Application of RNA Interference for the Control of

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Application of RNA Interference for the Control of Female Reproductive Functions Alexander V. Sirotkin1,2,* 1 2

Institute of Animal Genetics and Reproduction, Animal Production Research Centre Nitra, 951 41 Luianky near Nitra, Slovakia; Department of Zoology and Anthropology, Constantine the Philosopher University, 949 74 Nitra, Slovakia, Abstract: RNA interference, a recently discovered new mechanism controlling gene expression via small RNAs, was shown to be involved in the characterization and control of basic ovarian cell functions. The main classes of small RNAs, as well as their expression in ovaries have been described. Furthermore, the successful application of RNA interference for the study and control of basic ovarian functions (fertility, proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation and related ovarian cell malignant transformation) and production of recombinant proteins has been demonstrated. Application of RNA interference in reproductive biology and medicine can be successful in three main areas - (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and (3) treatment of reproductive disorders or their particular indexes. Problems of improvement of small RNA delivery to target ovarian cells and potent RNA interference-related approaches for the treatment of ovarian disorders (especially of ovarian cancer) have been discussed.

Keywords: RNA interference, ovarian, fertility, proliferation, apoptosis, secretion, oocyte maturation, RNA delivery, cancer. 1. FUNCTION AND CLASSIFICATION OF SMALL RNAS Post-transcriptional gene regulation includes different RNArelated mechanisms including recently discovered RNA interference. RNA interference (post-transcriptional gene silencing) is a conserved biological regulatory process by which long doublestranded RNAs (dsRNAs) silence the expression of target genes. This process induces the resistance of cell to exogenous pathogenic nucleic acids and regulates the expression of own protein-coding genes. In biomedical research, RNA interference has been developed as a means to manipulate gene expression experimentally and to probe gene function on a whole-genome scale. The history of RNA interference discovery is described in several special reviews [1-4]. The term "RNA interference" was first used by researchers studying nematode Caenorhabditis elegans. In these studies synthetic dsRNA induced silencing of some worm genes [5,6]. Thereafter, the treatment with dsRNA induced gene silencing in fruit flies Drosophila melanogaster embryos [2,7]. It was found that dsRNA added to Drosophila embryo lysates is processed to 21-23 nucleotide species - small interfering RNAs (siRNAs), that the homologous endogenous mRNA is cleaved only in the region corresponding to the introduced dsRNA, and that this cleavage occurred at 21-23 nucleotide intervals corresponding to siRNAs [8,9]. Over the last few years, these RNA interference strategies have been used as reverse genetics tools in Drosophila organisms, embryo lysates, and cells to characterize various loss-offunction phenotypes [4,10]. Synthetic siRNAs introduced by transient transfection were found to effectively induce RNA interference in either invertebrate or mammalian cells in a sequencespecific manner [1,3,4,9,11-13]. Current mechanisms of RNA interference are described in detail in comprehensive reviews [4,9,13-17]. Shortly, the RNAi pathway is initiated by the enzyme Dicer, which cleaves long dsRNA molecules into short fragments of ~20 nucleotides. The sense strand is cleaved and degraded, whereas the antisense strand of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which *Address correspondence to this author at the Institute of Animal Genetics and Reproduction, Animal Production Research Centre Nitra, 951 41 Luianky near Nitra, Slovakia; Tel: +421-37-6546335; Fax: +421-37-6546480; E-mail: [email protected] 1873-4286/12 $58.00+.00

occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induce cleavage by Argonaute, the catalytic component of the RISC complex. RISCs may then promote epigenetic silencing through RNA-directed DNA methylation (translational repression), by target RNA cleavage or both. The main steps of small RNA production are shown in Fig. (1). Several types of small RNA molecules - small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), piwi-interacting RNA (piwiRNA or piRNAs), small nucleolar RNAs (snoRNAs) and ribozymes - are central to RNA interference. Some endogenous small RNAs (miRNAs, piRNAs, snoRNAs, endo-siRNAs, piRNAs, ribozymes) commonly occur in the cell nuclei or cytoplasm, others, exogenous (shRNAs, synthetic siRNAs) are man-made. Structure, characteristics and action of different small RNAs are described in several special reviews [11, 12, 14-18]. Shortly, all these small RNAs can bind to specific other RNAs (products of specific genes) and either increase or decrease their activity. A small hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn. The shRNA hairpin structure is cleaved by the cellular machinery into small interfering RNA (siRNA), which is then bound to the RNAinduced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. Mature microRNAs (miRNA) are structurally similar to siRNAs produced from dsRNA, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modification. An miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA which is processed, in the cell nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein Pasha. The dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex, thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing. Piwiinteracting RNAs (piRNA) form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes are characterized by lack of primary sequence conservation and increased complexity. Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that guide chemical modifications (methylation and pseudouridylation) of other RNAs. A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an © 2012 Bentham Science Publishers

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Alexander V. Sirotkin

Fig. (1). The main steps of small RNA generation and action within the cell (from http: //withfriendship.com/images/h/36741/the-rna-interferencepathway.jpg). Explanations are in the text.

RNA molecule that catalyzes a chemical reaction (hydrolysis of one of their own phosphodiester bonds, of bonds in other RNAs, increase aminotransferase activity of the ribosome a.o.). From the viewpoints of basic studies and practical application, now the most useful are shRNAs, their products siRNAs and miRNAs. The function of siRNAs differs from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences, i.e. they can affect the expression of several genes, and their specificity is limited. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a

single, specific target. Therefore, the main studies requiring specific gene silencing, were performed on synthetic siRNA or their precursors, shRNA [11,12,18,19]. Analysis of available literature (see below) shows, that application of RNA interference in the reproductive biology and medicine is successful in three main areas - (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for the regulation of reproductive processes and (3) treatment of reproductive disorders or their particular indexes.

RNA Interference and Ovarian Functions

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2. BASIC INTRA-OVARIAN PROCESSES The principal functional unit of the ovary is the follicle, which goes through several stages of development [20-22] (see Fig. (2)): stage of primordial, primary, secondary, tertiary and preovulatory Graafian follicle. During follicular development, both nuclear (meiosis) and cytoplasmic maturation of intrafollicular oocyte occur. At different stages of follicullogenesis, follicular selection induces degeneration/atresia and death of the majority of follicles, whilst only small proportion of follicles ovulates and releases mature oocyte. Fate of follicle and its development and differentiation are determined by antagonistic processes of ovarian cell proliferation and atresia, which in turn are controlled by extrafollicular (release of gonadotropins and other hormones) and intrafollicular (receptors to hormones, secretes of ovarian somatic cells, oocytes, leukocytes a.o.) factors [20,23,24]. In some cases, ovarian cells are transformed to tumor cells characterized by increased proliferation, decreased apoptosis and altered response to upstream regulators [25-27]. In the periovulatory period, mature follicle undergoes lutenization and in some mammals transforms to Corpus luteum, which is involved in the regulation of ovarian cyclicity and gravidity [21,22]. All these processes can be regulated directly or indirectly by small RNAs. RNA interference has high potential impact in the control of reproduction and treatment of reproductive disorders. Nevertheless, only few reviews [19,28-30] on particular aspects of this subject (mainly in relation to ovarian cancer treatment) have been published yet. Furthermore, due to the fast growing body of new evidence concerning effects and areas of application of small RNAs for the control of ovarian substances and processes, these reviews require permanent updating. The present paper represents an update and substantial expansion of our previous review [30] by new publications appeared in the last two years. This paper reviews the

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currently available data concerning the involvement and application of shRNAs, siRNA and miRNAs in the control of ovarian cell proliferation, apoptosis, secretory activity, development of corpus luteum and oocyte maturation. The available literature does not contain direct evidence concerning involvement of other kinds of small RNAs - piRNAs, snoRNAs and rybozymes in the control of particular ovarian functions yet, although the presence and silencing activity of piRNAs in insect ovarian cells has been demonstrated [31-33]. In addition, some aspects of small RNA delivery and application for the production of recombinant proteins by using ovarian cells have been discussed. 3. EXPRESSION OF SMALL RNAS IN THE OVARY In the ovary, approximately 400 small RNAs were identified, whilst 122 miRNAs were unique for reproductive organs suggesting their involvement in reproductive functions [34]. A substantial number of ovarian tissue-specific siRNAs, miRNAs and piRNAs are demonstrated in the ovarian follicular cells [35], Corpis luteum [36], oocytes and Cumulus oophorus [37-42]. Furthermore, the expression of particular small RNAs depends on the stage of ovarian and follicular cycle and ovarian health. For example, LH/hCG and cAMP-induced preovulatory changes include up-regulation of miR-132 and miR-212 [35]. Ovarian follicular dominance leading to survival and development and ovulation of dominant follicle and its oocyte, in contrast to atresia and death of subordinate follicleoocyte complex, is associated with discrimination of expression of a number of genes under the influence of small RNAs [43,44]. Development of processes, that have been associated with PCOS [45] and ovarian cancer [45-52] is associated with different patterns of expression of numerous small RNAs. Moreover, the small RNA profile could have diagnostic and prognostic value for the development of ovarian cancer [50,51]. The ovarian state-related changes in small RNAs might be induced by either up- or down-regulation of

luteogenesis

luteolysis ovulation

folliculogenesis

Fig. (2). The cyclic changes in ovarian structures - targets of small RNAs.(from en.wikipedia.org/wiki/Folliculogenesis). Explanations are in the text.

