Amanda N. Kallen

The Imprinted H19 LncRNA Antagonizes Let-7 MicroRNAs

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.

Gender differences in cardiovascular disease: hormonal and biochemical influences.

Objective: Atherosclerosis is a complex process characterized by an increase in vascular wall thickness owing to the accumulation of cells and extracellular matrix between the endothelium and the smooth muscle cell wall. There is evidence that females are at lower risk of developing cardiovascular disease (CVD) as compared to males. This has led to an interest in examining the contribution of genetic background and sex hormones to the development of CVD. The objective of this review is to provide an overview of factors, including those related to gender, that influence CVD. Methods: Evidence analysis from PubMed and individual searches concerning biochemical and endocrine influences and gender differences, which affect the origin and development of CVD. Results: Although still controversial, evidence suggests that hormones including estradiol and androgens are responsible for subtle cardiovascular changes long before the development of overt atherosclerosis. Conclusion: Exposure to sex hormones throughout an individual’s lifespan modulates many endocrine factors involved in atherosclerosis.

Cardiovascular disease and ovarian function.

Purpose of review Coronary heart disease (CHD) is the leading cause of death in the aging female population in the developed world. Ovarian endocrinology plays an important role in modulating a womans CHD risk. We herein present an overview of our current understanding of CHD risk profile in the context of ovarian physiology and senescence. Recent findings Endogenous ovarian estrogen has long been recognized to offer cardiac benefit and vascular protection against atherosclerosis. Existing data, however, do not allow for an extrapolation of the recognized cardioprotective implications of the reproductive-age endogenous estrogenic milieu to the use of exogenous estrogen in postmenopausal women. Ongoing efforts are targeting the concept that when reintroduced proximate to onset of ovarian senescence, exogenous estrogen may retard the process of atherogenesis. Until this hypothesis is substantiated, cardioprotection must not be an indication for initiating hormone therapy in menopausal women. Summary Ovarian hormones modulate the processes of atherosclerosis and the mechanisms underlying CHD. The female reproductive hormones offer a cardioprotective milieu that is rapidly attenuated with the cessation of ovarian function (be it following natural menopause or after medical or surgical ovarian extirpation). The role of exogenous hormone therapy, and the nuances of timing and duration of exposure, are still being elucidated.

Poor ovarian response in women undergoing in vitro fertilization is associated with altered microRNA expression in cumulus cells

OBJECTIVE To analyze the association of micro-ribonucleic acid (miRNA) expression with the number of oocytes retrieved, in women undergoing in vitro fertilization (IVF). DESIGN Experimental study. SETTING Academic medical center. PATIENT(S) A total of 189 women undergoing IVF-intracytoplasmic sperm injection (ICSI). INTERVENTION(S) Pooled cumulus cells were collected. MAIN OUTCOME MEASURE(S) Poor responders were identified as patients who produced fewer oocytes than the 25th percentile of their respective age group. MicroRNAs were extracted from cumulus cells, and an miRNA microarray was performed, comparing poor responders (n = 3) to non-poor responders (n = 3). Expression of miR-21-5p (active strand of miR-21) and miR-21-3p was tested in poor responders (n = 21) and non-poor responders (n = 29), using reverse transcription real-time polymerase chain reaction (qRT-PCR). Regulation of miR-21-5p and miR-21-3p, in human granulosa-like tumor (KGN) cells, by estradiol (E2), was tested in vitro. RESULT(S) MicroRNA microarray analysis showed up-regulation of 16 miRNAs and down-regulation of 88 miRNAs in poor responders. Notably, miR-21 was significantly up-regulated 5-fold in poor-responder samples. Analysis using qRT-PCR confirmed that miR-21-5p expression was significantly up-regulated in poor responders, whereas miR-21-3p expression was significantly lower, suggesting that elevated miR-21-5p expression in cumulus cells is not regulated at the pre-miR-21 level in poor responders. Both miR-21-5p and miR-21-3p were increased in KGN cells in response to higher doses of E2; their expression was not affected at lower E2 concentrations. CONCLUSION(S) We found that poor response to IVF is associated with altered miRNA expression in cumulus cells, specifically with elevated expression of miR-21-5p, and that this elevated expression is independent of lower serum E2 levels in poor responders.

