Purpose: This study was performed to identify the detailed mechanisms by which miR-296-3p functions as a tumor suppressor to prevent lung adenocarcinoma (LADC) cell growth, metastasis, and chemoresistance.<br /><br />Experimental Design: The miR-296-3p expression was examined by real-time PCR and in situ hybridization. MTT, EDU incorporation, Transwell assays, and MTT cytotoxicity were respectively performed for cell proliferation, metastasis, and chemoresistance, western blotting was performed to analyze the pathways by miR-296-3p and HDGF/DDX5 complex. The miRNA microarray and luciferase reporter assays were respectively used for the HDGF-mediated miRNAs and target genes of miR-296-3p. The ChIP, EMSA assays, and Co-immunoprecipitation combined with mass spectrometry and GST pull-down were respectively designed to analyze the DNA-protein complex and HDGF/DDX5/β-catenin complex.<br /><br />Results:We observed that miR-296-3p not only controls cell proliferation and metastasis, but also sensitizes LADC cells to Cisplatin (DDP) in vitro and in vivo. Mechanistic studies demonstrated that miR-296-3p directly targets PRKCA to suppress FAK-Ras-c-Myc signaling thus stimulating its own expression in a feedback loop that blocks cell cycle and epithelial-mesenchymal transition (EMT) signal. Furthermore, we observed that suppression of HDGF-β-catenin-c-Myc signaling activates miR-296-3p, ultimately inhibiting the PRKCA-FAK-Ras pathway. Finally, we found that DDX5 directly interacts with HDGF and induces β-catenin-c-Myc, which suppresses miR-296-3p and further activates PRKCA-FAK-Ras, cell cycle, and EMT signaling. In clinical samples, reduced miR-296-3p is an unfavorable factor that inversely correlates with HDGF/DDX5, but not PRKCA. <br /><br />Conclusions:Our study provides a novel mechanism that the miR-296-3p-PRKCA-FAK-Ras-c-Myc feedback loop modulated by HDGF/DDX5/β-catenin complex attenuates cell growth, metastasis, and chemoresistance in LADC.
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