(P18-061-23) Let-7g-5p Regulates Mammary Epithelial Cell Function and Is Modulated by Polyphenols

Objectives: Concern over poor lactation performance is the primary reason ~60% of women do not meet their breastfeeding goals and understanding the biology behind poor lactation performance is critical to improving both maternal and infant health outcomes. miRNAs are small nucleotides (~22 bases) that target specific mRNAs for degradation, thereby post-transcriptionally regulating gene expression. Importantly, miRNAs secreted into milk may serve as a “liquid biopsy” of mammary gland function. We previously showed elevated levels of the miRNA let-7g-5p are associated with low milk supply (LMS) and inversely related to fruit intake. As a result, we hypothesize let-7g-5p reduces expression of key mRNA targets, impairing differentiation and increasing oxidative stress, and that polyphenols restrict let-7g-5p expression, reduce oxidative stress, and promote differentiation.

Methods: Mammary epithelial (HC11) cells were transfected with hsa-let-7g-5p miRNA mimic (let7g^OX) and overexpression was verified by RT-qPCR. Effects on two predicted mRNA targets, insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) and prolactin receptor (PRLR) were assessed by immunoblotting. To assess effects of polyphenols, HC11 cells were treated with grape seed extract (GSE; 0-100 ug/ml) for 24 h and cell morphology was visualized, cell number was enumerated, and let-7g-5p expression was measured by RT-qPCR. Oxidative stress in response to H₂O₂ was measured using H2DCFDA.

Results: Transfection with let-7g-5p mimic increased let-7g-5p levels and downregulated PRLR and IMP1. Surprisingly, let7g^OX protected cells from H₂O₂-induced oxidative stress. Effects of GSE were phenotype-dependent; GSE decreased let-7g-5p expression, impaired cell spreading and reduced cell number (~60%) in proliferating cells but promoted a cuboidal morphology and increased cell number (~200%) in differentiated cells.

Conclusions: Our data provide evidence that let-7g-5p regulates key mRNAs in MECs, suggesting wide-ranging effects on lactation. Moreover, polyphenols regulate let-7g-5p levels and affect MECs in a phenotype-specific manner, suggesting manipulation of let-7g-5p and polyphenols may be potential therapeutic options for women with LMS.

Funding Sources: U Mass Lowell Research Incentive Funds