The online version of this article (https://doi.org/10.1186/s13046-017-0670-6) contains supplementary material, which is available to authorized users.
Recent studies have shown that interferon-γ (IFN-γ)-induced galectin-9 expression in Kupffer cells plays an essential role in modulatingthe microenvironment of hepatitis-associated hepatocellular carcinoma (HCC). However, whether or not IFN-γ induces galectin-9 expression in HCC cells, its biological role and regulatory mechanism in HCC development and progression are poorly defined.
Quantitative PCR and western blotting analysis were used to detect galectin-9 and EZH2 levels in HCC cell lines stimulated with IFN-γ. Bioinformatics analysis and luciferase reporter assay were utilized to confirmthe binding ofmiR-22 to the 3′ untranslated region (3’-UTR) of galectin-9. The methylation status of miR-22 promoter was analyzed by MSP (Methylation specific PCR) and BSP (bisulfite sequencing PCR), while chromatin immunoprecipitation (ChIP) assay identify the occupation status of EZH2 and H3K27me3 at the promoter. Furthermore, the effect of ectopic expression of galectin-9 and miR-22 on cell proliferation, migration, invasion and cell apoptosis was assessed by using CCK-8, transwell assays and flow cytometric analysis, respectively.
IFN-γ induces up-regulation of galectin-9 and EZH2 in HCC cell lines. Galectin-9 is a target of miR-22 and EZH2 facilitates galectin-9 expression by tri-methylation of H3K27 on miR-22 promoter but not hyper-methylation status of DNA. MiR-22 overexpression suppressed HCC cell growth, invasion, and metastasis both in vitro and in vivo. Interestingly, galectin-9 also exhibited antitumor effects, and restoring galectin-9 expression in miR-22 overexpressing cells strengthened its antitumor effects.
These findings indicated that EZH2 facilitates galectin-9 expression by epigenetically repressing miR-22 and that galectin-9, which is known as an immunosuppressant, also functions as a tumor suppressor in HCC.
Additional file 1: Primers. (DOCX 16 kb)13046_2017_670_MOESM1_ESM.docx
Additional file 2: Western blotting showed that miR-22 mimic transfection could abolish the increases in galectin-9 expression induced by EZH2 overexpression, while miR-22 inhibitor transfection could enhance the increases. (TIFF 1501 kb)13046_2017_670_MOESM2_ESM.tif
Additional file 3: EZH2 suppressed miR-22 transcription by DNA hyper-methylation-independent histone methylation. a, MSP analysis was conducted in HCC cell lines and tissues to determine the methylation status of the CpG island within the MIR22HG promoter. b, The methylation statuses of 28 CpG sites within the CpG island located within the core region of MIR22HG were analyzed by bisulfite sequencing. Ten clones were selected from each group, and their methylation statuses were determined. The value for the HepG2 cells was 1.07%, and the value for the HepG2 cells transfected with EZH2 was 1.79%. The value for the Hep3B cells was 1.79%, and the value for the Hep3B cells transfected with EZH2 was 2.14%. (TIFF 1755 kb)13046_2017_670_MOESM3_ESM.tif
Additional file 4: ChIP-seq data from ENCODE (encyclopedia of DNA elements) database showed enrichment peaks of EZH2 and H3K27me3 at the promoter region of MIR22HG. (TIFF 312 kb)13046_2017_670_MOESM4_ESM.tif
Additional file 5: Supplementary methods. (DOCX 14 kb)13046_2017_670_MOESM5_ESM.docx
Heusschen R, Griffioen AW, Thijssen VL. Galectin-9 in tumor biology: a jack of multiple trades. Biochim Biophys Acta. 2013;1836:177–85. PubMed
Golden-Mason L, Rosen HR. Galectin-9: diverse roles in hepatic immune homeostasis and inflammation. Hepatology. 2017;
SL A, Wong CC, Lee JM, Fan DN, Tsang FH, Ng IO, et al. Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis. Hepatology. 2012;56:622–31. CrossRef
Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. elife. 2015;12:4.
Imaizumi T, Kumagai M, Sasaki N, Kurotaki H, Mori F, Seki M, et al. Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells. J Leukoc Biol. 2002;72:486–91. PubMed
- EZH2 promotes hepatocellular carcinoma progression through modulating miR-22/galectin-9 axis
- BioMed Central
Journal of Experimental & Clinical Cancer Research
Elektronische ISSN: 1756-9966
Neu im Fachgebiet Onkologie
Mail Icon II