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

<div><p>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 microRNA-148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGF-β)/SMAD2 signal pathway in HCC cell lines (HepG2, Huh-7, and MHCC97H). Indeed, GLA inhibited the activations/expressions of both TGFβ-induced and the endogenous SMAD2. Further, GLA improved the expression of miR-148a in a dose/time-dependent manner. MiR-148a, which targeted the <i>SMAD2</i>-3′UTR, decreased the expression and function of SMAD2. Knockdown of miR-148a abolished the GLA-induced inhibition of TGF-β/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-β/SMAD2 signal pathway, which may help to identify potential targets for the therapies of HCC.</p></div

GLA attenuates the CSCs-like properties in HCC cells.

<p>(<b>A</b>) HepG2 cells were treated by 0, 10, or 20 µM GLA for 72 h. RT-PCR analyses of <i>CD44</i>, <i>EpCAM</i>, <i>CD133</i>, <i>CD90</i>, <i>Oct-4</i>, and <i>BMI-1</i>; (<b>B</b>) Huh-7 and MHCC97H cells were treated by 0 or 20 µM GLA for 72 h. RT-PCR analyses of <i>CD44</i> and <i>EpCAM</i>; (C and D) HepG2 and Huh-7 cells were treated by 0 or 20 µM GLA for 72 h. (<b>C</b>) Free-floating, viable spheres formed by cells (bar = 250 µm); (<b>D</b>) Sphere quantitation (mean ± SD, n = 3); (E and F) HepG2 and MHCC97H cells were treated by 0 or 20 µM GLA for 72 h. (<b>E</b>) Colony formation in the soft agar (bar  = 250 µm); (<b>F</b>) Colony numbers (mean ± SD, n = 3). **p<0.01 compared with medium control cells (student's <i>t</i> test).</p

GLA inhibits the SMAD2 by miR-148a in HCC cells.

<p>(A–C) After HepG2 cells were pre-transfected by anti-con or anti-miR-148a for 12 h, they were exposed to 0 or 20 µM of GLA for 72 h. (<b>A</b>) qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3); (<b>B</b>) Luciferase reporter assays analysis of the effects of miR-148a on <i>SMAD2</i> 3′UTR; (<b>C</b>) RT-PCR analyses of <i>SMAD2</i> and <i>snail</i> (top), and Western blots analyses of p-SMAD2 and SMAD2 (bottom). **p<0.01 compared with medium control cells, and ^##p<0.01 compared with HepG2 cells treated by GLA alone or with anti-con-transfected HepG2 cells treated by GLA (ANOVA followed by Dunnett's <i>t</i> test). (D and E) Huh-7 cells were transfected by con-mimic or miR-148a-mimic for 12 h. (<b>D</b>) qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3); (<b>E</b>) RT-PCR analyses of <i>SMAD2</i> and <i>snail</i> (top), and Western blots analyses of SMAD2 (bottom); **p<0.01 compared with Huh-7 cells transfected by con-mimic (student's t test). (F and G) After MHCC97H cells were transfected by anti-miR-148a for 12 h, they were cultured in fresh DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin for another 24 h, followed by transfected with con-mimic or miR-148a-mimic for 12 h. (<b>F</b>) qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3); (<b>G</b>) RT-PCR analyses of <i>SMAD2</i>. **p<0.01 compared with MHCC97H cells transfected by anti-con, ^##p<0.01 compared with MHCC97H cells transfected by anti-miR-148a plus con-mimic (ANOVA followed by Dunnett's <i>t</i> test).</p

GLA improves the expression of miR-148a in HCC cells.

<p>(<b>A</b>) The target sequences of miR-148a in the 3′-UTR of <i>SMAD2</i>; (<b>B</b>) HepG2 cells were treated by 0, 10, or 20 µM GLA for 24 h. qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3); (<b>C</b>) HepG2 cells were treated by 0 or 20 µM GLA for 0, 8, 16, 24, or 48 h. qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3); (<b>D</b>) Huh-7 and MHCC97H cells were treated by 0 or 20 µM GLA for 24 h. qRT-PCR analyses of the expression of miR-148a (mean ± SD, n = 3). **p<0.01 compared with medium control cells (student's <i>t</i> test).</p

Additional file 5: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Table S2. Comparison of efficacy between study YO28390 (Chinese patients) and the BRIM-2 and BRIM-3 studies (predominantly Caucasian patients). (DOCX 18 kb

Additional file 1: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Schedule of assessments. (DOCX 19 kb

Additional file 5: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Table S2. Comparison of efficacy between study YO28390 (Chinese patients) and the BRIM-2 and BRIM-3 studies (predominantly Caucasian patients). (DOCX 18 kb

Additional file 2: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Figure S1. Vemurafenib Ctrough concentrations (mean ± SD) after day 28 in the pharmacokinetics and expansion cohorts. SD standard deviation, CV coefficient of variation. (PDF 71 kb

Additional file 4: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Table S1. Comparison of pharmacokinetic parameters between study YO28390 (Chinese patients) and study NP25163 (predominantly Caucasian patients). (DOCX 19 kb

Additional file 3: of Vemurafenib in Chinese patients with BRAFV600 mutation–positive unresectable or metastatic melanoma: an open-label, multicenter phase I study

Figure S2. Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival (OS). (PDF 80 kb