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Dicer and Drosha, the key regulators of small RNA formation. At least, transfection-induced deletion of either Dicer or Argonaute proteins in mice resulted in dramatic changes in the expression of more than 1500 genes in the ovary, increased primordial follicle pool endowment, accelerated early follicle recruitment and induced degeneration of advanced follicles [53], reduced ovulation [54], developmental competence of oocytes [41,54-57], as well as induced infertility [36,54]. Moreover, the activity of Dicer and Drosha was positively associated with response to chemotherapy and final clinical outcome in ovarian cancer patients [49]. These observations suggest, that small RNAs and their regulators could be applicable indexes for the characterization and prediction of the ovarian state. It is important to note, that different miRNA signatures of ovarian tumors may distinguish these tumor subtypes. These differences suggest, that small RNAs can control different processes (proliferation, apoptosis, survival, inter- and intracellular communication, immune response) and their regulators (production and reception of hormones, growth factors, cytokines, their receptors, post-receptor intracellular signaling pathways and other effectors) in normal and pathologic ovarian cells. 4. APPLICATION OF RNA INTERFERENCE FOR THE CONTROL OF OVARIAN FUNCTIONS 4.1. RNA Interference and Fertility Specific depletion of Dicer resulted in the blockade of small RNA (miRNAs and sirRNAs) formation in murine ovarian cells. These changes were associated with retardation in uterine and oviductal development and function as ovarian function and infertility Female infertility was shown to be the result of a primary oviductal defect and reduced ovarian function - natural and gonadotropininduced ovulation rates, loss of miRNA within the ovarian granulosa cells, reduced vascularization and progesterone release by Corpora lutea resulted in an inability to sustain pregnancies. Some of these defects were able to be prevented by exogenous administration of two miRNA (miR-17-5p and let-7b) [28,36]. Furthermore, inactivation of Dicer1 in the follicular granulosa cells led to an increased primordial follicle pool endowment, accelerated early follicle recruitment and subsequent follicle degeneration. These changes were associated with alteration in the expression of some follicle development-related genes, such as Amh, Inhba, Cyp17a1, Cyp19a1, Zps, Gdf9 and Bmp15 [53]. Furthermore, the decreased expression of Dicer and Drosha, in epithelial ovarian cancers is associated with poor clinical outcome in patients [49]. It is not to be excluded, that these key RNA interference enzymes could be useful not only for the control of reproduction, but also for the prediction and treatment of ovarian malignant transformation. These studies indicate the importance of Dicer and Dicerinduces small RNA formation in the control of female reproduction, fertility and maybe their pathology at the level of ovarian follicullogenesis, luteogenesis, oviduct and uterus, however, the role of endogenous siRNA versus miRNA mediated gene regulation has not been directly addressed in ovarian somatic cells. More specific and potent tool to control ovarian function and fertility is the knock-down of some regulatory ovarian molecules by dsRNA and siRNAs. A good example of this could be ability of injections of siRNAs down-regulating gene encoding transcription factor heterogeneous nuclear ribonucleoprotein K (Hnrnpk) to block multiple events important for rat ovarian follicle development - development and nuclear reorganization of oocytes, primordial and primary follicles. These changes were associated with the down-regulation of 41 and 22 other genes and alterations in nuclear apoptosis, which could be considered as potential downstream mediators of Hnrnpk effect [58]. Another example of multiple action of siRNA construct shows a report of An et al. [59]. In their experiments, the transfection of

Alexander V. Sirotkin

ovarian tumor cells with siRNA against insulin-like growth factor receptor (IGFR) suppressed tumor cell proliferation in in vitro and tumor growth, cellular proliferation, angiogenesis as well as promoted tumor cellular apoptosis. RNA interference-induced knock-down of specific genes and molecules was shown to be useful to control follicullogenesis in healthy ovaries and to understand its regulatory molecules. For example, the number of follicles significantly declined when mouse ovaries were treated with the shRNA against Partitioning-defective protein 6 (PAR6) [60]. Experiments with injections of dsRNA to mosquitoes demonstrated, that RNA interference could be an efficient tool to affect not only mammalian, but insect reproduction as well. In these experiments dsRNAs-induced knock-down of both insulin receptor and target of rapamycin (TOR)-dependent intracellular signaling mechanism resulted in inhibition in blood digestion, vitellogenesis and egg laying in mosquitoes [61]. RNA-interference-induced blockade of other metabolic factor, leucine aminopeptidase (HlLAP) suppressed oocyte maturation in parthenogenetic ixodic tick Haemaphysalis longicornis [62]. Experiments of Poulton et al. [63] demonstrated, that miRNA-induced blockade of Notch signaling pathway results in delayed follicle cell differentiation and defects in the cell cycle in Drosophila ovaries. Finally, RNAi studies showed that the ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells in red flour beetle, Tribolium castaneum [64]. 4.2. RNA Interference and Ovarian Cell Proliferation A number of small RNAs could be involved in the control of normal ovarian cells. For example, in our genome-wide screen, eleven out of 80 tested miRNA constructs were stimulated, and 53 miRNAs inhibited the expression of marker of granulosa cell proliferation [65]. Furthermore, a number of siRNAs silencing different the protein kinases were able to either up- or down-regulate proliferation of ovarian granulosa cells [66]. The ability of small RNAs to suppress ovarian cell cycle indicated, that RNA interference could be a useful tool to suppress the over proliferating ovarian cancer cells. Since cell proliferation is controlled by a number of mitogenic factors (growth factors, protein kinases and transcription factors), the reported studies were focused on silencing of different physiological mitogens. One of the first successful attempts to use RNA interference for combating cancer was the development of miRNA inhibiting the expression of proto-oncogene, human epidermal growth factor receptor type 2 (HER-2). Transfection of ovarian cancer cell with this miRNA decreased the cell number in an effective way, than blockade of HER-2 with corresponding antisense construct [67]. Thereafter it was demonstrated, that the suppression of HER2 by siRNA inhibits ovarian cancer cell growth and increases their sensitivity to chemotherapeutic treatment [68]. After report of Tsuda et al. [67], a small RNA-induced suppression of a number of other mitogenic factors has been tested for understanding their physiological role and/or inhibiting ovarian cancer cell proliferation. Vascular endothelial growth factor (VEGF) is a classical promoter of ovarian cell cancerogenesis. Decrease in the production of VEGF and its receptor through RNA interference-induced inhibition of tissue inhibitor of metalloproteinase 1 (TIMP) and suppressed the proliferation of human ovarian granulosa cells [69]. VEGF receptor (VGFR-2) small siRNA transfection remarkably also decreased invasiveness of ovarian carcinoma cells [70]. Moreover, the inhibition of VEGF production by anti-VEGF siRNA suppressed cell proliferation, expression of anti-apoptotic protein surviving and activated both nuclear and cytoplasmic/mitochondrial apoptosis [71]. Surprisingly, knock-down of VEGF receptors by shRNA increased the VEGF expression and had an opposite, stimu-

