2 -liganded ER interacts directly with a specific DNA sequence called the estrogen response element (ERE = 5'-AGGTCAnnnTGACCT-3'), historically located in the promoter region and currently established to be located at great distances from the transcription start site including in the 3' flanking regions of target genes [30][31][32][33][34]. DNA binding increases ER interaction with basal transcription factors and coregulator proteins (reviewed in [35]). Fig. (1) depicts essential features of genomic ER action. EREs are enriched in genes upregulated by E 2 -ER , at least in MCF-7 cells [36]. ER can also be activated by phosphorylation of ser118 in the N-terminal A/B domain that activates activation function-1 (AF-1) in the absence of ligand binding [37]. At least in HeLa cells transfected with fluorescent fluorescent-tagged ER (GFP-or CFP-ER ), E 2 causes rapid intracellular and intranuclear movement of ER to form punctuate nuclear speckles that appear to indicate ER -nuclear matrix interaction [38][39][40]. In addition to direct ER-ERE binding, ER also activates transcription via a "tethering mechanism" in which ER interacts directly with transcription factors, e.g. Sp1 [41] and AP-1 [42], bound to their response elements. These DNA-protein and protein-protein interactions recruit coactivator/chromatin remodeling complexes resulting in histone modifications that lead to nucleosomal remodeling, increased accessibility to the DNA template for RNA polymerase II interaction, and increased target gene transcription (reviewed in [43][44][45]).
By definition, coactivators are proteins that interact directly with transcription factors to enhance transcription [46]. It is important to note that the term coactivator or corepressor is used when referring to an ER (or other NR) coregulator is gene-, cell-type, and context-specific [47]. This indicates that proteins classified as coactivators can also repress transcription and corepressors such as SMRT are gene-and cell-specific coactivators for ER [48]. Coactivators promote the assembly of the transcription initiation complex in part by altering chromatin structure and 'loosening' DNA-histone interactions, facilitated by increased histone lysine residue acetylation, methylation, ubiquitination, or sumoylation [49]. Once the transcription initiation complex is complete, RNA polymerase II (RNA pol II) is recruited to the transcription start site and begins transcription. By my count, at least 60 different ER coactivators and 23 corepressors have been functionally identified (reviewed in [43,50,51], see also http://www.nursa.org/index.cfm). The current model predicts that ERE-bound, agonist-liganded ER recruits coactivator proteins to enhance gene expression [52]. In contrast for those genes at which tamoxifen (TAM) is an antagonist of ER transactivation, the LBD of TAM-occupied ER does not interact with coactivators due to key conformational differences between agonist and antagonistoccupied ER in AF-2 [53]. TAM-occupied ER interacts with corepressors, e.g. NCoR or SMRT [54][55][56][57][58], that recruit histone deacetylase (HDAC) complexes thus keeping chromatin condensed and blocking transcriptional activation. Antiestrogen ICI 182,780 (Fulvestrant)-occupied ER is targeted to the 26S proteasome for degradation [40,59]. In contrast, although ICI 182,780 inhibits ER -mediated transcription, it stabilizes the ER protein [60].
Premenopausal women have a lower risk of developing cardiovascular disease [106,107] and hypertension than men or post-menopausal women and estrogens are thought to be responsible for regulating peripheral resistance [108] as well as effects in the myocardium [106]. Many of the cardioprotective activities of E 2 may be mediated by nongenomic signaling. Cumulative studies show that a subpopulation of intact ER is associated with the endothelial PM and with caveolae [61]. Recent electron microscopy studies revealed nuclear, cytoplasmic, and plasma membrane localization of both ER and ER in human umbilical vein endothelial cells (HUVEC) [109]. ER [79,110] has been shown to interact with caveolin-1 (Cav-1) which serves as a structural core for interaction of plasma-membrane-associated proteins including the -subunit of G-proteins, Ha-Ras, Src-kinases, eNOS, epidermal growth factor (EGF) receptors, and some protein kinase-C isoenzymes [110]. E 2 -ER interaction within caveolae leads to G i activation, MAPK and Akt signaling, and perturbation of the local Ca +2 environment, leading to eNOS phosphorylation and NO production [61]. Endothelial PM-associated ER is coupled via a G i to MAPK and eNOS [111].
In addition to PM-associated ER, GPR30 has reported to serve as a membrane estrogen receptor because it binds E 2 with high affinity (Kd = 2.7nM) and activates adenylate cyclase, thus increasing cAMP levels [70,73,[131][132][133][134]. GPR30 is distinct from ER and ER in that ICI 182,780 and tamoxifen also bind GPR30 with high affinity and mimic the effects of E 2 [133]. Although the role of GPR30 in MCF-7 and SKBr3 breast cancer cells has been questioned [62], it appears likely that GPR30 is a bone fide membrane estrogen receptor in some cell types [135][136][137][138][139][140][141].

