Functional characterization of the promoter of carbonyl reductase 1 gene in porcine endometrial cells

Abstract

Prostaglandins (PGs) play a critical role in porcine reproduction, of which prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 (CBR1) catalyzes the conversion of PGE2 to PGF2α. A high ratio of PGE2:PGF2α is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor κB (NF-κB) is involved in the process. Bioinformatic analysis has shown that NF-κB is a possible factor bound to two cis-regulatory elements in CBR1 promoter. In this study, we cloned the 2997 bp (−2875/+122) of the promoter, and constructed six 5'-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 (−1640/+7) exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 (−2089/+7). The activity of P1, P2, and P3 (−1019/+7) was highly significantly higher than that of others (P<0.01), suggesting that two positive regulatory elements were likely present in the regions of −1640/−1019 and −1019/−647. The results also showed that the −1640/−647 region was indispensable for the promoter. The results of chromatin immunoprecipitation (ChIP) demonstrated that the NF-κB subunit p65 binds to one site around −1545/−1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 (P<0.05) in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBR1 expression. These results indicated that NF-κB (p65) could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-κB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.

中文概要

题目

CBR1 基因启动子在猪子宫内膜细胞的功能研究

目的

研究CBR1 基因启动子在猪子宫内膜细胞的表达调控机制。

创新点

发现CBR1 基因启动子在猪子宫内膜受到炎性因子核转录因子kappa B(NF-κB)成员p65 调控。p65 对该启动子具有正向调节作用,但是对于CBR1 基因的表达并不是必需的。

方法

通过双荧光素酶报告基因载体确定CBR1 基因启动子转录活性区,通过染色质免疫沉淀(ChIP)技术确定p65 能够结合CBR1 基因启动子,通过超表达和干扰表达实验证实p65 对CBR1 基因启动子的调控作用。

结论

猪CBR1 基因启动子−1640/−647 区对于其转录活性是必需的,在−1545/−1531 区存在p65 的结合位点。p65 在猪子宫内膜细胞中促进了CBR1 基因mRNA的表达,但是干扰p65则不会造成CBR1基因mRNA 表达量下降,推断p65 不是CBR1 基因表达的必需因素。