Carboxypeptidase A6 suppresses the proliferation and invasion of colorectal cancer cells and is negatively regulated by miR-96-3p

Background: Colorectal cancer (CRC) is a common malignant tumor, and this study aims to explore the role and the regulatory mechanism of carboxypeptidase A6 (CPA6) in CRC cells. Methods: Specific shRNA targeting CPA6 mRNA was transfected into NCM460 and HT29 cells to down-regulate CPA expression, and expression plasmid was transfected into HCT116 cells to exogenously overexpress CPA6. The dual luciferase assay was used to detect the direct binding of miR-96-3p to CPA6 3'UTR. Phosphorylation and activation of Akt were detected using Western blot. Cells were treated with miR-96-3p mimics, Akt inhibitor (MK-2206) or agonist (SC79) for rescue experiments. The cell functions were evaluated using CCK-8, clone formation, transwell, and Western blot assays. Xenograft tumor assay was also used to analyze the effect of altered CPA6 expression on tumor growth. Results: Knockdown of CPA6 promoted the proliferation, clone formation, migration, and invasion of NCM460 and HT29 cells in vitro, and the tumor growth of nude mouse xenograft tumor in vivo. Moreover, over-expression of CPA6 significantly inhibited the malignant proliferation and invasion of HCT116 cells in vitro, and the tumor growth of xenograft tumor in vivo. Furthermore, miR-96-3p could directly regulate CPA6 expression by targeting its 3' UTR, and miR-96-3p mimics rescued the inhibitory effects of CPA6 overexpression on the malignant proliferation and invasion of CRC cells. Finally, CPA6 knockdown enhanced Akt/mTOR phosphorylation and activation, while CPA6 overexpression inhibited Akt/mTOR activation. The regulatory effect of CPA6 on Akt/mTOR signaling was naturally regulated by miR-96-3p. Akt inhibitor or agonist rescued the effects of CPA6 knockdown or overexpression on proliferation and EMT of colon cancer cells. Conclusion: CPA6 has a significant tumor suppressive effect on CRC by inhibiting the activation of Akt/mTOR signaling, and miR-96-3p negatively regulates the expression of CPA6.