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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2017 Vol.18 No.7 P.626-634
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.
Key words: Pig; Carbonyl reductase 1 (CBR1); Promoter; NF-κB; Endometrium
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。
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DOI:
10.1631/jzus.B1600225
CLC number:
S828.2
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On-line Access:
2017-07-05
Received:
2016-05-18
Revision Accepted:
2016-09-12
Crosschecked:
2017-06-16