253 GENE SILENCING OF CYCLOOXYGENASE-2 mRNA BY RNA INTERFERENCE IN BOVINE CUMULUS–GRANULOSA CELLS

Abstract

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F_2± (PGF_2±) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF_2± concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF_2± concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF_2± concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells.

This study was supported by the Japan Society for the Promotion of Science for Young Scientists.