title: Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells.
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 cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) 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 Cox-2 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 Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 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 Cox-2 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(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) 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 Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.
authors.affiliation: Research Team for Effects of Climate Change on Agriculture, National Agricultural Research Center for Kyushu Okinawa Region, Kumamoto, Japan.
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Miki
authors.initials: M
authors.lastname: Sakatani
authors.forename: Shuji
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Okuda
authors.forename: Masashi
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002417:Cattle; D054885:Cumulus Cells / Q000502:physiology*; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D015237:Dinoprost / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006107:Granulosa Cells / Q000502:physiology*; D008967:Molecular Biology / Q000379:methods*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D015237:Dinoprost; D051546:Cyclooxygenase 2