MicroRNAs (miRNAs)
Evidence from the Encyclopedia of DNA project (EN-CODE) has revealed surprising new information about the human genome. For example, although the protein coding ER and ER are located in the cytoplasm and nucleus, bound to Caveolin-1 in caveolae in the plasma membrane and inside mitochondria. For genomic (nuclear) ER activity, E 2 binds and activates ER resulting in dimerization, ERE binding or interaction with other transcription factors, e.g. AP-1 bound to DNA, coregulator and chromatin remodeling complex recruitment, chromatin remodeling, and increased transcription of target genes. For nongenomic/membrane-initiated estrogen signaling, E 2 binds ER in caveolae in the plasma membrane [112,215]. ER interacts with G-proteins, the p85 subunit of PI3K, c-Src, and Cav-1 to initiate PI3K/AKT and MAPK signaling cascades [61,216]. ER interacts with MNAR [127] and Shc [89] in the cytoplasm. ER interacts with the EGF-and IGF-1 receptors in plasma membranes. In mitochondria, ER interact with the D-loop of mtDNA [217,218].
regions account for only 2% of the total DNA in the human genome, surprisingly, 80-93 % of the genome is "expressed" [142][143][144]. The transcribed RNAs are largely conserved between humans and mice suggesting that these noncoding RNAs (ncRNAs) have important functions. Evidence of the importance of the various types of ncRNAs was recently reviewed [145] and includes roles in cancer, diabetes, and coronary disease, all aging-associated disorders. Among the small ncRNAs are microRNAs (miRNAs). The importance of miRNAs is highlighted by the fact that the 2008 Albert Lasker Award for Basic Medical research was awarded to Drs. Victor Ambros [146] and Gary Ruvkun [147] who discovered and characterized the first miRNAs in C. elgans and Dr. David Baulcombe who discovered let-7 miRNA in plants [148] (see also http://www.laskerfoundation.org/). miRNAs are a class of naturally-occurring, small, noncoding RNA molecules that are related to, but distinct from, small interfering RNAs (siRNAs) [9][10][11]. About half of miRNAs are expressed from introns of protein-coding transcripts and miRNAs have 5' and 3' sequence features that form boundaries including transcription start sites, CpG islands, and transcription factor binding recognition elements [149]. miRNAs may be differentially processed from the sense and antisense strands of the same hairpin RNA or transcripts from the same locus, thus expanding the number of miRNAs from a single genomic locus [145].
The pathway of mature miRNA biogenesis is depicted in Fig. (2). miRNA genes are mostly transcribed by RNA polymerase II into primary-micro-RNAs (pri-miRNAs) that are capped and polyadenylated [150]. Pri-microRNAs contain self-base-pairing stem-loop structure that is necessary for critical processing within the nucleus by Drosha, an endonuclease of the RNAse III family, and its cofactor DGCR8 into short (60 to 70 nt) imperfect hairpin structure precursor-miRNAs (pre-miRNAs) [151]. These pre-miRNAs are exported from the nucleus by exportin and Ran-GTP. Pre-miRNAs are processed by the cytoplasmic RNAse II enzyme Dicer to form mature ~22 nt transiently double-stranded miRNA duplexes that are transferred to Argonaute proteins (Ago1, Ago2, Ago3, and Ago4 [152]) in the RNA-induced silencing complex (RISC), leading to unwinding of the duplexes to form single stranded microRNAs. RISC guides RNA silencing with the miRNA binding either to the 3' untranslated region (3' UTR) or to the open reading frame (ORF) of its target mRNA [153][154][155][156]. Most commonly, because of imperfect complimentarity of the base pairing between the miRNA and the 3'UTR, the RISC complex causes translational repression by RISC interaction with eIF6 which prevents assembly of 80S ribosomal assembly [157] or by inhibition of translation [16]. Thus, miRNA-mRNA 3'UTR interaction results in a decrease in target protein, not mRNA. The 7 to 8 nucleotide region of basepairing between the 5' end of the mature miRNA and the mRNA is called the 'seed sequence'. Base pairing of the miRNA-RISC complex within the ORF requires almost perfect complimentarity for its mRNA target and the mRNA is either degraded or translation is blocked [150]. The miRNA-containing ribonucleoprotein particle (miRNP)-silenced mRNA is directed to the Pbodies, where the mRNA is either released from its inhibi- Fig. (2). Model of miRNA biogenesis and function. Primary transcripts of microRNAs (pri-miRNAs) are transcribed by RNA polymerase II, processed by the RNAse III enzyme, Drosha and its cofactor DGCR8, to precursor microRNAs (pre-miRNAs) and are then exported from the nucleus by Exportin/RAN-GTP [150]. In the cytoplasm, pre-miRNAs are processed by the RNAse III enzyme, Dicer to form mature ~22 nt transiently double-stranded miRNA duplexes that are transferred to Argonaute proteins (Ago1, Ago2, Ago3, and Ago4 [152]) in the RNAinduced silencing complex (RISC), leading to unwinding of the duplexes to form single stranded miRNAs. The mature miRNAs bind either to the 3' untranslated region (3' UTR) or to the open reading frame (ORF) of its target mRNA [153][154][155][156]. Binding of miRNA/RISC complex with the 3'UTR causes translational repression [16]. Thus, miRNA-mRNA 3'UTR interaction results in a decrease in target protein, not mRNA. tion upon a cellular signal and/or actively degraded [158]. Comparative genomics analyses have revealed that over 45,000 miRNA binding sites within human 3'UTRs that are conserved above background levels [159]. This number was reported to indicate that more than 60% of human proteincoding genes have been under selective pressure to maintain pairing to miRNAs [159]. Recent evidence indicates that miRNAs may also increase translation of select mRNAs in a cell cycle-dependent manner [160].

