Proteomics Insights into the Gene Network of cis9, trans11-Conjugated Linoleic Acid Biosynthesis in Bovine Mammary Gland Epithelial Cells

SIMPLE SUMMARY: Cis9, trans11-conjugated linoleic acid (c9, t11-CLA) is a bioactive compound that is mainly found in ruminant products and has a variety of beneficial biological functions in human health. However, the gene network of c9, t11-CLA biosynthesis in the mammary gland is still not well documented. Therefore, this study aimed to elucidate the stearoyl-coenzyme A desaturase (SCD1)-dependent gene network of c9, t11-CLA biosynthesis in MAC-T cells from the perspective of energy metabolism. The results showed that the deficiency of stearoyl-coenzyme A desaturase (SCD1), led by CAY10566, blocked the biosynthesis of c9, t11-CLA. In total, 60 SCD1-related proteins that were mainly involved in energy metabolism pathways were primary screened by Tandem mass tag-based quantitative proteomics analysis. Moreover, 17 proteins were validated under the parallel reaction monitoring analysis. Then, 11 genes involved in energy metabolism pathways were verified to have negative relationships with SCD1 after small RNA interference analysis. Based on the above results, we concluded that the 11 genes involved in energy metabolism pathways had an impact on the SCD1-dependent molecular mechanism of c9, t11-CLA biosynthesis. This study provided a fresh understanding of the gene network of c9, t11-CLA biosynthesis in the mammary gland from a metabolism perspective. ABSTRACT: The objective of the study was to elucidate the stearoyl-coenzyme A desaturase (SCD1)-dependent gene network of c9, t11-CLA biosynthesis in MAC-T cells from an energy metabolism perspective. The cells were divided into the CAY group (firstly incubated with CAY10566, a chemical inhibitor of SCD1, then incubated with trans-11-octadecenoic acid, (TVA)), the TVA group (only TVA), and the control group (without CAY, TVA). The c9, t11-CLA, and TVA contents were determined by gas chromatography. The mRNA levels of SCD1 and candidate genes were analyzed via real-time PCR. Tandem mass tag (TMT)-based quantitative proteomics, bioinformatic analysis, parallel reaction monitoring (PRM), and small RNA interference were used to explore genes involved in the SCD1-dependent c9, t11-CLA biosynthesis. The results showed that the SCD1 deficiency led by CAY10566 blocked the biosynthesis of c9, t11-CLA. In total, 60 SCD1-related proteins mainly involved in energy metabolism pathways were primarily screened by TMT-based quantitative proteomics analysis. Moreover, 17 proteins were validated using PRM analysis. Then, 11 genes were verified to have negative relationships with SCD1 after the small RNA interference analysis. Based on the above results, we concluded that genes involved in energy metabolism pathways have an impact on the SCD1-dependent molecular mechanism of c9, t11-CLA biosynthesis.