Casticin inhibits stemness of hepatocellular carcinoma cells via disrupting the reciprocal negative regulation between DNMT1 and miR-148a-3p


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Abstract

Casticin (CAS) is a polymethyl flavonoid from Fructus viticis and has multiple pharmacological activities, including anticancer. However, whether the molecular mechanism underlying CAS represses stemness characteristics in hepatocellular carcinoma (HCC) cells involves intervention in the reciprocal negative regulation between DNA methyltransferase 1 (DNMT1) and miR-148a-3p has not yet been reported. In this study, the effect of CAS on stemness characteristics of HCC cells and its mechanism were investigated. Results showed that CAS selectively reduced the viabilities of HCC cells but not L02 cells, as determined by CCK-8 assay. Importantly, the sub-cytotoxic concentrations of CAS could inhibit the stemness characteristics in HCC cells, as demonstrated by the expression of stemness biomarkers (CD44, EpCAM, Bmi1, Nanog, and Oct4), sphere forming assay, RT-qPCR, and Western blotting. In addition, CAS repressed DNMT1 activity and expression and increased miR-148a-3p. The effect of CAS on stemness characteristics was abolished by stable DNMT1 overexpression. MiR-148a-3p overexpression enhanced the reduction of CAS on stemness characteristics. DNMT1 overexpression promoted miR-148a-3p promoter hypermethylation as detected by methylation-specific PCR (MSP), which repressed its expression. Conversely, miR-148a-3p repressed DNMT1 expression by specific site binding to 3′-UTR of DNMT1 mRNA, as determined by luciferase assay. Moreover, the combination of CAS and agomir-148a-3p had robust effects on tumor suppression as compared to the sole activity of either molecule in nude mouse xenograft experiments in vivo. The findings suggested that CAS could inhibit stemness characteristics in HCC cells by interruption of the reciprocal negative regulation between DNMT1 and miR-148a-3p.Graphical abstractHighlightsThe interaction between DNMT1 and miR-148a-3p promoted stemness in HCC cells.Casticin disrupted the interaction between DNMT1 and miR-148a-3p.Casticin inhibited stemness by disrupting interaction between DNMT1 and miR-148a-3p.Casticin reduced DNMT1 activity in HCC cells partly by repression of miR-148a-3p.Casticin elevated miR-148a-3p by its promoter demethylation via inhibiting DNMT1.

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