RNA Interference and Ovarian Functions

latory effect on mouse ovarian carcinoma cells in vivo. It did not decrease, but increase the aggressivity of ovarian tumor [72]. Opioid growth factor (OGF, [Met(5)]-enkephalin) and the OGF receptor (OGFr) are present in ovarian cancer cells, whilst small RNA-induced silencing of OGFr and its targets, p16 and p21 promoted proliferation in cultured ovarian cancer cells [73]. Some tumors produce increased amounts of growth factor endothelin-1 (ET-1), its expression can be implicated in the progression of various cancers, including ovarian carcinoma. Small interfering RNA targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to the inhibition of ET-1 peptide secretion, expression of proliferation markers, decreased invasiveness, increased production of adhesion molecules, improved adhesion to basal lamina proteins, reduced expression of a mesenchymal marker, but not alter viability of cultured ovarian carcinoma cells [74]. Although recombinant human erythropoietin (EPO) is used for the treatment of anemia, EPO use may be associated with decreased survival in cancer patients. Specific inhibition of EPO receptor expression using a shRNA resulted in markedly reduced proliferation and invasiveness of cancer cells and ovarian tumor growth in in vitro and in vivo [75]. An increased proliferation of cancer cells can be due to the overexpression of epidermal growth factor receptor (EGFR). The knockdown of the expression of EGFR by siRNA reduced proliferation and abolished the antiproliferative effect of GnRH analog in ovarian carcinoma cells [76]. Another known growth factor stimulating cancerogenesis is insulin-like growth factor. As it was mentioned above, transfection of ovarian tumor cells with siRNA against insulin-like growth factor receptor suppressed tumor cell proliferation in vitro and tumor growth, cellular proliferation, angiogenesis in vivo [59]. Hormone/growth factor adiponectin can be involved in energy homeostasis and other functions in different cells including ovarian ones. Blockade of adiponectin receptor resulted in a decrease in the expression of proliferation marker in normal granulosa cells (Pierre et al., 2009). Ovarian cancer cell proliferation can be promoted by estrogens, whilst RNA interference can down-regulate estrogen receptor coactivator and estrogen-postreceptor signaling substance [78]. Follicle-stimulating hormone (FSH) can promote ovarian cancerogenesis by stimulating ovarian cell proliferation and inhibition apoptosis. FSH exerts this action via the promotion of survivin production. Knock-down of survivin via RNA interference prevents stimulatory action of FSH on ovarian cancerogenesis [79]. Dopamine is a known promoter of ovarian cancerogenesis. siRNAs down regulating the expression of dopamine receptor 2 incorporated into nanoparticles were shown to be useful for the inhibition of ovarian carcinoma growth in vivo [80]. Fibroblast Growth Factors (FGFs) have been implicated in malignant transformation, tumor mitogenesis, angiogenesis and chemoresistance. Using FGFR-specific shRNAi we demonstrated that reductions in FGFR2 inhibited proliferation, increased G(2)/M arrest of ovarian cancer cell lines in vitro, increased their sensitivity to cisplatin and reduced growth rates of ovarian tumor xenografts [81]. Interferon (IFN) alpha and its mediators, IFN regulatory factor 9 (IRF9) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are physiological inhibitors of normal and cancer cell proliferation. IRF9-RNA and TRAIL receptor (TRAIL-R2) RNA interference inhibited the antiproliferative activity of IFNalpha and TRAIL suggesting that these molecules could be potentially used for control of normal and malignant ovarian cells [82].

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Bmi-1 is an oncogenic factor overexpressed in ovarian cancer cells. miRNAs miR-15a and miR-16, directly target the Bmi-1 3' untranslated region and significantly correlate with Bmi-1 protein levels in the ovarian cancer patients and cell lines. Furthermore, Bmi-1 protein levels are downregulated in response to miR-15a or miR-16 expression and lead to significant reduction in ovarian cancer cell proliferation and clonal growth. These findings suggest the development of therapeutic strategies by restoring miR-15a and miR-16 expression in ovarian cancer and in other cancers that involve the upregulation of Bmi-1 [83]. Janus kinases 1 and 2 via stimulation of cyclins and cyclindependent kinases can promote the entry of cells to mitosis (transition from G1 to S phase and from G2 to M phase). Silencing of Janus kinases [84], cyclin [85] or cyclin-dependent kinases [86,87] can down-regulate the proliferation-related markers and induce cell cycle arrest of cultured human cancer cells. In-vivo administration of siRNA down-regulating an other oncogene, EphA2, substantially reduced tumor burden, angiogenesis and proliferation of mouse ovarian cancer cells [88]. PI3 kinases play a key role in promotion of proliferation and survival of both normal and cancer cells, as well as of tissue vascularization. shRNA silencing catalytic unit of PI3 kinase was able to inhibit both these processes in cultured human ovarian tumor cells and ovarian carcinoma xenografts [89]. Another important protein kinase regulating malignant transformation is NF-kappaB kinase. Inactivation of inhibitor of NFkappaB kinase beta by the corresponding shRNA blocked the proliferation, invasion, and adhesion of murine cancer cells [90]. MAP/ERK1,2 kinase is an important mediator of mitogen action on both normal and cancer ovarian cells. Reduction in the expression of nucleoporin 153, promoter of MAPK/ERK1,2 translocation to the nucleus, by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells [91]. Finally, siRNAs against other protein kinase, PKG-Ialpha kinase, caused inhibition of DNA synthesis/proliferation of cultured human carcinoma cells [92]. In ovarian carcinoma cell lines, enhancer of zeste homolog 2 (EZH2) knockdown by RNA interference led to a G(1) phase cell cycle arrest, reduced cell growth/proliferation and inhibited cell migration and/or invasion in in vitro. In addition, EZH2 knockdown was found to reduce the expression of transforming growth factorbeta1 (TGF-beta1), regulator of cancerogenesis [93]. Transcription factor cAMP response element-binding protein (CREB), which can be overexpressed in some ovarian tumors, was found to promote ovarian surface epithelial cell survival and proliferation. To determine whether CREB regulated proliferation and/or apoptosis in the ovarian tumor cell line, CREB expression was suppressed using RNA interference. Decreased CREB expression significantly reduced the ovarian tumor cell proliferation, while there was no effect on apoptosis in these cells [94]. Another transcription factor, FOXG1 and its target p21, mediated stimulátory effect of TGF beta on ovarian cancer cells. The natural overexpression of FOXG1 was correlated with high-grade ovarian cancer, promoted ovarian cancer cell proliferation and inhibited proliferation blocker p21. Conversely, the shRNA-induced FOXG1 knockdown resulted in the decrease of cell proliferation due to increase in p21 expression [95]. Exposure to the siRNA for transcription factor POU6F1 reduced the expression of G protein-coupled receptors involved in tumor cell proliferation, mobolity and invasibility of clear cell adenocarcinoma cell lines and tumors in mice ovaries [96, 97]. Another transcription factor, signal transducer and activator of transcription 3 (STAT3) plays an important role in the formation of many tumors including ovarian cancer. STAT3 shRNA specifically suppressed STAT3 expression at both mRNA and protein levels. At

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the same time, expressions of Bcl-xL, cyclin D1, and c-myc were down-regulated, whereas the cleaved caspase 3 was up-regulated. In addition, STAT3 knockdown inhibited anchorage-independent growth and induced apoptosis in CAOV3 cells, and decreased tumor growth in nude mice implanted with ovarian cancer cells [98]. Later Cai et al. [99] confirmed an anti-proliferative action of STAT3 using RNA interference. In their experiments the STAT3 siRNA down-regulated the expression of proliferation-related substances cyclin D1, survivin, and VEGF in ovarian cancer cells. X-linked Inhibitor of Apoptosis Protein (XIAP) is upregulated in various malignancies, including human ovarian carcinomas, it promotes invasion, metastasis, growth, and survival of malignant cells. Down-regulation of XIAP gene by shRNA inhibited proliferation and tumorigenicity of the human ovarian carcinoma cells [100]. Stress can induce the growth of ovarian tumor via the promotion of norepinephrine output, which in turn stimulates ovarian cancerogenesis through increase in formation of transcription factor AP1/FosB and then of interleukin 8 (IL8). This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using the corresponding siRNAs. These treatments also reduced the vascularization of mouse ovarian tumors [101]. Impairment of murine ovarian carcinoma cell proliferation was also observed after the administration of siRNAs against two survival-inducing molecules, survivin and metallothionein-IIa (MTIIA) [102]. The proliferative and colony-formation ability of two ovarian cancer cell lines was substantially also reduced under the influence of shRNA -induced knock-down of Nin one binding protein (NOB1p), a crucial molecule in the maturation of the 20S proteasome and protein degradation [103]. An interesting approach to stop cancer cell proliferation was reported by Luo et al. [104]. They used shRNAs to down-regulate the main rate-limiting subunit of telomerase, human telomerase reverse transcriptase (hTERT), enzyme affecting cell cycle at S phase, apoptosis through either telomeric effect or via transcription factors p53 and p21. This treatment induced S-phase and growth arrest in cultured ovarian carcinoma cell. Comparison of cell lines with different expression of p53 and p21 demonstrated, that this effect of shRNA against hTERT has not been probably mediated by these transcription factors. Another approach to affect ovarian cancer cell proliferation is influencing mitogens not directly, but indirectly, via regulators of their stability. For example, it was observed, that ovarian expression of enzyme glyceraldehyde-3-phosphate dehydrogenase GAPDH is associated with the expression of colony-stimulating factor-1 (CSF-1), and that GAPDH binds and stabilizes CSF-1. GAPDH siRNA reduced both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA and decreased CSF-1 mRNA half-lives by 50% [105]. 4.3. RNA Interference and Ovarian Cell Apoptosis Apoptosis (programmed cell death) is induced by a cascade of regulators and effectors including growth factors and their pro- and antiapoptotic intracellular proteins. RNA interference can play an important role in the control of these molecules and of apoptosis. In our experiments, 11 of the 80 miRNAs tested promoted apoptosis (expression of apoptosis-related molecule Bax, pro-apoptotic and anti-cancer transcription factor p53 and other apoptotic markers like TdT and caspase), while 46 miRNAs reduced it in human ovarian cells [65]. A number of siRNAs targeting protein kinases were able to either promote or suppress the expression of apoptosis marker of ovarian granulosa cells [66]. Numerous attempts have been made to use the ability of small RNAs to affect apoptosis for reducing the viability of ovarian cancer cells and inducing their death. For this

