Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling

Background: Isoliquiritigenin (ISL), a natural flavonoid isolated from the root of licorice (Glycyrrhiza uralensis), has shown various pharmacological properties including anti-oxidant, anti-inflammatory and anti-cancer activities. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been reported as post transcriptional regulators with altered expression levels in melanoma. This study aims to investigate the anti-melanoma effect of ISL and its potential mechanism. Methods: We investigated the effect of ISL on the proliferation and apoptosis of melanoma cell lines with functional assays, such as CCK-8 assay, colony formation assay and flow cytometry. The protein level of apoptosis related genes weie measured by western blotting. High throughput genome seguencing was used for screening differentially expressed miRNAs of melanoma cell lines after the treatment of ISL. We performed functional assays to determine the oncogenic role of miR-301b, the most diffeientially expressed miRNA, and its target gene leucine rich repeats and immunoglobulin like domains 1 (LRIG1), confirmed by bioinformatic analysis, luciferase reporter assay, western blotting and immunohistochemical assay in melanoma. Immunocompromised mouse models were used to determine the role of miR-301b and its target gene in melanoma tumorigenesis in vivo. The relationship between miR-301b and LRIG1 was further verified in GEO data set and tissue specimens. Results: Functional assays indicated that ISL exerted significant growth inhibition and apoptosis induction on melanoma ceils. MiR-301b is the most differentially expressed miRNA after the treatment of ISL and significantly downregulated. The suppressive effect of ISL on cell growth is reversed by ectopic expression of miR-301b. Intratumorally administiation of miR-301b angomir enhances the inhibitoiy effect of ISL on tumor growth in vivo. Bioinformatic analysis showed that miR-301b may target LRIG1, miR-301b suppresses the luciferase activity of reporter constructs containing 3'UTR of LRIG1 as well as the expression level of LRIG1. And the anti-cancer effect of ISL is mitigated when LRIG1 is silenced in vivo and in vitro. Analysis of the melanoma samples obtained from patients shows that LRIG1 is negatively correlated with miR-301b. Conclusions: ISL may inhibit the proliferation of melanoma cells by suppressing miR-301b and inducing its target LRIG1.