MicroRNA-6809-5p mediates luteolin-induced anticancer effects against hepatoma by targeting flotillin 1

Abstract

Background: Luteolin (3,4,5,7-tetrahydroxy flavone) is a natural flavonoid abundant in fruits and vegetables. Although luteolin has shown pro-apoptotic activity in hepatocellular carcinoma (HCC) cells, the underlying molecular mechanism has not yet been clarified.

Purpose: The aim of this study is to identify novel miRNAs involved in the action of luteolin in HCC cells and to explore the biological roles of these miRNAs.

Methods: The effect of luteolin on HCC cell growth was assessed using CCK-8 colony formation assay, flow cytometric analysis in vitro, and a xenograft model in vivo. miRNA expression profiles were assessed using next-generation sequencing. Differentially expressed miRNAs were validated by quantitative PCR. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of miR-6809-5p to the 3'-untranslated region (3'-UTR) of flotillin 1 (FLOT1). Furthermore, the effects of ectopic FLOT1 and miR-6809-5 expression on cell proliferation, colony formation, and cell apoptosis were also assessed. Western blotting analysis was used to detect activation of multiple signaling molecules including Erk1/2, p38, JNK, and NF-κB/p65 in the MAPK pathway.

Results: It was found that luteolin significantly inhibited HCC growth and caused apoptosis and cell cycle arrest at the G0/G1 phase in Huh7 cells, at the G2/M phase in HepG2 cells in vitro. Tumorigenic studies revealed that luteolin treatment significantly suppressed HCC growth in vivo. miR-6809-5p was upregulated by luteolin. Overexpression of miR-6809-5p suppressed HCC cell growth, while knockdown of miR-6809-5p reversed the anticancer effect of luteolin. With regards to its signaling mechanism, miR-6809-5p directly targets FLOT1in HCC cells. Enforced expression of FLOT1 prevented miR-6809-5p-mediated growth suppression. Downregulation of FLOT1 exerted growth-suppressive effects on HCC cells. Multiple signaling pathways including Erk1/2, p38, JNK, and NF-κB/p65 were inactivated by miR-6809-5p overexpression or FLOT1 downregulation.

Conclusion: These findings indicated that miR-6809-5p mediates the growth-suppressive activity of luteolin in HCC, which is causally linked to FLOT1 downregulation. Induction of miR-6809-5p may provide therapeutic benefits in the treatment of HCC.

Keywords: Flotillin 1; Hepatocellular carcinoma; Luteolin; MAPK pathway; miR-6809-5p.