Midkine as a driver of age-related changes and increase in mammary tumorigenesis [bulk RNA-seq]

Aging is one of the pivotal risk factors for cancer, notably in breast cancer with diagnosis striking at the average age of 62. However, the intricate mechanisms underlying aging and breast cancer susceptibility remain unclear. In this study, we depicted a comprehensive single-cell landscape of gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) of mammary glands from different aged rats. Mechanically, we revealed midkine, a growth factor secreted by basal epithelial cells, which might mediates the age-related mammary changes, as a validation, we treated young rats with midkine for a month and performed the single-cell transcriptome analysis on those mammary glands. We found midkine could largely mediates the transcriptional shift and hyperproliferation of aged epithelial cell by activating PI3K/AKT-SREBF1 signaling. Furthermore, we find the aging-related accumulation of midkine could largely mediated aging-related changes of mammary gland and promoting the tumorigenesis of breast tumors proved using a well-established Nitroso-N-methylurea (NMU)-induced breast cancer rat model. Our finding identify a promising biomarker and intervention target for both mammary aging and tumorigenesis. For NMU-induced mammary tumor experiment, Fischer rats were treated with midkine protein at 300 ug/kg 3 times/ week for two weeks and followed by a single intraperitoneal injection (i.p.) of 50 mg/kg N-nitroso N-methylurea (NMU). Then, rats were treated with midkine protein for another two weeks with same injection frequency, followed by another single NMU injection. Rats were checked three times/ week, and tumor growth was monitored using caliper measurements. Maximum tumor size burden allowed for rats is 4 cm and this was not exceeded in any of the experiments. All the remaining rats were sacrificed upon the last tumor in midkine treatment group reached the size limit. Rats were euthanized by CO2 inhalation. All the tumors from vehicle and midkine treatment groups were saved for bulk RNA-seq analysis.