Gene Silencing of Cyclooxygenase-2 mRNA by RNA Interference in Bovine Cumulus-Granulosa Cells

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Abstract

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of <i>cyclooxygenase-2</i> (<i>Cox-2</i>) mRNA and prostagrandin F<sub>2</sub><sub>α</sub> (PGF <sub>2</sub><sub>α</sub>) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of <i>Cox-2</i> mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of <i>Cox-2</i> mRNA expression was measured and the culture medium was collected to determine the PGF<sub>2</sub><sub>α</sub> concentration by enzyme immunoassay. The <i>Cox-2</i> mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the <i>Cox-2</i> mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF<sub>2</sub><sub>α</sub> concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF<sub>2</sub><sub>α</sub> concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of <i>Cox-2</i> with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.<br>

Journal

  • Journal of Reproduction and Development

    Journal of Reproduction and Development 53(6), 1305-1311, 2007-12-01

    THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

References:  29

Cited by:  1

Codes

  • NII Article ID (NAID)
    10020173139
  • NII NACSIS-CAT ID (NCID)
    AA10936678
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    09168818
  • NDL Article ID
    9327217
  • NDL Source Classification
    ZR22(科学技術--農林水産--畜産)
  • NDL Call No.
    Z54-H305
  • Data Source
    CJP  CJPref  NDL  J-STAGE 
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