Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.

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

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.