Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

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. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.

[1]  F. Le Provost,et al.  MicroRNA involvement in mammary gland development and breast cancer. , 2006, Reproduction, nutrition, development.

[2]  Wei Yan,et al.  Cloning and expression profiling of small RNAs expressed in the mouse ovary. , 2007, RNA.

[3]  F. Slack,et al.  MicroRNAs in search of a target. , 2006, Cold Spring Harbor symposia on quantitative biology.

[4]  X. Cui,et al.  A link between the interleukin‐6/Stat3 anti‐apoptotic pathway and microRNA‐21 in preimplantation mouse embryos , 2009, Molecular reproduction and development.

[5]  Q. Pan,et al.  The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression. , 2007, Molecular human reproduction.

[6]  S. Hillier,et al.  Gonadotropic control of ovarian follicular growth and development , 2001, Molecular and Cellular Endocrinology.

[7]  N. Tsuda,et al.  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. , 2005, International journal of oncology.

[8]  Wei Wang,et al.  MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. , 2007, Cancer research.

[9]  B. Joseph,et al.  The mitochondrial death pathway: a promising therapeutic target in diseases , 2009, Journal of cellular and molecular medicine.

[10]  M. Tena-Sempere,et al.  Novel expression and functional role of ghrelin in chicken ovary , 2006, Molecular and Cellular Endocrinology.

[11]  Xu Ma,et al.  MiR-125b Expression Affects the Proliferation and Apoptosis of Human Glioma Cells by Targeting Bmf , 2009, Cellular Physiology and Biochemistry.

[12]  Mauro Biffoni,et al.  The miR-15a–miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities , 2008, Nature Medicine.

[13]  P. Gammell.  MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells , 2007, Cytotechnology.

[14]  G. Maga,et al.  Proliferating cell nuclear antigen (PCNA): a dancer with many partners , 2003, Journal of Cell Science.

[15]  Jae Hoon Kim,et al.  MicroRNA Expression Profiles in Serous Ovarian Carcinoma , 2008, Clinical Cancer Research.

[16]  J. Kotwica,et al.  Leptin Directly Controls Secretory Activity of Human Ovarian Granulosa Cells: Possible Inter-Relationship with the IGF/IGFBP System , 2005, Hormone Research in Paediatrics.

[17]  Huan Yang,et al.  MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. , 2008, Cancer research.

[18]  C. Lee,et al.  MicroRNA and cancer – focus on apoptosis , 2008, Journal of cellular and molecular medicine.

[19]  Junxia Chen,et al.  MicroRNA‐21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice , 2009, FEBS letters.

[20]  H. Hollema,et al.  Aromatase in the context of breast and endometrial cancer. A review. , 2006, Minerva endocrinologica.

[21]  Giovanni Vanni Frajese,et al.  The Inhibition of the Highly Expressed Mir-221 and Mir-222 Impairs the Growth of Prostate Carcinoma Xenografts in Mice , 2008, PloS one.

[22]  M. Lindsay,et al.  Expression profiling in vivo demonstrates rapid changes in lung microRNA levels following lipopolysaccharide-induced inflammation but not in the anti-inflammatory action of glucocorticoids , 2007, BMC Genomics.

[23]  Giovanni Vanni Frajese,et al.  miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1* , 2007, Journal of Biological Chemistry.

[24]  Thomas Tuschl,et al.  The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members , 2008, Development.

[25]  Charles D. Johnson,et al.  Genome-scale microRNA and small interfering RNA screens identify small RNA modulators of TRAIL-induced apoptosis pathway. , 2007, Cancer research.

[26]  H. Hermeking.  The miR-34 family in cancer and apoptosis , 2010, Cell Death and Differentiation.

[27]  A. Kazlauskas,et al.  Growth factor‐dependent signaling and cell cycle progression , 2001, Chemical reviews.

[28]  D. Ovcharenko,et al.  Identification of MicroRNAs controlling human ovarian cell steroidogenesis via a genome‐scale screen , 2009, Journal of cellular physiology.

[29]  C. Croce,et al.  MicroRNA signatures in human ovarian cancer. , 2007, Cancer research.

[30]  D. Ovcharenko,et al.  Protein kinases controlling PCNA and p53 expression in human ovarian cells , 2009, Functional & Integrative Genomics.

[31]  C. Croce,et al.  miR-15a and miR-16-1 in cancer: discovery, function and future perspectives , 2010, Cell Death and Differentiation.

[32]  R. Stouffer,et al.  Molecular control of ovulation and luteinization in the primate follicle. , 2007, Frontiers in bioscience : a journal and virtual library.

[33]  M. Osborn,et al.  Immunocytochemistry of Frozen and of Paraffin Tissue Sections , 2006 .

[34]  P. Foster,et al.  Regulation of microRNA by antagomirs: a new class of pharmacological antagonists for the specific regulation of gene function? , 2007, American journal of respiratory cell and molecular biology.

[35]  S. Hammes,et al.  Ovarian Steroids: The Good, the Bad, and the Signals that Raise Them , 2006, Cell cycle.

[36]  B. Fauser,et al.  Follicle-stimulating hormone and advanced follicle development in the human. , 2001, Archives of medical research.

[37]  Dirk Winkler,et al.  miR-34a as part of the resistance network in chronic lymphocytic leukemia. , 2008, Blood.