Alexander V. Sirotkin

purpose, both stimulation of pro-apoptotic substances and inhibition of anti-apoptotic factors were tested. Gene silencing by cyclooxygenase-2 (COX-2) specific siRNA in cultured epithelial ovarian cancer cells impaired the phosphorylation of its mediator of action, AKT kinase, resulting in decreased downstream signaling leading to cell growth inhibition and induction of apoptosis [106]. Epidermal growth factor (EGF) expression is elevated in ovarian cancer cells. The siRNA-induced suppression of epidermal growth factor (EGF) receptor (EGFR) was able to promote ovarian cancer cell apoptosis and sensitivity of cancer cells to chemotherapy suggesting that small RNA targeting EGFR can be a potential anti-cancer drug [107, 108]. siRNA targeting other EGF receptor (HER-2) was able to promote apoptosis and increase the sensitivity of ovarian carcinoma cells to chemotherapeutic agent [ 68 ]. Treatment with shRNA down-regulating FGF, promoter of ovarian cancerogenesis (see above) can induce apoptosis and reduce ovarian cancerogenesis both in in vivo and in vitro [81]. As it was mentioned above, the siRNA-induced blockade of insulin-like growth factor receptor promoted apoptosis of mouse ovarian carcinoma cells too [59]. Some growth factors are working through the activation of PI3 kinases, which play a key role in the maintenance of cell viability via suppression of apoptosis. shRNA-induced silencing of catalytic PI3 kinase subunit induced apoptosis in human cancer cells [89]. Another protein kinase involved in the control of ovarian cancer cell survival could be focal adhesion kinase (FAK), which closely correlated with the phosphorylation of the target kinase Akt. Depletion of FAK content by RNA interference was also associated with the inhibition of Akt activation and survival of ovarian cancer ascites [109]. XIAP mentioned above is one of the most important members of the inhibitors of apoptosis family and cancer-related substance. It promotes the survival of malignant cells, and it also confers resistance to some chemotherapeutic drugs. Down-regulation of XIAP gene by shRNA inhibited not only proliferation and tumorigenicity, but also promoted apoptosis and chemosensitivity of the human ovarian carcinoma cells to chemotherapeutic agents [100]. Inhibition by using siRNA B-cell lymphoma protein, Bcl-X(L), which binds and inactivates inductor of cytoplasmic apoptosis Bax, increased the sensitivity of ovarian cancer cell lines to chemotherapeutic agent [110,111]. Bcl-2 can be bound and inactivated by p53. In agreement with these observations, reduction of p53 inhibitor MDM2 by RNA interference increases resistance to DNA-damageinduced apoptosis [112]. On the other hand, action of Bcl-2 can be promoted by Myeloid cell leukemia sequence 1 (BCL2-related) (MCL-1). Multi-targeted RNAi strategy directed against these two anti-apoptotic proteins was able to induce complete death of human cancer cell population [111]. Another approach to promote ovarian cancer cell apoptosis can be the inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1). Both in-vitro and in-vivo administration of siRNA to Parp1 into mouse inhibited ovarian carcinoma cell growth, primarily by the induction of apoptosis and extended the survival of mice bearing tumors derived from ovarian cancer cells [113]. Physiological inhibitors of apoptosis and promoters of ovarian and non-ovarian cell viability are peptides of survivin family. siRNAs targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA). These siRNAs were able to inhibit murine carcinoma growth in in vitro and in vitro [114]. The ability of siRNAs against either survivin or to metallothionein-IIa (MT-IIA) prevents murine ovarian carcinoma cell survival and proliferation by promotion of apoptosis has been demonstrated by Tarapore et al. [102]. An inhibition of production of

RNA Interference and Ovarian Functions

VEGF, which can prevent cancer cell apoptosis via inhibition of anti-apoptotic protein survivin, suppressed surviving expression and activated both nuclear and cytoplasmic/mitochondrial apoptosis [71]. FSH can reduce apoptosis in both healthy and cancer ovarian cells just via FSH-induced survivin production. RNA interferenceinduced knock-down of survivin prevents anti-apoptotic action of FSH [115]. Therefore, small RNAs targeting survivin could be an efficient tool to promote apoptosis by ovarian cancer cells and, therefore, to treat ovarian malignant transformation. The ability of siRNA against human telomerase reverse transcriptase (hTERT) to induce apoptosis via transcription factors p53 and p21 has been demonstrated by Luo et al. [104]. Transcription factor STAT3 signaling can be a promising molecular target for the induction of apoptosis and ovarian cancer therapy. At least, the STAT3 siRNA down-regulated the expression of cyclin D1, survivin, and VEGF in ovarian cancer cells both at the transcription and translation levels. Inhibition of STAT3 and its related genes was accompanied by the growth suppression and induction of apoptosis in cancer cells in vitro [99]. The ability of siRNAs against transcription factor heterogeneous nuclear ribonucleoprotein K (Hnrnpk) to affect apoptosis in rat ovarian somatic cells [58] has been mentioned above. 4.4. RNA Interference and Ovarian Secretory Activity As it was seen above, ovarian cell proliferation, apoptosis, differentiation and other functions are regulated by the system of hormones and growth factors. Therefore, ovarian cell functions and treatment of ovarian disorders could be affected through changes in hormones and growth factors secretion and reception. Blockade of Dicer and related small RNAs can reduce progesterone release by Corpus luteum [36]. Numerous small RNA can be involved in the control of ovarian secretory activity. For example, the genome-wide screen of miRNAs affecting steroid hormones release by human ovarian granulose cells revealed, that thirty-six out of 80 tested miRNA constructs resulted in the inhibition of progesterone release, and 10 miRNAs promoted it. Transfection of cells with antisense constructs to two selected miRNAs blocking progesterone release and 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 [116]. Furthermore, both stimulátory and inhibitory effects of a number of siRNAs silencing protein kinases on the release of progesterone and IGF-I by cultured human granulosa cells were found [117]. The usability of RNA interference as an efficient tool to affect ovarian steroidogenesis was confirmed by Wu et al [118]. In their experiments, siRNAs against transcription factor liver receptor homolog-1 (LRH1), and not against steroidogenic factor-1 (SF-1) were able to reduce basal and testosterone-induced expression of aromatase (Cyp19) and P450 side-change cleavage, two enzymes crucial for the normal ovarian steroidogenesis and function, in rat ovaries. Steroid hormones play an important role in the control of different ovarian functions and in steroid-dependent cancers and PCOS. For example, an increased activity of 17alpha-hydroxylase/ 17, 20-lyase can be the cause of androgen excess in patients with polycystic ovary syndrome (PCOS) [119]. To understand control mechanisms of steroid release and to reduce cancerogenic effects of steroid hormones, small RNAs were successfully used for switchoff of production of steroids and their regulators. Production of steroidogenic acute regulatory protein (STAR), progesterone and estradiol by granulosa cells can be facilitated by small RNAsinduced silencing of their physiological inductors, adiponectin receptors (AdipoR1 and AdipoR2) [77], 17alpha-hydroxylase/17, 20lyase [119] or transcription factors GATA4 and GATA6 [120]. Besides steroid hormones, RNA interference was used to affect other, non-steroid hormones, which are important from physiologi-