Altered miRNA Expression in Breast Cancer
The spectrum of miRNAs expressed in solid tumors, i.e., prostate, colon, stomach, pancreas, lung, and breast, is different from normal tissues [177]. Although the precise sequence of events leading to breast tumors is not understood, lifetime exposure to estrogens is widely accepted as a major risk factor for the development of breast cancer [179]. Some investigators have documented that E 2 is carcinogenic in human breast epithelial cells [180][181][182]. However, epidemiological evidence disputing the carcinogenicity of E 2 in humans has been published [183]. Surprisingly, there are no published studies evaluating the effect of E 2 on global miRNA expression in breast cancer cells.
Aberrant patterns of miRNA expression have been reported in human breast cancer [12, 13, 18, 20, 21, 151, 162, 164-170, 176-178, 184-191] and recently reviewed [150]. The first miRNA study in breast cancer indicated differential expression of miRNAs in concordance with other wellestablished markers of breast cancer stage and patient prognosis including ER and PR expression, tumor stage, number of positive lymph nodes, and vascular invasion [20]. Different miRNA expression profiles were also associated with ErbB2+ versus ER+ tumors [22]. More recently, patients whose breast tumors showed reduced miR-126, miR-206, or miR-335 were found to have reduced survival, regardless of ER or ErbB2 status [18].
A number of genes involved in breast cancer progression have been identified by in silico analysis to be targets of miRNAs that are deregulated in breast cancer [192] and some have been experimentally proven. A recent study reported that miR-21 expression was reduced in breast tumors and that antisense to miR-21 suppressed MCF-7 breast cancer cell growth in vitro and as tumor xenografts in mice by regulating Bcl-2 [13]. Interestingly, we recently reported that overexpression of miR-21 in MCF-7 cells increased soft agar colony formation, reflecting increased tumorigenicity of these cells [193]. We demonstrated that miR-21 binds to a seed element in the 3'-UTR of the programmed cell death 4 (PDCD4) gene and reduces Pdcd4 protein expression [193].

Estrogenic Regulation of miRNA Expression
A PubMed search for estrogen AND miRNA revealed 27 papers. However, in that list and in total there are, to my knowledge, only 6 studies in which miRNA regulation by E 2 has been directly examined (see below). Indeed, although a software application that will retrieve all miRNA:mRNA functional pairs in an experimentally derived set of genes was recently developed and used to identify E 2 -regulated mRNA genes in breast cancer [196], this paper does not experimentally address miRNA changes regulated by E 2 .

