A positive TGF-β/miR-9 regulatory loop promotes the expansion and activity of tumour-initiating cells in breast cancer.

Abstract

MicroRNA-9 (miR-9) has previously been described as a dual-functional RNA during breast cancer progression and its roles need to be clarified thoroughly. A miR-9 knockout mode of mouse breast cancer, the MMTV-PyMT model (PyMT-miR-9-/- ), combined with different human breast cancer cell lines were used to evaluate the effects of miR-9 on breast cancer initiation, progression and metastasis. Lin-NECs (Neoplastic mammary epithelial cells) and pNECs (Pre-neoplastic mammary epithelial cells) were isolated and subjected to tumour-initiation assay. Whole-mount staining of mammary gland and histology was performed to determine mammary gland growth. Tumour-initiating analysis combining a series of in vitro experiments were carried out to evaluate miR-9 roles in tumour-initiating ability. RNA-sequencing of human breast cancer cells, and mammary glands at hyperplastic stages and established tumours in PyMT and PyMT-miR-9-/- mice, ChIP and luciferase report assays were conducted to reveal the underlying mechanisms. MiR-9 is ectopically expressed in breast cancer and its level is negatively correlated with the prognosis, especially in basal-like breast cancer patients. Additionally, miR-9 is essential for breast cancer progression by promoting the expansion and activity of tumour-initiating cells (TICs) in preneoplastic glands, established tumours and xenograft modes. Mechanistically, the activity of TICs hinges on a positive TGF-β/miR-9 regulatory loop mediated by the STARD13/YAP axis. These findings demonstrate that miR-9 is an oncogenic miRNA rather than a tumour-suppressor in breast cancer, calling for rectification of the model for this conserved and highly abundant miRNA.