Background: This study aimed to explore the potential regulatory mechanisms of brain metastasis and to identify novel underlying targets of lung cancer with brain metastasis.
Methods: Exosomes were isolated from the plasma of lung cancer patients with or without brain metastasis and low or high metastatic lung cancer cells, and small RNA from plasma-derived exosomes were sequenced. Differentially expressed miRNAs (DE-miRNAs) were identified. Human brain microvascular endothelial cells (HBMECs) were transfected with miR-550a-3-5p mimics or inhibitors and exosomes. Cell viability, migration, and apoptosis/cycle were determined using Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. Western blotting was used to measure the expression of the associated proteins. Finally, a dual-luciferase reporter gene assay was performed to confirm the miR-550a-3-5p target.
Results: Transmission electron microscopy, NanoSight, and western blotting showed that exosomes were successfully isolated and cell-derived exosomes could be taken up by HBMECs. Sequencing identified 22 DE-miRNAs which were enriched in the MAPK, chemokine, PPAR, and Wnt signaling pathways. MiR-550a-3-5p was significantly enriched in brain metastatic exosomes. Cellular experiments showed that miR-550a-3-5p and exosome enrichment significantly inhibited cell viability and migration, promoted apoptosis, and regulated the cell cycle of HBMECs compared with the controls (P < 0.05). Compared with the controls, high levels of both miR-550a-3-5p and exosomes markedly upregulated cleaved-PARP expression, but downregulated the expression of pRB, CDK6, YAP1, CTGF, and CYR61 (P < 0.05). Finally, YAP1 was confirmed to bind directly to miR-550a-3-5p.
Conclusion: Our results indicate that miR-550a-3-5p and YAP1 may be novel potential targets for controlling brain metastasis.
Keywords: Brain metastasis; HBMECs; Lung cancer; YAP1; miR-550a-3-5p.
© 2021. The Author(s).