The miR-124-3p/Neuropilin-1 Axis Contributes to the Proliferation and Metastasis of Triple-Negative Breast Cancer Cells and Co-Activates the TGF-β Pathway

Abstract

Triple-negative breast cancer (TNBC) accounts for 90% of breast cancer-associated mortality. Neuropilin-1 (NRP-1) acts as a non-tyrosine kinase receptor for several cellular signaling pathways involved in the proliferation and metastasis of cancer cells. However, the miRNAs that regulate NRP-1 expression and the underlying mechanisms in TNBC cells remain unclear. In the present study, we found that TNBC cells expressed higher levels of NRP-1 than non-TNBC cells. Stable transfectants depleted of NRP-1 were generated from two TNBC cell lines, human MDA-MB-231 and mouse 4T1 cells. NRP-1 depletion significantly suppressed the proliferation of TNBC cells by arresting the cell cycle at phase G0/G1 by upregulating p27 and downregulating cyclin E and cyclin-dependent kinase 2. NRP-1 depletion also repressed cell migration and epithelial-mesenchymal transition (EMT) by inducing the upregulation of E-cadherin and the downregulation of N-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9, and reducing MMP-2 and MMP-9 activities as detected by gelatin zymography assay. By applying multiple miRNA-target prediction tools, we screened potential miRNAs with binding sites with the 3’-untranslated region of the NRP-1 gene and selected 12 miRNA candidates, among which miR-124-3p displayed the most vigorous activity to downregulate NRP-1 as validated by luciferase assay and miRNA transfection assay. By downregulating NRP-1, miR-124-3p mimics inhibited the proliferation, migration, and invasion of TNBC cells, and antagomiR-124-3p could partially abolish the effects of NRP-1 depletion. In the animal experiments, NRP-1 depletion inhibited tumorigenesis and liver metastasis of TNBC cells, while miR-124-3p mimics inhibited the growth of established TNBC tumors. In the mechanistic exploration, we revealed that NRP-1 co-interacted with transforming growth factor (TGF)-β to activate the TGF-β pathway, which regulates EMT-related molecules. In summary, the present results indicate that the miR-124-3p/NRP-1 axis contributes to the proliferation and metastasis of TNBC cells and co-activates the TGF-β pathway, suggesting that these molecules may present as potential therapeutic targets and valuable biomarkers for TNBC.

Lovastatin Inhibits EMT and Metastasis of Triple-Negative Breast Cancer Stem Cells Through Dysregulation of Cytoskeleton-Associated Proteins

“…Metastatic burden was evaluated by counting the metastatic nodules on the surface of each liver. Micrometastasis in the liver tissues were quantified based on the literature (36). All animal studies were approved by the Hunan Normal University Animal Care Committee.…”

The Role of MicroRNA in DNA Damage Response

“…In addition to the typical cell cycle regulators mentioned above, some novel proteins have also been found to participate in the regulation of G0/G1 phase by miRNAs, enriching the regulatory network of miRNAs on cell cycle. For instance, triple negative breast cancer cells expressed high level of Neuropilin-1 (NRP-1) ( Zhang et al, 2021 ). MiR-124-3p bound to 3′-UTR region of NRP-1 mRNA, blocked cell cycle at G0/G1 phase and inhibited cell proliferation ( Zhang et al, 2021 ).…”

“…For instance, triple negative breast cancer cells expressed high level of Neuropilin-1 (NRP-1) ( Zhang et al, 2021 ). MiR-124-3p bound to 3′-UTR region of NRP-1 mRNA, blocked cell cycle at G0/G1 phase and inhibited cell proliferation ( Zhang et al, 2021 ). After that, knockdown of NRP-1 increased the level of p27 and suppressed cyclin E and CDK2 ( Zhang et al, 2021 ).…”

“…MiR-124-3p bound to 3′-UTR region of NRP-1 mRNA, blocked cell cycle at G0/G1 phase and inhibited cell proliferation ( Zhang et al, 2021 ). After that, knockdown of NRP-1 increased the level of p27 and suppressed cyclin E and CDK2 ( Zhang et al, 2021 ). It is not difficult to conjecture that the novel proteins may act as mediators between miRNAs and cell cycle, and affect the downstream cyclins, CDK inhibitors and CDKs family.…”

Inhibitory effect of the novel tyrosine kinase inhibitor DCC-2036 on triple-negative breast cancer stem cells through AXL-KLF5 positive feedback loop

“…Around 24% of breast cancer patients were diagnosed with TNBC [ 2 , 3 ], which correlates with a higher rate of relapse and chemoresistance, as well as shorter overall survival. Additionally, TNBC constitutes 90% of breast cancer-related mortality [ 4 ]. In this case, discovering and researching effective therapies and drugs for TNBC is an urgent need as well as a major challenge in the field of breast cancer treatment.…”