E 2 Regulation of miRNAs in Animal Studies
The effect of E 2 in miRNA expression has been examined in zebrafish [197], August Copenhagen Irish (ACI) rats [198], and mouse splenocytes [199]. A recent review of miRNA expression in female mammalian reproductive tissues described transgenic and knockout mouse models and findings related to changes in miRNAs in the ovary and uterus in response to deletion of Dicer [200], LH, and during development (immature versus mature mice) [201]. Changes in miRNA expression in mouse uterus during implantation have been cataloged [202]. Importantly, the authors of this review concluded that the expression, regulation, and function of miRNAs within specific tissues and cells still needs to be determined [201].
A study of the effect of E 2 on miRNA expression in the adult (3 mos) zebrafish male (Danio rerio) identified altered expression of 38 miRNAs in the whole body homogenates [197]. E 2 was added to the aquariums at a final concentration of 5 g/liter (18 nM) and although various times of treatment were analyzed, most miRNA changes in response to E 2 were observed after 12 h. miRNAs were regulated by E 2 in a tissue-specific manner with E 2 downregulating miRNAs in the liver and increasing miRNA expression in the skin of the zebrafish. For example, miR-122 was decreased by E 2 in skin, but increased in gills, intestine. and liver. Among the most up-regulated miRNAs were miR-196b and let-7h, and miR-130c and miR-101a were the most down-regulated. The authors identified Hoxb8a as a target of miR-196b and showed that E 2 , by increasing miR-196b, decreased Hoxb8a [197]. The authors concluded that miR-196b may serve as "a biomarker of exposure to environmental estrogens and endo-crine-disrupting chemicals that fish may encounter in their aquatic environment" [197].

E 2 Regulation of miRNAs in Human Cell Lines
A study identifying miRNAs expressed in myometrial and leiomyoma smooth muscle cells (MSMC and LSMC) using microarray and real time PCR reported that E 2 inhibited the expression of miR-21 in MSMC and LSMC, whereas E 2 increased and inhibited miR-26a in MSMC and LSMC, respectively [210]. In contrast, ICI 182,780 increased the expression of miR-20a and miR-21 in MSMC and LSMC, and miR-26a in MSMC, while inhibiting the expression of miR-26a in LSMC [210]. No mechanistic studies or mRNA target gene studies were performed to identify the mechanism(s) involved in these cell-specific differences in miRNA regulation by E 2 and ICI or their downstream targets.
To identify E 2 regulated miRNAs in a classical estrogenresponsive human breast cancer cell line, we treated ERpositive MCF-7 cells with 10nM E 2 or EtOH (vehicle con-trol) for 6 h to identify primary E 2 target miRNAs. RNA was harvested, labeled either with Cy3 or Cy5, and hybridized with two identical, dual-color miRNA microarrays from LC Sciences. This array contained probes to detect mature miRNA sequences as well as precursor (pre)-miRNAs in the Sanger miRNA registry 7.0 (http://microrna.sanger.ac.uk/ sequences/). The differentially expressed transcripts that were consistent on both chips are summarized in Table 1. 38 miRNA genes were regulated by E 2 : 9 were reduced and 29 were increased. A summary about what is known about each of these E 2 -responsive miRNAs in terms of breast cancer and estrogen-responsiveness is included in Table 1.

