Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells
Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells
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DOI：
10.1262/jrd.19050
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发表时间：
2007-12-01
影响因子：
1.8
通讯作者：
Takahashi, Masashi
中科院分区：
文献类型：
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作者：
Kobayashi, Shu-Ichi;Sakatani, Miki;Takahashi, Masashi
利用RNA干扰（RNAi）抑制特定基因表达是用于靶基因的功能分析的有价值的工具。然而，有很少的信息可用于RNAi的基因功能分析与反刍动物卵泡细胞的生殖生理学。因此，本研究的目的是评估小干扰RNA（siRNA）对牛卵丘-颗粒（CG）细胞中环氧合酶-2（考克斯-2）mRNA表达和木兰素F-2 α（PGF（2 α））产生的干扰效果。从抽吸的卵泡收集牛CG细胞并进行培养。达到融合后，进行了两个实验。在实验1中，为了研究siRNA的有效浓度，将0、100、250和500 pM的考克斯-2 siRNA分别导入CG细胞。24 h后，通过RT-PCR和实时定量PCR测量考克斯-2 mRNA的表达量。在实验2中，为了研究siRNA有效干扰和考克斯-2活性所需的时间，引入250 pM siRNA 0、3、6、12和24 h。培养后，测定考克斯-2 mRNA表达量，收集培养基，通过酶免疫测定法测定PGF（2 α）浓度。100 pM siRNA作用CG细胞24 h后，考克斯-2 mRNA的表达无明显变化，但250和500 pM考克斯-2 siRNA显著降低了考克斯-2 mRNA的表达。250 pM的siRNA在6 h后对细胞的mRNA表达有明显的抑制作用，12 h后抑制作用更加明显。另一方面，在siRNA导入后12小时，培养基中的PGF（2 α）浓度没有显著差异，然而，在siRNA导入后24小时，与相同时间点的对照相比，PGF（2 α）浓度显著降低。这些结果表明，利用siRNA沉默考克斯-2基因能够分析牛CG细胞中特定基因的功能和表达。
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-2 alpha (PGF(2 alpha)) 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(2 alpha) 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(2 alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2 alpha) 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.