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cal or clinical viewpoints. For example, knockdown of hypoxiainducible transcription factor-1 (HIF-1alpha) suppressed the expression of VEGF, promoter of malignancy in human cancer cells [79], whilst suppression of tissue inhibitor of metalloproteinase 1 (TIMP2) promoted it [69]. SiRNA-induced blockade of cyclooxygenase-2 (Cox-2), enzyme of prostaglandin biosynthesis, suppressed the release of prostagrandin F2alpha by bovine cumulusgranulosa cells. Thus, RNA interference helps to understand the role of prostaglandins in ovarian functions [121], for example their role in the promotion of ovarian cell proliferation and in inhibition of their apoptosis mentioned above [106]. Hormones are the key regulators of all ovarian functions. Therefore, the small RNA interference-induced changes in ovarian hormones release and reception could be used for the control of other, non-secretory ovarian functions. Some examples of the control of ovarian proliferation and apoptosis through small RNA targeting hormones, growth factors [74,77,81,106,119,122] and their receptors [5-7,68,69,73-75,77,78,80,82,95,107,122] were presented above. 4.5. RNA Interference and Ovarian Luteogenesis Blockade of small RNAs formation in Dicer-null mice prevented the formation of a capillary structure and subsequent development of mice Corpus luteum. These changes were associated with blockade in the formation of a number of miRNAs including miR17-5p and let7b, miRNAs that participate in angiogenesis by regulating the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1. Injection of miR17-5p and let7b into the ovaries of Dicer(d/d) mice partially normalized the tissue inhibitor of metalloproteinase 1 expression and luteal angiogenesis. These data indicate that the development and function of the ovarian Corpus luteum is a physiological process that appears to be regulated by miRNAs [36]. 4.6. RNA Interference and Oocyte Maturation The involvement of RNA silencing pathways in the control of mouse oocytes and early embryos as well as the use of RNA silencing for experimental inhibition of gene expression in these structures has been suggested [38]. Oocytes after deletion of Dicer and Argonaute, which blocked the formation of miRNAs, has reduced the expression of a number of oocyte-specific genes and they failed to progress through the first cell division because of disorganized spindle formation suggesting a mandatory role of miRNAs in maternal control of oocyte maturation [28,41,55,57,123]. On the other hand, there is great of evidence, that deletion of Dicer or DGCR8, an enzyme specific for miRNA biosynthesis, had no adverse effect on the maturation of mouse oocytes [28,124]. There are some examples of successful use of RNA interference targeting regulators of oocyte nuclear maturation, growth and zona pellucida formation, in the control of oocyte functions, although practical application of this process has been reported only on insect oocytes yet. Meiosis activating sterol, produced directly by lanosterol 14-ademethylase (CYP51) during cholesterol biosynthesis has been shown to promote the initiation of oocyte meiosis. The knockdown of CYP51 expression via siRNAs inhibited FSH-, but not LHinduced meiosis in mouse oocytes [122]. Furthermore, cultured mouse oocyte-granulosa cell complexes injected with siRNAs of mouse zona pellucida glycoprotein A (ZpA) had reduced the growth and survival and abnormal morphology of zona pellucida. These data suggest that mouse ZPA protein contributes to oocyte growth and zona pellucida formativ, and that its targeting by small RNAs can influence these processes [125]. In insects, vitellogenin uptake is an important prerequisite of egg formation. Vitellogenin uptake depends on the presence and activity of vitellogenin receptor (VgR). Adult ixodid tick, Haemaphysalis longicornis Neumann injected by dsRNA against VgR had not developed into fully mature oocytes and laid abnormal eggs

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[126]. Small RNA-induced silencing of the VgR in virgin queens of fire ant, Solenopsis invicta Buren through RNA interference abolished egg formativ [127]. As it was mentioned above, injections of dsRNAs against insulin receptor and TOR-dependent intracellular mechanisms were able to inhibit vitellogenesis and egg formation in mosquitoes too [61]. RNA interference (dsRNA) was also used to silence catalase gene expression in female sand fly. The dsRNAinduced depletion of catalase led to a significant increase of mortality and a reduction in the number of developing fly oocytes [128]. The RNA interference could be useful not only for blockage, but also for the promotion of oocyte maturation, when it is necessary. For example, single injection of dsRNA against gonad-inhibiting hormone in both domesticated and wild female shrimps Penaeus monodon broodstock induced ovarian maturation and eventual spawning [ 129 ]. Therefore, the first published reports demonstrated usefulness of RNA interference for both understanding of the mechanisms regulating oocyte maturation and for the regulation of reproduction and fertility. 6. RNA INTERFERENCE AND RECOMBINANT PROTEIN PRODUCTION Industrial production of recombinant protein represents important and faulty growing branch of biotechnology. RNA interference can be used for increased cellular productivity and the quality of produced recombinant proteins. Chinese hamster ovary (CHO) cells are the most important mammalian cell line used in producing licensed biopharmaceuticals these days. It was reported, that the silencing of apoptosis-associated gene expression, protein glycosylation-associated gene expression, lactate dehydrogenase involved in the cellular metabolism, and dihydrofolate reductase used for gene amplification can promote viability and productivity of CHO cells. Furthermore, such approach can be extended to silence multiple targets involved in different cellular pathways for changing the global gene regulation in cells, as well as the targets related to microRNA molecules for cellular self regulation. Thus, RNA interference can be used in different cells used in the biotechnological industry for protein production [130]. 5. IMPROVEMENT OF SMALL RNA DELIVERY TO THEIR TARGETS Despite fast progress in the application of small RNAs, they are still used more in in-vitro laboratory experiments, than in in-vivo systems, most important site of their biological and therapeutic use. This is due to problems with efficient delivery of small RNA to avoid small RNA degradation and to ensure their specific binding to desirable target cells. Up to date in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. These processes can depend on the kind of delivered small RNAs and their carriers. For example, delivery of small RNAs highly depends on vector [18]. Furthermore, shRNAs, siRNA and miRNAs can have comparable silencing activity, but different stability of silencing or sensitivity to Dicer and Drosha [18, 131]. Early attempts at the delivery of “naked” siRNA, high-pressure siRNA injections, and intratumoral injections have been investigated with limited success. Viral systems of delivery can be more efficient and not very toxic [49,119]. Nevertheless, reported significant morbidity and mortality in murine models when administering shRNA viral vectors were reported. It is suggested that exogenous delivery of viral vector shRNA constructs may “overwhelm” the endogenous miRNA competition for RNAi maturation. In cells with low machinery expression, these scenarios could have detrimental effects in host toxicity and therapeutic efficacy [49]. Complexation of therapeutic nucleic acids with cationic polymers, cationic lipids, or their combination, represents a main strategy of genetic drug used in in vivo now. Owing to a tendency of ovarian cancer to spread through the abdominal cavity, a delivery

Alexander V. Sirotkin

system should allow an intraperitoneal mode of administration. Therefore, the clinical application of RNAi may rely on a combination of biosciences and nanotechnology: in particular, identifying optimal small interfering RNAs (siRNAs) against optimal target genes and developing an efficient system for siRNA delivery into the cancer cells [29,49]. Lipidoids are a new class of lipid-based molecules that are used to form novel nanoparticle formulations for the systemic delivery of RNAi therapeutics. siRNAs encapsulated in lipidoid formulations can induce potent, specific and durable effects when administered in multiple animal species including mice, rats and nonhuman primates [49,101,113,132]. The successful use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the specific receptor to be blocked (EphA2 receptor) to deliver small interfering RNAs (siRNAs) targeting EGFR has been reported [108]. Recently Tanaka et al. [88] reported on a novel efficient approach to overcome limitations of in-vivo small RNA delivery by using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA). The packaging RNA (pRNA) of bacteriophage phi29 DNApackaging motor, which has the capability to assemble into multivalent nanoparticles, has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) The folate receptor-targeted delivery of siRNA by the folate-pRNA dimer emphasizes the cell-specific aspect of this system. Therefore, the phi29 pRNA/siRNA nanoparticles show great promise as a highly potent system for the delivery of therapeutic agents [102]. Furthermore, specific binding of small RNAs to ovarian cancer cells could be improved by the conjugation of these RNAs with polyethylene glycol (PEG) and LHRH, which promote the specific binding and transfection of ovarian cells. Such siRNA-PEG-LHRH complex was successfully used for targeting VEGF in ovarian cancer cells [133]. Taken together, the available data concerning RNA interference in relation to ovarian functions suggest, that small RNAs, when adequately administrated, are useful for the characterization and prediction of physiological and pathological state of the ovary, for the regulation of signaling molecules controlling reproductive processes and the treatment of ovarian disorders including malignant transformation. 7. CONCLUSIONS AND POSSIBLE FUTURE DIRECTIONS RNA interference provides a new and very promising tool for the study and control of basic ovarian functions (proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation resulted fertility and reproductive efficiency, as well as related ovarian cell malignant transformation), as well as for the production of recombinant proteins. Application of RNA interference in the reproductive biology and medicine can be successful in three main areas - (1) characterization and prediction of physiological and pathological state (association between particular small RNA and physiological or pathological processes), (2) application of small RNAs for regulation of reproductive processes and (3) treatment of reproductive disorders or their particular indexes. Control of reproductive processes, prevention and treatment of their pathological changes (cancer, PCOS a.o.) could be affected via different targets cells (ovarian follicles, Corpora lutea, oocytes), processes (ovarian cell proliferation, apoptosis, secretory activity, luteogenesis, oocyte maturation and response of these processes to upstream regulators) and signaling molecules (cell cycle and apoptosis-related substances, hormones, growth factors, their receptors, protein kinases, transcription factors and genes). The main targets of small RNAs in the ovary (intraovarian signaling substances and processes related

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Small RNAs

+

+

+

+

Hormones, growth factors, their receptors and regulators, protein kinases, transcription factors HIF1

ET1, Bmi-1, EPO

TIMP2

CREB, JAK, FOX61

NFkBk GAPDH

+ STAT3, PKG, EZH2 VEGF

+

+

MAPK, AP1, IL8, EphA2

Estrogen, EGF, PI3K, XIAP Hnrnpk, IGFR, COX2, FAK PARP1, hTERT

CSF1

-

+ PAP6, mTOR, Notch POUGF1, EcR, USP

+

+

+

Promoters of proliferation

Apoptosis-related substances

+

+

Atresia, luteolysis, prevention Follicle growth, luteogenesis, cancerogenesis

of cancer

Fig. (3). The main intra-ovarian targets (signaling molecules and processes related to proliferation and apoptosis), whose control by small RNAs has been demonstrated. This picture is based on the materials described in this reviews. For explanations of abbreviations, more detailed description of processes and references see the corresponding parts of the text (modified from Sirotkin, 2010).

