1Joint Laboratory of Modern Agricultural Technology International Cooperation, Ministry of Education, Jilin Agricultural University, Jilin, Changchun, 130118, China
2Key Laboratory of Animal Production, Product Quality and Security, Ministry of Education, Jilin Agricultural University, Jilin, Changchun, 130118, China
3Jilin Province Engineering Laboratory for Ruminant Reproductive Biotechnology and Healthy Production, College of Animal Science and Technology, Jilin Agricultural University, Changchun, 130118, China
*Corresponding Author: Jing Zhao, Wenfa Lu, Joint Laboratory of Modern Agricultural Technology International Cooperation, Ministry of Education, Jilin Agricultural University, Jilin Changchun130118, China, Email: jlndzjing@126.com
#Co-first authors.
Cow mastitis is a major disease that affects dairy industry worldwide. Although the inflammatory response induced by lipopolysaccharide (LPS) in bovine mammary epithelial cells (BMECs) is similar to the response to pathogenic bacteria, the underlying regulatory mechanisms remain unclear. This study aimed to clarify the effect of DNA methylation on LPS-induced expression of tumour necrosis factor alpha (TNF-α) in BMECs.
The mammary epithelial cells were treated with LPS and DNA methylation inhibition 5-Aza-2'-deoxycytodine (5Aza). Expression of TNF-α, IL-6, BNBD-5, DNA methyltransferases (DNMT1, DNMT2, DNMT3A and DNMT3B) and DNA methylation at TNF-α regions, were analyzed using quantitative realtime PCR (qRT-PCR), Elisa and bisulfite sequencing PCR (BSP).
Our results showed that LPS significantly increased the expression of inflammatory factors, including interleukin-6 (IL-6), bovine neutrophil beta-defensins (BNBD-5) and TNF-α. Further, we observed that the DNA methylation inhibitor, 5-Aza- 2′-deoxycytosine (5-Aza), enhanced LPS-induced TNF-α mRNA expression. In addition, we found that LPS treatment significantly decreased the methylation levels of specific CpG sites in the TNF-α promoter (at-245 and-323) and inhibited the expression of DNA methyl transferases (DNMT1, DNMT2, DNMT3A and DNMT3B). Our results indicate that LPS promotes the expression of TNF-α in BMECs by inhibiting DNA methylation in the gene promoter region.
Bovine mammary epithelial cells, DNA methylation, LPS, TNF-α
