Identification of microRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen.
TL;DR: It is demonstrated that miRNAs can control reproductive functions resulting in enhanced or inhibited release of ovarian progestagen, androgen and estrogen, the first demonstration that miRNA‐mediated effects could be potentially used for regulation of reproductive processes.
Abstract: The aim of our studies was to identify miRNAs affecting the release of the major ovarian steroid hormones progestagen, androgen and estrogen by human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different gene constructs encoding human pre-miRNAs on release of progesterone, testosterone and estradiol was evaluated by enzyme immunoassay. In addition, effect of two selected antisense constructs blocking corresponding miRNA on progesterone release was tested. Efficiency of transfection (incorporation transfection reagent) and silencing of marker substances (GAPDH mRNA, GAPDH and CREB-1) were validated by fluorescent microscopy, real-time reverse transcription-PCR analysis and immunocytochemical analysis. Thirty-six out of 80 tested miRNA constructs resulted in inhibition of progesterone release in granulosa cells, and 10 miRNAs promoted progesterone release. Transfected of cells with antisense constructs to two selected miRNAs blocking progesterone release induced increase in progesterone output. Fifty-seven miRNAs tested inhibited testosterone release, and only one miRNA enhanced testosterone output. Fifty-one miRNAs suppressed estradiol release, while none of the miRNAs tested stimulated it. This is the first demonstration that miRNAs can control reproductive functions resulting in enhanced or inhibited release of ovarian progestagen, androgen and estrogen. We hypothesize that such miRNA-mediated effects could be potentially used for regulation of reproductive processes, including fertility, and for treatment of reproductive and other steroid-dependent disorders.
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TL;DR: This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-beta1-induced GC proliferation and ovarian estrogen release.
Abstract: Many members of the TGF-beta superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-beta1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-beta-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-beta1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFbeta superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-beta1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-beta1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-beta1. In addition, both miR-224 and TGF-beta1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-beta1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.
262 citations
TL;DR: It is shown that micro-RNA378 (miR-378) is spatiotemporally expressed in porcine granulosa cells, the cells that generate estradiol in the ovary during follicular development, in an inverse manner compared with the expression of aromatase.
Abstract: Estradiol is a steroid hormone that not only plays an important role in ovarian follicular development but also is associated with many reproductive disorders. Owing to the importance of aromatase in the production of estradiol, the regulation of aromatase gene expression at the transcriptional level has been an extensive area of study for over two decades. However, its regulation at the posttranscriptional level has remained unclear. Here, we show that micro-RNA378 (miR-378) is spatiotemporally expressed in porcine granulosa cells, the cells that generate estradiol in the ovary during follicular development, in an inverse manner compared with the expression of aromatase. In vitro overexpression and inhibition experiments revealed that aromatase expression, and therefore estradiol production, by granulosa cells, is posttranscriptionally down-regulated by miR-378. Furthermore, site-directed mutation studies identified two binding sites in the 3′-untranslated region (3′-UTR) of the aromatase coding sequence that are critical for the action of miR-378. Interestingly, overexpression of the aromatase 3′-UTR enhanced aromatase expression at the protein level in granulosa cells, possibly mediated by the binding of miR-378 within this region, thereby reducing the binding of this micro-RNA to the endogenous aromatase 3′-UTR.
190 citations
TL;DR: The role of estrogen-regulated miRNA expression, the target genes of these miRNAs, and the role of miRNAAs in health and disease is a 'hot' area of research that will yield new insight into molecular mechanisms of estrogen action.
Abstract: MicroRNAs (miRNAs) are short, noncoding RNAs that generally base-pair within the 3' untranslated region of target mRNAs causing translational inhibition and/or mRNA degradation. Estradiol (E(2)) and other estrogen receptor (ER) ligands suppress or stimulate miRNA expression in human breast cancer cells, endometrial cells, rat mammary gland, and mouse uterus, and post-translationally regulate protein expression. Aberrant miRNA expression is implicated in estrogen-related breast and endometrial cancers, and several miRNAs downregulate ERα. The role of estrogen-regulated miRNA expression, the target genes of these miRNAs, and the role of miRNAs in health and disease is a 'hot' area of research that will yield new insight into molecular mechanisms of estrogen action.
181 citations
Cites background from "Identification of microRNAs control..."
..., but none that stimulated E2 release [58]....
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TL;DR: This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of mi RNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen.
Abstract: Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre-miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir-15a and mir-188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir-15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir-188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre-mir-10a, mir-105, and mir-182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome-wide miRNA screen.
