Plumbagin Nanoliposomes Suppress HepG2 Cell Proliferation, Migration, and Invasion by Accelerating miR-16-5p Expression and Inhibiting the VEGFA/EMT Pathway

Abstract

It has been reported that plumbagin (PL) can inhibit tumor cell growth and induce apoptosis, but the underlying mechanisms remain unclear. In this study, plumbagin nanoliposomes were prepared, and Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays were performed to determine the effects of PL on HepG2 cell proliferation. Transwell and wound healing assays were also used to investigate the effects of PL on HepG2 cell motility. Additionally, quantitative real-time polymerase chain reaction (qRT–PCR) was carried out to confirm the expression of microRNAs (miRNAs) under PL treatment, in which miR-16-5p showed the most substantially elevated profile among all miRNAs. After transfection with mimics or inhibitors of miR-16-5p or treatment with PL alone, the efficiency of up- and downregulation of miR-16-5p was determined by qRT–PCR in HepG2 cells. Furthermore, EdU, Transwell, and wound healing assays were used to explore the effects of up- and downregulation of miR-16-5p in HepG2 cells. Next, bioinformatic analysis was used to predict the potential target genes of miR-16-5p, along with further validation by dual luciferase reporter assays, qRT–PCR, and western blotting. To investigate the roles of PL and miR-16-5p in vivo, HepG2 cells were infected with lentivirus of miR-16-5p mimics or inhibitor, or the negative control (NC), and stable expression tumor cell lines were established. Then, a tumor mouse model was constructed and PL nanoliposomes were administered to evaluate their therapeutic effect. The volume and weight of tumor were recorded and analyzed and immunohistochemical assays were applied to determine the tumor growth and motility changes. The results showed that PL nanoliposomes suppressed HepG2 cell proliferation and migration in a dose-dependent manner. Mechanistically, PL enhanced miR-16-5p expression and inhibited HepG2 cell growth and migration by targeting the VEGFA/EMT pathway both in vitro and in vivo. Our study demonstrated that PL can inhibit the malignant functions of HepG2 cells by enhancing miR-16-5p expression, which functions as a tumor suppressor gene through targeting the VEGFA/EMT pathway.