to proliferation and apoptosis) are summarized in Fig. (3). This presented summary based on the publications reviewed above which reflects only the first steps of application of RNA interference in the reproductive biology. Further, more efficient application of RNA interference for such purposes could require (1) exact definition of small RNAs targeting and regulating these sites, (2) better understanding of interrelationships between different small RNAs and between different signaling molecules affected by these RNAs, (3) development of reliable methods of quantification of small RNAs to use them as quantitative markers to characterize and to predict the state of particular tissue and cell, (4) understanding, whether not only production, but also effect of small RNAs could vary depending on the species, target tissue, stage of ovarian cycle and health identification (4) improvement of small RNA delivery to target ovarian cells, (5) development of small RNA preparations with an increased stability and efficiency, as well as of efficient small RNA carriers and protectors, (6) study and application of several synergic small RNAs affecting the same or related targets to increase biological effect of treatment, (7) to find new areas of application of RNA interference, which were not properly examined previously (reproduction of farm animals, development of human contraceptives, improvement technologies of assisted reproduction, treatment of infertility and age-related reproductive disorders etc.). Furthermore, (8) since miRNAs are targeting several genes and their products, the chemical modification of miRNAs could enable development on the basis of one miRNA several miRNAs with more specialized and efficient action. Finally, (9) treatment of some disorders with combination of small RNAs together with sense and antisense cDNA constructs and classical pharmaceutical drugs

could be more promising, than the treatment with single molecule. All these and other possible approaches not listed here require however profound studies of chemical, biological and pharmacological aspects of production, delivery, metabolism and effects of small RNAs. DISCLOSURE Part of information included in this article has been previously published in "Journal of Cellular Physiology Volume 225, Issue 2, pages 354-363, November 2010. REFERENCES [1] [2] [3] [4] [5] [6]

[7] [8]

Cogoni C, Macino G. Post-transcriptional gene silencing across kingdoms. Genes Dev 2000; 10: 638-43. Guru T. A silence that speaks volumes. Nature 2000; 404: 804-08. Hammond SM, Caudy AA, Hannon GJ. Post-transcriptional Gene Silencing by Double-stranded RNA. Nature Rev Gen 2001; 2: 1109. Hannon GJ. RNA interference. Nature 2002; 418: 244-51. Guo S, Kempheus KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 1995; 81: 611-20. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806-11. Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA. Nature Biotech 2000 18: 896-8. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: Doublestranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25-33.

334 Current Pharmaceutical Design, 2012, Vol. 18, No. 3 [9] [10]

[11] [12]

[13] [14] [15] [16] [17] [18]

[19] [20] [21] [22]

[23] [24] [25]

[26] [27]

[28] [29] [30] [31]

[32]

[33] [34]

[35] [36]

Hutvagner G, Zamore PD. RNAi: nature abhors a double-strand. Curr Opin Genetics & Development 2002; 12: 225-32. Schmid A, Schindelholz B, Zinn K. Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila. Trends Neurosci 2002; 25: 71-4. Sui G, Soohoo C, Affar E-B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 5515-20. Yu J-Y, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 6047-52. Stefani G, Slack F. MicroRNAs in search of a target. Cold Spring Harb Symp Quant Biol 2006; 71: 129-34. Seto, A.G., R.E. Kingston, N.C. Lau. The coming of age for piwi proteins. Molecular Cell 2007; 26: 603-9. Farazi TA, Juranek SA, Tuschl T. The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 2008; 135: 1201-14. Doran T., Helliwell C (eds.) RNA Interference: Methods for Plants and Animals (Principles and Protocol Series). Cabi 2008; 272 pp. Thompson AJ, Patel K. Antisense inhibitors, ribozymes, and siRNAs. Clin Liver Dis 2009; 13: 375-90. Amarzguioui M, Rossi JJ, Kim D. Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Lett 2005; 579: 5974-81. Mangala LS, Han HD, Lopez-Berestein G, Sood AK. Liposomal siRNA for ovarian cancer. Methods Mol Biol 2009; 555: 29-42. Webb R, Campbell BK. Development of the dominant follicle: mechanisms of selection and maintenance of oocyte quality. Soc Reprod Fertil Suppl 2007; 64: 141-63 Oktem O, Oktay K. The ovary: anatomy and function throughout human life. Ann N Y Acad Sci 2008; 1127: 1-9. Tingen C, Kim A, Woodruff TK . The primordial pool of follicles and nest breakdown in mammalian ovaries. Mol Hum Reprod 2009; 15: 795-803. Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in immature granulosa cells that regulate target gene expression: Branching out from protein kinase A. Cell Signal 2006; 18: 1351-9. Pangas SA. Growth factors and ovarian development. Semin Reprod Med 2007; 25: 225-34. Deng CX. BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution. Nucleic Acids Res 2006; 34: 1416-26. Li YF, Hu W, Fu SQ, Li JD, Liu JH, Kavanagh JJ. Aromatase inhibitors in ovarian cancer: is there a role? Int J Gynecol Cancer 2008; 18: 600-14. Yoshida S, Furukawa N, Haruta S, et al. Expression profiles of genes involved in poor prognosis of epithelial ovarian carcinoma: a review. Int J Gynecol Cancer 2009; 19: 992-997. Christenson LK. MicroRNA control of ovarian function. Anim Reprod 2010; 7: 129-33. Malek A, Tchernitsa O. Evaluation of targets for ovarian cancer gene silencing therapy: in vitro and in vivo approaches. Methods Mol Biol 2010; 623: 423-36. Sirotkin AV. RNA interference and ovarian functions. J Cell Physiol 2010; 225: 354-63. Desset S, Buchon N, Meignin C, Coiffet M, Vaury C. In Drosophila melanogaster the COM locus directs the somatic silencing of two retrotransposons through both Piwi-dependent and -independent pathways. PLoS One 2008; 3: e1526 Malone CD, Brennecke J, Dus M, et al. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 2009; 137: 522-35. Saito K, Inagaki S, Mituyama T, et al. A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature 2009; 461: 1296-9. Ro S, Song R, Park C, Zheng H, Sanders KM, Yan W. Cloning and expression profiling of small RNAs expressed in the mouse ovary. RNA 2007; 13: 2366-80. Fiedler SD, Carletti MZ, Hong X, Christenson LK. Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells. Biol Reprod 2008; 79: 1030-7. Otsuka M, Zheng M, Hayashi M, et al. Impaired microRNA processing causes corpus luteum insufficiency and infertility in mice. J Clin Invest 2008; 118: 1944-54.

Alexander V. Sirotkin [37]

[38]

[39] [40]

[41] [42]

[43] [44]

[45]

[46]

[47] [48] [49] [50] [51]

[52] [53] [54]

[55] [56] [57]

[58]

[59]

[60] [61]