miRNAs Regulating ER Expression or Activity
miRNAs can influence estrogen-regulated gene expression by directly reducing ER mRNA stability or translation. Four miRNAs have been reported to reduce ER protein levels (Fig. 3). Two miR-206 recognition sites were identified in the 3'UTR of ER and transfection of an expression vector for miR-206 in MCF-7 cells reduced both mRNA and protein levels of ER [162]. Treatment of MCF-7 cells with 1nM E 2 or the ER agonist PPT (10nM) reduced miR-206 levels by ~ 80%. In contrast the ER agonist DPN (10nM) increased miR-206 expression by ~ 60%. Interestingly, the investigators found that miR-206 levels were significantly higher in ER -negative MDA-MB-231 cells than in MCF-7 cells, suggesting a mechanism for miR-206 in repressing ER protein levels in MDA-MB-231 cells. The authors suggested that miR-206 may function in a mutually negative feedback loop to temporally regulate ER expression and ductal/lobuloalveolar proliferation [162]. More recent studies showed that miR-206 is inversely correlated with ER expression, but not ER , in human breast tumors [211]. Further, transfection of MCF-7 human breast cancer cells with an expression plasmid for pre-miR-206 reduced ER mRNA expression ~ 25%, reduced the basal expression levels of PR, cyclin D1, and pS2 (all well-established ER -regulated genes), and inhibited cell proliferation with or without E 2 [211]. miR-221/222 was recently reported to be higher in ER negative than ER positive breast cancer cell lines and human breast tumors [212]. Two miR-221 and miR-222 seed elements were identified in the 3'UTR of ER and transfection of miR-221 and miR-222 suppressed ER protein, but not mRNA in ER positive MCF-7 and T47D cells. Conversely, knockdown of miR-221 and miR-22 in ERnegative MDA-MB-468 partially restored ER protein expression and increased tamoxifen-induced apoptosis [212]. miR-22 regulates ER protein expression in a pancreatic cancer cell line [213]. In a study to identify curcumin gene targets, curcumin increased miR-22 by 65% in BxPC-3 human pancreatic carcinoma cells [213]. One of the predicted 3'UTR gene targets of miR-22 was ESR1 (ER ) [213]. Follow-up studies showed that curcumin reduced ER protein expression in BxPC-3 cells and that transfection of an antisense RNA oligonucleotide of miRNA-22 into BxPC-3 cells increased ER protein by ~ 1.9-fold. Thus, miR-22 regulates ER protein levels and the authors suggest a role for ER as anti-tumorigenic in pancreatic cancer. Expression was higher in ER + than ER -tumors [12].
let-7f -0.25 let-7f was > in node negative versus positive human breast tumors [22] and higher in ER + tumors [12]. let-7f in mammary gland was reduced by E2 treatment of female ACI rats [198]. Higher in PR+ than PR-tumors, but higher in ER -than ER + breast tumors [12]. miR-106b is overexpressed in breast tumors compared to normal breast and miR-106b reduced p21 mRNA and protein and thus stimulates G1-S cell cycle progression in human mammary epithelial cells [224]. miR-106b in mammary gland was increased by 6 wks of E2 treatment of female ACI rats [198].

E 2 Regulation of Ago2 and ER in Human Breast Cancer Cell Lines
Argonaut-2 (Ago2), the catalytic subunit of the RISC complex that mediates miRNA-dependent cleavage/degradation in mammals [154,170,214], expression is higher in ER -negative, HER2-positive (basal) than ER -positive/ HER2 negative (luminal) human breast cancer cell lines and tumors [14]. E 2 and the ER -agonist PPT, but not the ERagonist DPN, increased Ago2 protein expression in MCF-7 cells [14]. Further studies showed that EGF acts through the MAPK pathway to increase Ago2 protein stability, but there were no studies examining the mechanism by which E 2 and PPT, presumably through ER , increase Ago2 protein levels. Surprisingly, Ago2 overexpression in MCF-7 cells increased ER protein levels by 3-fold, despite also increasing miR-206 that reduces ER . The authors concluded that this "discordant" finding indicates that there is a greater concentration of miRNAs than target proteins involved in ER suppression than those that target ER itself" [14].

CONCLUSION
Estrogen signaling plays a critical role in regulating reproduction, lactation, bone density, cardiovascular function, neuronal signaling, immune function, and homeostasis in a wide variety of tissues. The reduction in serum E 2 in postmenopausal women is involved in a number of ageassociated disorders. Research on the mechanisms by which E 2 and other estrogens regulate diverse physiological effects has established both genomic and nongenomic mechanisms involving ER , ER , and GPR30 in signal transduction (Fig.  1). miRNAs are small, non-coding RNAs that bind to the 3' UTR of target mRNAs and either block the translation of the message or bind the ORF and target the mRNA transcript to be degraded. Although there are a number of studies identifying miRNA changes in breast tumors and comparing ERpositive versus ER -negative miRNA signatures for their potential use as biomarkers, there are few studies identifying E 2 -responsive miRNAs in any normal or neoplastic tissues or cell models. In those few studies that have identified E 2induced alterations in miRNA expression, there is little, if any, mechanistic detail elaborated for the E 2 effect (s) on miRNA expression. Further, it appears that E 2 regulates miRNA expression in a cell-type-dependent manner. Thus, identification of E 2 -regulated miRNAs and the function of miRNAs within specific tissues and cells still remains to be determined.

ACKNOWLEDGEMENT
Thanks to Dr. Nalinie S. Wickramasinghe who treated the MCF-7 cells and isolated the RNA for the miRNA microarray study included in Table 1. This study was supported by NIH R21 CA124811 to CMK.