The Repressive Effect of miR-148a on TGF beta-SMADs Signal Pathway Is Involved in the Glabridin-Induced Inhibition of the Cancer Stem Cells-Like Properties in Hepatocellular Carcinoma Cells

et al. (2014) The Repressive Effect of miR-148a on TGF beta-SMADs Signal Pathway Is Involved in the Glabridin-Induced Inhibition of the Cancer Stem Cells-Like Properties in Hepatocellular Carcinoma Cells. PLoS ONE 9(5): e96698. doi:10.1371/journal.pone.0096698 The Repressive Effect of miR-148a on TGF beta-SMADs Signal Pathway Is Involved in the Glabridin-Induced Inhibition of the Cancer Stem Cells-Like Properties in Hepatocellular Carcinoma Cells Fei Jiang 0 Juan Mu 0 Xingxing Wang 0 Xianqing Ye 0 Lu Si 0 Shilong Ning 0 Zhong Li 0 Yuan Li 0 Lian-Yue Yang, Xiangya Hospital of Central South University, China 0 Department of Nutrition and Food Hygiene, The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University , Nanjing, Jiangsu , People's Republic of China Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of cancer stem cells (CSCs)-mediated post-surgical recurrence. For this reason, targeting the CSCs or the cancer cells with CSCs-like properties has become a new approach for the treatment of HCC. GLA exhibits anti-tumor effects in that it attenuates the proliferation, migration, invasion, and angiogenesis of human cancer cells. However, the functions of GLA in the regulation of CSCs-like properties in HCC cells, and the molecular mechanisms underlying in remain obscure. Here we found that GLA attenuated the CSCs-like properties by the microRNA148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGF-b)/SMAD2 signal pathway in HCC cell lines (HepG2, Huh-7, and MHCC97H). Indeed, GLA inhibited the activations/expressions of both TGFb-induced and the endogenous SMAD2. Further, GLA improved the expression of miR-148a in a dose/time-dependent manner. MiR-148a, which targeted the SMAD2-39UTR, decreased the expression and function of SMAD2. Knockdown of miR-148a abolished the GLA-induced inhibition of TGF-b/SMAD2 signal pathway and the CSCs-like properties in HCC cells. Our study found a novel mechanism that GLA inhibits the CSCs-like properties of HCC cells by miR-148a-mediated inhibition of TGF-b/SMAD2 signal pathway, which may help to identify potential targets for the therapies of HCC. - Funding: This work was supported by National Natural Science Foundation of China (81171987, 30972507) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Hepatocellular carcinoma (HCC) is the most common liver malignancy and the third leading cause of cancer-related mortality worldwide [1]. Current standard practices for treatment of HCC, surgical resection and chemotherapy are less than satisfactory because of metastasis and post-surgical recurrence [1]. A concept proposed to explain the characteristics of neoplastic tissues is the existence of self-renewing, stem-like cells, called cancer stem cells (CSCs) [2]. CSCs have been identified in various human cancers, including HCC. Within a tumor, CSCs, which constitute a small portion of the neoplastic cells, are defined by their capacity to produce new tumors [3]. For this reason, targeting the cancer cells with CSCs-like properties has become the new way for the treatment of human liver cancers. The root of glycyrrhiza glabra (licorice) has been used for many centuries in Asia and Europe as an antioxidant, antidote, demulcent, expectorant and a remedy for allergic inflammation, as well as a flavoring and sweetening agent [4]. Glabridin [GLA, (R)-4-(3, 4-dihydro-8, 8-dimethyl)-2H, 8H-benzo [1, 2-b: 3, 4-b] dipyran-3yl)-1, 3-benzenediol] is a polyphenolic flavonoid and a main constituent in the hydrophobic fraction of licorice extract [5]. In addition to estrogenic effects, GLA exhibits a wide range of biological activities including neuro-protective, cardiovascularprotective, anti-inflammatory, etc [57]. Recent studies indicate that GLA presents anti-tumor effects, with attenuation of the proliferation, migration/invasion, and angiogenesis in human cancer cells [8,9]; However, the effects of GLA on the CSCs-like properties in HCC cells, and the molecular mechanisms underlying in remain obscure. Transforming growth factor beta (TGF-b) is a critical regulator involved in the cell growth, differentiation, and development [10]. It is also the most potent hepatic pro-fibrogenic cytokine predominantly produced by activated mesenchymal cells upon chronic liver damage [11]. In the initiation and development of various tumors, TGF-b induces the epithelial-mesenchymal transition (EMT), which is a critical cellular event in the acquirement of CSCs-like properties [12,13]. Therefore, TGF-b inhibitors have been developed for anti-cancer therapies [14,15]. Current study indicates that inhibition of TGF-b blocks the generation of CSCs, which enhances the chemotherapy action against triple-negative breast cancer [16]. In the present study, we treated HCC cell lines (HepG2, Huh-7, and MHCC97H) by GLA Figure 1. GLA attenuates the CSCs-like properties in HCC cells. (A) HepG2 cells were treated by 0, 10, or 20 mM GLA for 72 h. RT-PCR analyses of CD44, EpCAM, CD133, CD90, Oct-4, and BMI-1; (B) Huh-7 and MHCC97H cells were treated by 0 or 20 mM GLA for 72 h. RT-PCR analyses of CD44 and EpCAM; (C and D) HepG2 and Huh-7 cells were treated by 0 or 20 mM GLA for 72 h. (C) Free-floating, viable spheres formed by cells (bar = 250 mm); (D) Sphere quantitation (mean 6 SD, n = 3); (E and F) HepG2 and MHCC97H cells were treated by 0 or 20 mM GLA for 72 h. (E) Colony formation in the soft agar (bar = 250 mm); (F) Colony numbers (mean 6 SD, n = 3). **p,0.01 compared with medium control cells (students t test). doi:10.1371/journal.pone.0096698.g001 to determine the early molecular changes, with emphases on CSCs-like properties and TGF-b pathway. Materials and Methods Cell culture and reagents HCC cell lines (HepG2 and Huh-7) and Human normal liver cell line (L-02) were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). MHCC97H cell line (HCC cells with a high migratory potential) was obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China. Cells were maintained in 5% CO2 at 37 uC in Dulbeccos Modified Eagle Medium (DMEM, Life Technologies/Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Life Technologies/Gibco), 100 U/ ml penicillin, and 100 mg/ml streptomycin (Life Technologies/ Gibco, Gaithersburg, MD). GLA ($98.0% purity) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used were of analytical grade or the highest grade available. Reverse-transcriptase (...truncated)