Watanabe T, Takeda A, Tsukiyama T, et al. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev 2006; 20: 1732-43. Watanabe T, Totoki Y, Toyoda A, et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 2008; 453: 539-43. Choi Y, Qin Y, Berger MF, Ballow DJ, Bulyk ML, Rajkovic A. Microarray analyses of newborn mouse ovaries lacking Nobox. Biol Reprod 2007; 77: 312-9. Tam OH, Aravin AA, Stein P, et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 2008; 453: 534-8. Tang F, Barbacioru C, Wang Y, et al. mRNA-Seq wholetranscriptome analysis of a single cell. Nat Methods 2009; 6: 37782. Tesfaye D, Worku D, Rings F, et al. Identification and expression profiling of microRNAs during bovine oocyte maturation using heterologous approach. Mol Reprod Dev 2009; 76: 665-77. Bonnet A, Dalbiès-Tran R, Sirard MA. Opportunities and challenges in applying genomics to the study of oogenesis and folliculogenesis in farm animals. Reproduction 2008; 135: 119-28. Forde N, Mihm M, Canty MJ, et al. Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle. Physiol Genomics 2008; 33: 193-204. Toloubeydokhti T, Bukulmez O, Chegini N. Potential regulatory functions of microRNAs in the ovary. Semin Reprod Med 2008; 26: 469-78. Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY. MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res 2007; 67: 8433-8. Iorio MV, Visone R, Di Leva G, et al. MicroRNA signatures in human ovarian cancer. Cancer Res 2007; 67: 8699-707. Nam EJ, Yoon H, Kim SW, et al. MicroRNA expression profiles in servus ovarian carcinoma. Clin Cancer Res 2008; 14: 2690-5. Merritt WM, Lin YG, Han LY, et al. Dicer, Drosha, and outcomes in patients with ovarian cancer. N Engl J Med 2008; 359: 2641450. Dahiya N, Morin P. MicroRNAs in Ovarian Carcinomas. Endocr Relat Cancer. 2009 Nov 10. [Epub ahead of print] Lodes MJ, Caraballo M, Suciu D, Munro S, Kumar A, Anderson B. Detection of cancer with serum miRNAs on an oligonucleotide microarray. PLoS One 2009; 4: e6229. Wang Y, Lee CG. MicroRNA and cancer--focus on apoptosis. J Cell Mol Med 2009; 13: 12-23. Lei L, Jin S, Gonzalez G, Behringer RR, Woodruff TK. The regulatory role of Dicer in folliculogenesis in mice. Mol Cell Endocrinol 2010; 315: 63-73. Nagaraja AK, Andreu-Vieyra C, Franco HL, et al. Deletion of Dicer in somatic cells of the female reproductive tract causes sterility. Mol Endocrinol 2008; 22: 2336-52. Murchison EP, Stein P, Xuan Z, et al. Critical roles for Dicer in the female germline. Genes Dev 2007; 21: 682-93 Tang F, Kaneda M, O'Carroll D, et al. Maternal microRNAs are essential for mouse zygotic development. Genes Dev 2007; 21: 644-8. Kaneda M, Tang F, O'Carroll D, Lao K, Surani MA. Essential role for Argonaute2 protein in mouse oogenesis. Epigenetics Chromatin 2009; 2: 9. Wang N, Zhang P, Guo X, Zhou Z, Sha J. Hnrnpk, a protein differentially expressed in immature rat ovarian development, is required for normal primordial follicle assembly and development. Endocrinology 2011; 152: 1024-35. An Y, Cai Y, Guan Y, et al. Inhibitory effect of small interfering RNA targeting insulin-like growth factor-I receptor in ovarian cancer OVCAR3 cells. Cancer Biother Radiopharm 2010; 25: 54552. Wen J, Zhang H, Li G, et al. PAR6, a potential marker for the germ cells selected to form primordia follicles in mouse ovary. PLoS One 2009; 4(: e7372. Gulia-Nuss M, Robertson AE, Brown MR, Strand MR. InsulinLike Peptides and tle Target of Rapamycin Pathway Coordinately Regulate Blood Digestion and Egg Maturation in the Mosquito Aedes aegypti. PLoS One 2011; 6: e20401.

RNA Interference and Ovarian Functions [62]

[63] [64]

[65] [66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78] [79]

[80] [81]

[82]

Hatta T, Tsuji N, Miyoshi T, et al. Leucine aminopeptidase, HlLAP, from the ixodid tick Haemaphysalis longicornis, plays vital roles in the development of oocytes. Parasitol Int 2010; 59: 286-9. Poulton JS, Huang YC, Smith L, et al. The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells. Development 2011; 138: 1737-45. Parthasarathy R, Sheng Z, Sun Z, Palli SR. Ecdysteroid regulation of ovaria growth and oocyte maturation in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol 2010; 40: 429-39. Sirotkin AV, Lauková M, Ovcharenko D, Brenaut P, Mlyncek M. Identification of microRNAs controlling human ovarian cell proliferation and apoptosis. J Cell Physiol 2010; 223: 49-56. Sirotkin AV, Ovcharenko D, Benco A, Mlyncek M. Protein kinases controlling PCNA and p53 expression in human ovarian cells. Funct Integr Genomics 2009b; 9: 185-95. Tsuda N, Kawano K, Efferson CL, Ioannides CG. Synthetic microRNA and double-stranded RNA targeting the 3'-untranslated region of HER-2/neu mRNA inhibit HER-2 protein expression in ovarian cancer cells. Int J Oncol 2005; 27: 1299-306. Lu YM, Zhang SL, Meng LR, Zhao YY. Influence of human epidermal growth factor receptor-2 siRNA on chemosensitivity to cisplatin of human ovarian carcinoma cells: an in vitro experiment. Zhonghua Yi Xue Za Zhi 2008; 88: 909-13. Bruegmann E, Gruemmer R, Neulen J, Motejlek K. Regulation of soluble vascular endothelial growth factor receptor 1 secretion from human endothelial cells by tissue inhibitor of metalloproteinase 1. Mol Hum Reprod 2009 Jul 7. [Epub ahead of print] PMID: 19584194 Wang FQ, Barfield E, Dutta S, Pua T, Fishman DA. VEGFR-2 silencing by small interference RNA (siRNA) suppresses LPAinduced epithelial ovarian cancer (EOC) invasion. Gynecol Oncol 2009 Sep 16. [Epub ahead of print] Zhou J, Gan N, Zhang W, Lu W, Xie X. Proliferation suppression and apoptosis of ovarian carcinoma cells induced by small interfering RNA against vascular endothelial growth factor. J Obstet Gynaecol Res 2010; 36: 232-8. Adham SA, Sher I, Coomber BL. Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells. Lab Invest 2010; 90: 709-23. Donahue RN, McLaughlin PJ, Zagon IS. Cell proliferation of human ovarian cancer is regulated by the opioid growth factor-opioid growth factor receptor axis. Am J Physiol Regul Integr Comp Physiol 2009; 296: R1716-25. Rayhman O, Klipper E, Muller L, Davidson B, Reich R, Meidan R. Small interfering RNA molecules targeting endothelin-converting enzyme-1 inhibit endothelin-1 synthesis and the invasive phenotype of ovarian carcinoma cells. Cancer Res 2008; 68: 926573. Paragh G, Kumar SM, Rakosy Z, Choi SC, Xu X, Acs G. RNA Interference-Mediated Inhibition Of Erythropoietin Receptor Expression Suppresses Tumor Growth And Invasiveness In A2780 Human Ovarian Carcinoma Cells. Am J Pathol 2009; 174: 150414. Guo J, Schally AV, Zarandi M, Varga J, Leung PC. Antiproliferative effect of growth hormone-releasing hormone (GHRH) antagonist on ovarian cancer cells through the EGFR-Akt pathway. Reprod Biol Endocrinol 2010; 8: 54. Pierre P, Froment P, Nègre D, et al. Role of adiponectin receptors, AdipoR1 and AdipoR2, in the steroidogenesis of tle human granulosa tumor cell line, KGN. Hum Reprod 2009 Aug 11. [Epub ahead of print] PMID: 19671624 Tian L, Wu Z, Zhao Y, et al. Differential induction of LRP16 by liganded and unliganded estrogen receptor alpha in SKOV3 ovarian carcinoma cells. J Endocrinol 2009; 202: 167-77. Huang Y, Hua K, Zhou X, et al. Activation of the PI3K/AKT pathway mediates FSH-stimulated VEGF expression in ovarian serous cystadenocarcinoma. Cell Res 2008; 18: 780-91. Moreno-Smith M, Lu C, Shahzad MM, et al. Dopamine Blocks Stress-Mediated Ovarian Carcinoma Growth. Clin Cancer Res 2011; 17: 3649-59. Cole C, Lau S, Backen A, et al. Inhibition of FGFR2 and FGFR1 increases cisplatin sensitivity in ovarian cancer. Cancer Biol Ther 2010; 10: 495-504. Tsuno T, Mejido J, Zhao T, Schmeisser H, Morrow A, Zoon KC. IRF9 is a Key Factor for Eliciting the Antiproliferative Activity of IFN-alpha. J Immunother 2009 Sep 11. [Epub ahead of print]

Current Pharmaceutical Design, 2012, Vol. 18, No. 3 [83]

[84] [85]

[86]

[87] [88]

[89]

[90]