Does Lin28 Antagonize miRNA-Mediated Repression by Displacing miRISC from Target mRNAs?

Lin28 is a developmentally regulated RNA-binding protein that plays important roles in diverse physiological and pathological processes including oncogenesis and brain synaptic function. These pleiotropic roles of Lin28 are mechanistically linked both to its ability to directly stimulate translation of genes involved primarily in cell growth and metabolism and to its ability to block biogenesis of a subset of miRNAs including the let-7 family of miRNAs. In the case of direct stimulation of gene expression, Lin28 binds to targeted mRNAs through recognition of Lin28-responsive elements (LREs) within mRNAs and recruits RNA helicase A (RHA) to promote translation. RHA belongs to the DEAD-box protein family of RNA helicases, which generally catalyze ATP-dependent unwinding of RNA duplexes or remodeling of ribonucleoprotein complexes (RNPs). Since any given mRNA can potentially be inhibited by miRNAs bearing complementary sequences, we hypothesize that binding of Lin28 to LREs not only nucleates the binding of multiple Lin28 molecules to the same mRNA, but also leads to remodeling of RNPs through recruitment of RHA and causes release of inhibitory miRNA-induced silencing complexes bound to the mRNA. This mode of action may contribute to Lin28-mediated stimulation of translation in both tumor and neuronal cells.

The Steroidogenic Acute Regulatory Protein (StAR) Is Regulated by the H19/let-7 Axis

The steroidogenic acute regulatory protein (StAR) governs the rate-limiting step in steroidogenesis, and its expression varies depending on the needs of the specific tissue. Tight control of steroid production is essential for multiple processes involved in reproduction, including follicular development, ovulation, and endometrial synchronization. Recently, there has been a growing interest in the role of noncoding RNAs in the regulation of reproduction. Here we demonstrate that StAR is a novel target of the microRNA let-7, which itself is regulated by the long noncoding RNA (lncRNA) H19. Using human and murine cell lines, we show that overexpression of H19 stimulates StAR expression by antagonizing let-7, which inhibits StAR at the post-transcriptional level. Our results uncover a novel mechanism underlying the regulation of StAR expression and represent the first example of lncRNA-mediated control of the rate-limiting step of steroidogenesis. This work thus adds to the body of literature describing the multiple roles in oncogenesis, cellular growth, glucose metabolism, and now regulation of steroidogenesis, of this complex lncRNA.

HOXA10 Regulates Expression of Cytokeratin 15 in Endometrial Epithelial Cytoskeletal Remodeling

Objective: The mammalian cytoskeleton is composed in part from keratin filaments which form a complex, highly dynamic intracellular network. We investigate the expression of cytokeratin 15 (CK15) in human endometrium and its regulation by HOXA10 in the human endometrial cell lines. Methods: Endometrial biopsies from throughout the menstrual cycle (N = 32) were evaluated for CK15 protein expression by immunohistochemistry using a mouse monoclonal antibody. The human endometrial epithelial cell line (Ishikawa) was transfected with pcDNA/HOXA10. Total RNA was isolated and quantitative real-time polymerase chain reaction was performed to determine expression levels of CK15. Results: In the peri-implantation window (days 16 through 23) CK15 protein expression in glandular epithelium of human endometrium decreased to 50% of proliferative phase expression levels. Expression of CK15 messenger RNA decreased by 99% (P < .05) after pcDNA/HOXA10 transfection of Ishikawa cells. The CK15 expression corresponded to the time of maximal secretory epithelial remodeling. Conclusion: Gene expression of CK15 is decreased in a HOXA10-dependent fashion in human endometrial epithelial cells. Expression decreases in the peri-implantation period concurrent with maximal HOXA10 expression. Dramatic changes in cellular architecture are necessary to achieve the secretory changes in the endometrial epithelium that bring about the implantation window. Alterations in CK15 likely facilitate these cytoskeletal changes, ultimately promoting endometrial receptivity.

