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

Abstract 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 were measured by western blotting. High-throughput genome sequencing 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 differentially 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 cells. 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 administration of miR-301b angomir enhances the inhibitory 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

Additional file 3: of Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling

Figure S1. ISL inhibits cell proliferation and induces cell apoptosis in melanoma cells in vitro. (A) MEWO cells were treated with ISL (0, 10, 20, 40, 80 μM) for 24 h, and cell viability was analyzed by CCK-8 assay. (B) MEWO cells were treated with 20 μM ISL, cell proliferation at indicated time (24, 48, 72 h) was measured by CCK-8 assay. (C, D) Flow cytometry analysis of apoptosis of MEWO cells after being treated with ISL (0, 10, 20 μM) for 24 h. (E, F) Representative images and quantification of colony formation of MEWO cells after being treated with ISL (0, 5, 10 Μm). (G, H)Western blot analysis of the protein level of apoptosis associated proteins(bcl-2, bax, parp, cleaved-caspase-3) in ISL treated A375 and A2058 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs ISL(0 μM) treated group. n = 3. (TIF 25527 kb

Additional file 4: of Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling

Figure S3. (A)RT-qPCR analysis of the mRNA level alteration of 7 common target genes of miR301b in A375 and A2058 cells after being transfected with miR301b mimic/NC or treated with miR301b inhibitor/NC. *P < 0.05 vs NC. (B)Design of luciferase reporters with the WT Akt3 3’UTR (Akt3–3’UTR WT) or the site-directed mutant Akt3 3’UTR (Akt3–3’UTR MUT). (C)RT-qPCR analysis of miR301b level in A375 and A2058 cells after being transfected with miR301b mimic/NC or treated with miR301b inhibitor/NC. **P < 0.01 vs NC. (D)RT-qPCR analysis of the mRNA level of LRIG1 in A375 and A2058 cells after being transfected with miR301b mimic/NC or treated with miR301b inhibitor/NC. **P < 0.01 vs NC.*P < 0.05 vs NC. (E, F)Western blot analysis of the protein level of apoptosis associated proteins(LRIG1, c-PARP, Bax, cleaved-caspase-3) in ISL treated A375 and A2058 cells which were transfected with si-LRIG1 or control(NC). *P < 0.05, **P < 0.01 vs PBS Treated in si-NC groups. #P < 0.05, ##P < 0.01 vs PBS Treated in si-LRIG1 groups. (G)Flow cytometry analysis of cell apoptosis in ISL treated A375 and A2058 cells which were transfected with si-LRIG1 or si-NC.*P < 0.05, **P < 0.01 vs PBS + si-NC. (H)Quantification of TUNEL positive cells in ISL treated A375 and A2058 cells which were transfected with si-LRIG1 or si-NC.*P < 0.05, **P < 0.01 vs PBS + si-NC. (TIF 25520 kb

Additional file 2: of Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling

Table S1. Sequences of mRNA PCR primers used in this study. (DOCX 19 kb

Additional file 1: of Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling

Figure S2. (A, B)RT-qPCR analysis of the mRNA level of miR301b in ISL treated A375 and A2058 cells which were transfected with miR301b or control(NC). *P < 0.05, **P < 0.01 vs PBS Treated group. (C, D)Western blot analysis of the protein level of apoptosis associated proteins(c-PARP, Bax, bcl-2, cleaved-caspase-3) in ISL treated A375 and A2058 cells which were transfected with miR301b or miR-NC. *P < 0.05, **P < 0.01 vs miR-NC Treated in PBS groups. #P < 0.05, ##P < 0.01 vs miR-NC Treated in ISL groups. (TIF 13582 kb