RNA-Seq analysis of pubertal mammary epithelial cells reveals novel n-3 polyunsaturated fatty acid transcriptomic changes in the fat-1 mouse model
Background/Objectives: Early exposure of nutrients during pubertal mammary gland development may reduce the risk of developing breast cancer later in life. Anticancer, n-3 polyunsaturated fatty acids (n-3 PUFA) are shown to modulate pubertal mammary gland development, however, the mechanisms of action remains unclear. Prior work focuses on effects at the whole tissue level, and little is known at the cellular level, such as mammary epithelial cells (MEC), which are implicated in cancer development. Methods: This pilot study examined the effects of lifelong n-3 PUFA exposure on the transcriptome by RNA-Seq in isolated MEC of pubertal (6–8-week-old) female fat-1 transgenic mice capable of de novo n-3 PUFA synthesis. edgeR and DESeq2 were used separately for the differential expression analysis of RNA sequencing data followed by Benjamini-Hochberg procedure for multiple testing correction. Results: Nine genes were found concordant and significantly different (p = 0.05) by both DESeq2 and edgeR methods. These genes were associated with multiple pathways, suggesting that n-3 PUFA stimulates estrogen-related signalling (Mllt10, Galr3, Nrip1) and a glycolytic profile (Soga1, Pdpr, Uso1) while offering protective effects for immune and DNA damage responses (Glpd1, Garre1, Rpa1) in MEC during puberty. Conclusions: This pilot study highlights the utility of RNA-Seq to better understand mechanistic effects of specific nutrients such as n-3 PUFA in a cell-specific manner. Thus, further studies are warranted to investigate cell-specific mechanisms by which n-3 PUFA influences pubertal mammary gland development and breast cancer risk later in life. Overall design: To investigate the transcriptomic effects of lifelong n-3 polyunsaturated fatty acid exposure in isolated mammary epithelial cells from female transgenic fat-1 mice compared to lifelong n-6 polyunsaturated fatty acid exposed wildtype (WT)  during puberty using bulk RNA-Seq. To reduce cofounding effects from dietary feeding, all mice maintained on their parental diet of 10% fat (w/w) from safflower oil that contains 70% of the essential n-6 PUFA linoleic acid, which transgenic fat-1 mice will endogenously utilize to synthesize individual n-3 PUFA in all tissues, including in the mammary gland.