175 citations
TL;DR: The reviewed literature indicates that BPA may be associated with infertility in women and may alter overall female reproductive capacity by affecting the morphology and function of the oviduct, uterus, ovary, and hypothalamus-pituitary-ovarian axis in animal models.
160 citations
Cites background from "Identification of microRNAs control..."
...BPA down-regulated miR-137 may decrease sex steroid hormone synthesis (112) and miR-765 may be associated with premature ovarian failure (113, 114)....
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References
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TL;DR: Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
32,946 citations
"Identification of microRNAs control..." refers background in this paper
...In humans, miRNA genes, which represent approximately 1–3% of genes, may regulate up to 90% of all genes (Bartel, 2004; Silveri et al., 2006; Stefani and Slack, 2006; Mattes et al., 2007; Farazi et al., 2008). miRNA precursors (pre-miRNAs) generated in the nucleus are exported to the cytoplasm,…...
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...Interestingly, mir-122 was able to reduce plasma cholesterol level and hepatic cholesterol synthesis (Bartel, 2004) suggesting that it could inhibit steroidogenesis via suppression of relatively independent processes in different tissues— inhibition of cholesterol synthesis in liver, transport of…...
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TL;DR: It is shown that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous mi R-375 function enhanced insulin secretion and may constitute a novel pharmacological target for the treatment of diabetes.
Abstract: MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375) Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375 Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes
2,064 citations
"Identification of microRNAs control..." refers background in this paper
...…production by megakaryoblasts (Guimaraes-Sternberg et al., 2006), plasma cholesterol level (Mattes et al., 2007) and on expression of peptides related to regeneration and insulin output in pancreatic cells (Poy et al., 2004; Plaisance et al., 2006; Baroukh et al., 2007; Joglekar et al., 2007)....
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TL;DR: In this paper, the authors presented novel methods for the diagnosis of ovarian cancer using at least one miR selected from miR-200b, miR -200b and miR −200c.
Abstract: The present invention provides novel methods for the diagnosis of ovarian cancer using at least one miR selected from miR-200b, miR-141, miR-199a, miR-140, miR-145 and miR-125b1miR-200c. The invention also provides methods of identifying anti-ovarian cancer agents and a kit for detecting ovarian cancer.
1,354 citations
TL;DR: It is proposed thatmiR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion through the regulation of BCL2 and CCND1.
Abstract: Two microRNAs, miR-15a and miR-16, localize to a chromosome region that is frequently deleted in cancer. Bonci et al. now show that these microRNAs have tumor suppressive effects in prostate cancer cells and regulate the expression of crucial oncogenic targets.
966 citations
"Identification of microRNAs control..." refers background in this paper
...…has been reported previously. miR-15a was shown to promote apoptosis through suppression of bcl-2 and other pro-proliferating and antiapoptotic substance and probably prevents proliferation and malignant transformation of lymphocytes and prostate cells (Bonci et al., 2008; Calin et al., 2008)....
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TL;DR: A high-throughput profiling of genes modulated by miR-15a/16-1 in a leukemic cell line model (MEG-01) and in primary CLL samples is produced and a significant enrichment in cancer genes that directly or indirectly affect apoptosis and cell cycle was found.
Abstract: MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human diseases, including cancer. Each miRNA is predicted to regulate hundreds of transcripts, but only few have experimental validation. In chronic lymphocytic leukemia (CLL), the most common adult human leukemia, miR-15a and miR-16-1 are lost or down-regulated in the majority of cases. After our previous work indicating a tumor suppressor function of miR-15a/16-1 by targeting the BCL2 oncogene, here, we produced a high-throughput profiling of genes modulated by miR-15a/16-1 in a leukemic cell line model (MEG-01) and in primary CLL samples. By combining experimental and bioinformatics data, we identified a miR-15a/16-1-gene signature in leukemic cells. Among the components of the miR-15a/16-1 signature, we observed a statistically significant enrichment in AU-rich elements (AREs). By examining the Gene Ontology (GO) database, a significant enrichment in cancer genes (such as MCL1, BCL2, ETS1, or JUN) that directly or indirectly affect apoptosis and cell cycle was found.
768 citations
"Identification of microRNAs control..." refers background in this paper
...…has been reported previously. miR-15a was shown to promote apoptosis through suppression of bcl-2 and other pro-proliferating and antiapoptotic substance and probably prevents proliferation and malignant transformation of lymphocytes and prostate cells (Bonci et al., 2008; Calin et al., 2008)....
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