miR-224 aggravates cancer-associated fibroblast-induced progression of non-small cell lung cancer by modulating a positive loop of the SIRT3/AMPK/mTOR/HIF-1α axis

Objectives: Cancer-associated fibroblast (CAF) is among the most important tumor-host microenvironment components by affecting tumor progression. This study explored the role of miR-224 in CAF-induced non-small cell lung cancer (NSCLC). Materials and methods: A CAF-NSCLC cell co-culture model was established, and the miR-224 expression in CAF was detected by reverse transcription-polymerase chain reaction (RT-PCR). Gain- and loss- of experiments of miR-224 were implemented to verify the effects of CAF on NSCLC cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and endothelial cell (EC) angiogenesis. Overexpressing genetic or pharmacological interventions were performed to explore the potential mechanisms of Sirtuins 3/AMP-activated protein kinase/mammalian target of rapamycin/hypoxia-inducible factor-1α (SIRT3/AMPK/mTOR/HIF-1α). Results: CAF enhanced the malignant phenotype of NSCLC cells and induced EC angiogenesis. miR-224 was significantly altered in CAFs. miR-224 up-regulation exacerbated NSCLC development mediated by CAFs, while miR-224 inhibition mostly reversed CAF-induced effects. Mechanistically, miR-224 targeted the 3’-untranslated regions (UTR) of SIRT3 mRNA, thereby inhibiting SIRT3/AMPK and activating mTOR/HIF-1α. Forced overexpression of SIRT3 up-regulated AMPK and inactivated mTOR/HIF-1α, while inhibiting HIF-1α markedly up-regulated SIRT3/AMPK and reduced mTOR phosphorylation. Interestingly, both Sirt1 overexpression and HIF-1α inhibition repressed miR-224 levels and miR-224-mediated promotive effects in NSCLC. Conclusion: The miR-224-SIRT3/AMPK/mTOR/HIF-1α axis formed a positive feedback loop in modulating CAF-induced carcinogenic effects on NSCLC.