[91] [92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

335

Bhattacharya R, Nicoloso M, Arvizo R, et al. MiR-15a and MiR-16 Control Bmi-1 Expression in Ovarian Cancer. Cancer Res 2009 Nov 10. [Epub ahead of print] Oktay K, Buyuk E, Oktem O, Oktay M, Giancotti FG. The c-Jun N-terminal kinase JNK functions upstream of Aurora B to promote entry into mitosis. Cell Cycle 2008; 7: 533-41. Todd MC, Spruill SC, Meerbrey KL. Small interference RNAmediated suppression of overexpressed cyclin E protein restores G1/S regulation in NIH-OVCAR-3 ovarian cancer cells. Int J Oncol 2009; 35: 375-80. Shin JS, Hong SW, Lee SL, et al. Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells. Int J Oncol 2008; 32: 435-9. Kulkarni AA, Kingsbury SR, Tudzarova S, et al. Cdc7 kinase is a predictor of survival and a novel therapeutic target in epithelial ovarian carcinoma. Clin Cancer Res 2009; 15: 2417-25. Tanaka T, Mangala LS, Vivas-Mejia PE, et al. Sustained small interfering RNA delivery by mesoporous silicon particles. Cancer Res 2010; 70: 3687-3696. Zhang X, Deng HX, Zhao X, et al. RNA interference-mediated silencing of the phosphatidylinositol 3-kinase catalytic subunit attenuates growth of human ovarian cancer cells in vitro and in vivo. Oncology 2009; 77: 22-32. Hernandez L, Hsu SC, Davidson B, Birrer MJ, Kohn EC, Annunziata CM. Activation of NF-kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian cancer. Cancer Res 2010; 70: 4005-14. Smith ER, Cai KQ, Smedberg JL, et al. Nuclear entry of activated MAPK is restricted in primary ovarian and mammary epithelial cells. PLoS One 2010; 5: e9295. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR. Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res 2010; 8: 578-91. Rao ZY, Cai MY, Yang GF, et al. EZH2 supports ovaria carcinoma cell invasion and/or metastasi via regulation of TGF-beta1 and is a predictor of outcome in ovaria carcinoma patiens. Carcinogenesis 2010; 31: 1576-83. Linnerth NM, Greenaway JB, Petrik JJ, Moorehead RA. cAMP response element-binding protein is expressed at high levels in human ovarian adenocarcinoma and regulates ovarian tumor cell proliferation. Int J Gynecol Cancer 2008; 18: 1248-57. Chan DW, Liu VW, To RM, Chiu et al. Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21(WAF1/CIP1) expression in ovarian cancer. Br J Cancer 2009; 146: 222-6 Yoshioka N, Suzuki N, Uekawa A, Kiguchi K, Ishizuka B. POU6F1 is tle transcription factor that might be involved in cell proliferation of clear cell adenocarcinoma of the ovary. Hum Cell 2009; 22: 94-100. Suzuki N, Yoshioka N, Uekawa A, et al. Transcription factor POU6F1 is important for proliferation of clear cell adenocarcinoma of the ovary and is a potential new molecular target. Int J Gynecol Cancer 2010; 20: 212-9. Huang F, Tong X, Fu L, Zhang R. Knockdown of STAT3 by shRNA inhibits the growth of CAOV3 ovarian cancer cell line in vitro and in vivo. Acta Biochim Biophys Sin (Shanghai) 2008; 40: 519-25. Cai L, Zhang G, Tong X, You et al. Growth inhibition of human ovarian cancer cells by blocking STAT3 activation with small interfering RNA. Eur J Obstet Gynecol Reprod Biol 2010; 148: 7380. Ma JJ, Chen BL, Xin XY. XIAP gene downregulation by small interfering RNA inhibits proliferation, induces apoptosis, and reverses the cisplatin resistance of ovarian carcinoma. Eur J Obstet Gynecol Reprod Biol 2009; 146: 222-6. Shahzad MM, Arevalo JM, Armaiz-Pena GN, et al. Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis. J Biol Chem 2010; 285: 35462-70. Tarapore P, Shu Y, Guo P, Ho S-M. Application of Phi29 motor pRNA for targeted therapeutic delivery of siRNA silencing metallothionein-IIA and survivin in ovarian cancers. Molecular Therapy 2011; 19: 386-94. Lin Y, Peng S, Yu H, Teng H, Cui M. RNAi-mediated downregulation of NOB1 suppresses the growth and colony-

336 Current Pharmaceutical Design, 2012, Vol. 18, No. 3

[104]

[105]

[106] [107]

[108] [109]

[110]

[111]

[112] [113]

[114]

[115] [116]

[117]

[118]

formation ability of human ovarian cancer cells. Med Oncol 2011 [Epub ahead of print] PubMed PMID: 21287298. Luo Y, Yi Y, Yao Z. Growth arrest in ovarian cancer cells by hTERT inhibition short-hairpin RNA targeting human telomerase reverse transcriptase induces immediate growth inhibition but not necessarily induces apoptosis in ovaria cancer cells. Cancer Invest 2009; 27: 960-70. Zhou Y, Yi X, Stoffer JB, et al. The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer. Mol Cancer Res 2008; 6: 1375-84. Uddin S, Ahmed M, Hussain A, et al. Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer. Int J Cancer 2010; 126: 382-94. Yotsumoto F, Yagi H, Suzuki SO, et al. Validation of HB-EGF and amphiregulin as targets for human cancer therapy. Biochem Biophys Res Commun 2008; 365: 555-61. Dickerson EB, Blackburn WH, Smith MH, Kapa LB, Lyon LA, McDonald JF. Chemosensitization of cancer cells by siRNA using targeted nanogel delivery. BMC Cancer 2010; 10: 10. Lane D, Goncharenko-Khaider N, Rancourt C, Piché A. Ovarian cancer ascites protects from TRAIL-induced cell death through alphavbeta5 integrin-mediated focal adhesion kinase and Akt activation. Oncogene 2010; 29: 3519-31. Witham J, Valenti MR, De-Haven-Brandon AK, et al. The Bcl2/Bcl-XL family inhibitor ABT-737 sensitizes ovarian cancer cells to carboplatin. Clin Cancer Res 2007; 13: 7191-8. Brotin E, Meryet-Figuière M, Simonin K, et al. Bcl-x(L) and MCL1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis. Int J Cancer 2009 Jul 24. [Epub ahead of print] Mancini F, Di Conza G, Pellegrino M, et al. MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway. EMBO J 2009; 28: 1926-39. Goldberg MS, Xing D, Ren Y, Orsulic S, Bhatia SN, Sharp PA. Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells. Proc Natl Acad Sci USA 2011; 108: 745-50. Vivas-Mejia PE, Rodriguez-Aguayo C, Han HD, et al. Silencing Survivin Splice Variant 2B Leads to Antitumor Activity in TaxaneResistant Ovarian Cancer. Clin Cancor Res 2011; 17: 3716-26. Huang Y, Jin H, Liu Y, et al. FSH inhibits ovarian cancer cell apoptosis by up-regulating survivin and down-regulating PDCD6 and DR5. Endocr Relat Cancer 2010; 18: 13-26. Sirotkin AV, Ovcharenko D, Grossmann R, Lauková M, Mlyncek M. Identification of microRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen. J Cell Physiol 2009; 219: 415-20. Sirotkin AV, Ovcharenko D, Mlynek M. Identification of protein kinases that control ovarian hormone release by selective siRNAs. J Molec Endocrinol 2010b; 44: 45-53. Wu YG, Bennett J, Talla D, Stocco C. Testosterone, not 5dihydrotestosterone, stimulates LRH-1 leading to FSH-independent expression of Cyp19 and P450scc in granulosa cells. Mol Endocrinol 2011; 25: 656-68.

Received: October 6, 2011

Accepted: November 30, 2011

Alexander V. Sirotkin [119]

[120]

[121]

[122]

[123]

[124] [125] [126]

[127]

[128]

[129]

[130] [131]

[132]

[133]

Li Y, Liang XY, Wei LN, et al. Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alphahydroxylase/17, 20-lyase activity in vivo. Reprod Biol Endocrinol 2009 7: 73. Hui YY, Lavoie HA. GATA4 reduction enhances 3',5'-cyclic adenosine 5'-monophosphate-stimulated steroidogenic acute regulatory protein messenger ribonucleic acid and progesterone production in luteinized porcine granulosa cells. Endocrinology 2008; 149: 5557-67. Kobayashi S, Sakatani M, Kobayashi S, Okuda K, Takahashi M. Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells. J Reprod Dev 2007; 53: 130511. Wang C, Xu B, Zhou B, et al. Reducing CYP51 inhibits FSHinduced resumption of mouse oocyte meiosis in vitro. J Lipid Res 2009 May 11. [Epub ahead of print] PMID: 19433477 Mattiske DM, Han L, Mann JR. Meiotic maturation failure induced by DICER1 deficiency is derived from primary oocyte ooplasm. Reproduction 2009; 137: 625-32. Suh N, Baehner L, Moltzahn F, et al. MicroRNA function is globally suppressed in mouse oocytes and early embryos. Curr Biol 2010; 20: 271-7. Hasegawa A, Koyama K. Contribution of zona proteins to oocyte growth. Soc Reprod Fertil Suppl 2007; 63: 229-35. Boldbaatar D, Battsetseg B, Matsuo T, et al. Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission of Babesia parasite. Biochem Cell Biol 2008; 86: 331-44. Lu HL, Vinson SB, Pietrantonio PV. Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virin queens. FEBS J 2009; 276: 3110-23. Diaz-Albiter H, Mitford R, Genta FA, Sant'Anna MR, Dillon RJ. Reactive oxygen species scavenging by catalase is important for female Lutzomyia longipalpis fecundity and mortality. PLoS One 2011; 6: e17486. Treerattrakool S, Panyim S, Udomkit A. Induction of ovarian maturation and spawning in Penaeus monodon broodstock by double-stranded RNA. Mar Biotechnik (NY) 2011; 13: 163-9. Wu SC. RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells. Biotechnology Advances 2009; 27: 417-22. Rahbek UL, Howard KA, Oupicky D, et al. Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides. J Gene Med 2008; 10: 81-93. Frank-Kamenetsky M, Grefhorst A, Anderson NN, et al. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA 2008; 105: 11915-20. Kim SH, Jeong JH, Lee SH, Kim SW, Park TG. LHRH receptormediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI. Bioconjug Chem 2008; 19: 2156-62.