Hormone therapy: A to Z.

Clinical Scenario A 65-year-old postmenopausal women comes into the office with concerns of vaginal dryness and hot flashes. She recounts numerous nights waking up drenched in sweat and days spent trying to find the coolest rooms at work. She reluctantly mentions that she and her partner have difficulty with intimacy because she has found intercourse to be painful because of vaginal dryness. She is distraught and frustrated seeking something to alleviate her menopause symptoms. As you begin a conversation about hormone therapy, she immediately becomes concerned and reluctant because she has seen many commercials indicating that this unsafe for women.

Endothelin Type A Receptor (ETA) Expression Is Regulated by HOXA10 in Human Endometrial Stromal Cells

Endothelin type A receptor (ETA) is a member of the superfamily of G protein-coupled receptors. Our laboratory conducted a microarray screen that identified ETA as target of HOXA10 transcriptional control in endometrium. Here, we confirm HOXA10-regulated ETA expression in endometrium. Endometrial biopsies were obtained from fertile reproductive-age individuals, and first trimester decidual samples were obtained at the time of elective termination. Immunohistochemistry (IHC) was used to identify ETA protein in endometrium as well as first trimester decidua. ETA was expressed in endometrial stromal cells throughout the menstrual cycle. ETA was also highly expressed in first trimester decidual cells. The regulatory relationship between HOXA10 and ETA was established by transient transfection analysis. The human endometrial stromal cell line (HESC) and the human endometrial epithelial cell line (Ishikawa) were transfected with pcDNA/HOXA10, HOXA10 small interfering RNA (siRNA), or respective controls. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to determine expression levels of HOXA10 and ETA in each group. ETA gene expression increased 9-fold (P < .05) after pcDNA/HOXA10 transfection of HESC. ETA was not regulated by HOXA10 in Ishikawa cells. We conclude that ETA is expressed in normal endometrium and decidua. Expression of this receptor is regulated by an essential mediator of endometrial receptivity, HOXA10. ETA may enhance the proliferative potential of endometrial cells in a manner similar to that seen in vascular smooth muscle cells. ET A likely acts as a molecular mechanism by which HOXA10 promotes stromal cell growth and prostaglandin production in both the implantation window and decidua.

A novel, noncoding-RNA-mediated, post-transcriptional mechanism of anti-Mullerian hormone regulation by the H19/let-7 axis†

Abstract In reproductive age women, the pool of primordial follicles is continuously depleted through the process of cyclic recruitment. Anti-Mullerian hormone (AMH) both inhibits the initial recruitment of primordial follicles into the growing pool and modulates the sensitivity of growing follicles to follicle stimulating hormone. Thus, AMH may be an important modulator of female infertility and ovarian reserve; however, the mechanisms regulating AMH remain unclear. To evaluate AMH levels in the absence of H19 lncRNA, H19 knockout (H19KO) mice were evaluated for analysis of ovarian AMH gene expression, protein production, and reproductive function, including assessment of follicle numbers and litter size analysis. To further investigate regulation of AMH by the H19/let-7 axis, let-7 binding sites on AMH were predicted, and in vitro studies of the effect of H19 knockdown/overexpression with let-7 rescue were performed. Lastly, response to superovulation was assessed via oocyte counts and estradiol measurements. The H19KO mouse demonstrates subfertility and accelerated follicular recruitment with increased spontaneous development of secondary, preantral, and antral follicles. Ovaries of H19KO mice have decreased AMH mRNA and protein, and AMH mRNA has a functional let-7 binding site, suggesting a plausible ncRNA-mediated mechanism for AMH regulation by H19/let-7. Lastly, in the absence of H19, superovulation results in higher estradiol and more oocytes, suggesting that H19 functions to limit the number of follicles that mature, produce estradiol, and ovulate. Thus, AMHs inhibitory actions are regulated at least in part by H19, likely via let-7, marking this ncRNA pair as important regulators of the establishment and maintenance of the follicular pool. Summary Sentence The long noncoding RNA H19 regulates AMH via let-7.