Table 1.
Sequences of qRT-PCR primers used for mRNA analysis.
Figure 1.
Expression levels of miR-200a and validation for stable miR-200a knockdown in WB cells.
(A) QRT-PCR analysis of the relative miR-200a levels in WB cells and three hepatoma cells (H-4-II-E, CBRH-7919, RH-35) compared with the normal liver cell line BRL. (B) Validation of miR-200a levels in WB cells lentivirally transfected with miR-200a antagomir (WB-anti-miR-200a) or negative control (WB-miR-NC) by qRT-PCR analysis. (C and D) Functional evaluation of down-regulated miR-200a on its validated target ZEB2 in WB cells using qRT-PCR (C) and western blot analysis (D). For A and B, data are normalized to U6 and represented as the mean ± SD; n = 5; **, p<0.01. For C, data are normalized to β-actin and presented as the mean ± SD; n = 4.
Figure 2.
Stable knockdown of miR-200a facilitates CSC-like phenotypes in WB cells.
(A) Growth curve of WB-miR-NC and WB-anti-miR-200a cells determined by cell counting. Data are expressed as the mean ± SD; n = 3; *, p<0.05. (B) Representative images of spheroids formed by WB-miR-NC and WB-anti-miR-200a cells in the spheroid formation assay (left, magnification×100). Number of spheroids formed in the primary, secondary and tertiary generations of suspension cultured WB-miR-NC or WB-anti-miR-200a cells (right). Data represent means from four randomly selected fields under the microscope, and error bars represent SD. *, p<0.05; **, p<0.01. (C and D) Apoptosis of WB-miR-NC and WB-anti-miR-200a cells measured by caspase-3/7 assay and Annexin V and PI staining. Data are expressed as the mean ± SD (C) and representative dot plots of apoptosis tests are shown (D); n = 5. (E) Expression of EpCAM, CD133, ABCG2, CK19, AFP, ALB and c-myc in WB-anti-miR-200a cells measured by qRT-PCR. Data are normalized to β-actin, shown relative to the level in WB-miR-NC cells and expressed as the mean ± SD; n = 3; *, p<0.05; **, p<0.01. (F) WB-miR-NC and WB-anti-miR-200a cells were treated with 10 ng/ml paclitaxel or 30 ng/ml doxorubicin for 48 h and then subjected to FACS with Annexin V and PI staining, respectively. Representative dot plots (left) and the mean percentage of apoptotic cells (± SD) from three independent experiments (right) are shown; *, p<0.05; **, p<0.01.
Figure 3.
Stable knockdown of miR-200a confers mesenchymal characteristics to WB cells.
(A) Morphological changes in WB-miR-NC and WB-anti-miR-200a cells (magnification×200). (B) Evaluation of in vitro migration abilities of WB-miR-NC and WB-anti-miR-200a cells by transwell migration assay. Representative images (upper, magnification×200) and the mean number of migrated cells (± SD) in five randomly selected fields counted under the microscope (lower) are shown; **, p<0.01. (C) Western blot analysis of epithelial (E-cadherin) and mesenchymal (N-cadherin and vimentin) markers in WB-miR-NC and WB-anti-miR-200a cells.
Figure 4.
miR-200a directly targets CTNNB1 and associates with Wnt/β-catenin pathway activity in WB cells.
(A) Predicted alignment of miR-200a and a potential binding site at the 3′-UTR of rat CTNNB1 mRNA (624–630 nt) by TargetScan. (B) Effect of miR-200a on CTNNB1 expression determined by a luciferase reporter assay. WB cells were co-transfected with anti-miR-200a (or anti-miR-control) and the pGL3-CTNNB1-wt (or pGL3-CTNNB1-mut) vector. Data are normalized by the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection and are shown as the mean ± SD; n = 3; **, p<0.01. (C) Expression of β-catenin, c-myc and cyclin D1 in WB-miR-NC and WB-anti-miR-200a cells detected by western blot analysis. (D) Cellular localization of β-catenin visualized by immunofluorescence staining in WB-miR-NC and WB-anti-miR-200a cells. Nuclear counterstaining was performed by DAPI (magnification×200). (E) The activity of β-catenin signaling determined by a luciferase reporter assay using the wild-type (TopFlash) or mutant (FopFlash) TCF4-reporter plasmids. Data are presented as fold increases in firefly luciferase over Renilla activity from three independent experiments; *, p<0.05.
Figure 5.
The anti-miR-200a effects are partially attenuated by silencing of CTNNB1 in WB-anti-miR-200a cells.
(A) The protein levels of β-catenin, c-myc and cyclin D1 measured by western blot analysis after siCTNNB1 transfection in WB-anti-miR-200a cells. (B) The β-catenin-mediated transcription activity determined by the Top/Fop ratio after siCTNNB1 transfection in WB-anti-miR-200a cells. Data are represented as the mean ± SD; n = 3; **, p<0.01. (C) Representative images of spheroids formed by WB-anti-miR-200a cells before and after siCTNNB1 transfection (left, magnification ? 100). Bar graph (right) represents the mean number of spheroids from five randomly selected fields under the microscope, and error bars represent SD. **, p<0.01. (D) Representative images of transwell migration before and after siCTNNB1 transfection in WB-anti-miR-200a cells (left, magnification × 200). Bar graph (right) represents the mean number of migrated cells (± SD) in five randomly selected fields counted under the microscope. **, p<0.01.
Figure 6.
Downregulation of miR-200a confers tumorigenicity to WB cells in vivo.
(A) Subcutaneous tumors developed by WB-miR-NC or WB-anti-miR-200a for 40 days post inoculation (left). Pictures of collected subcutaneous tumors were taken (middle) and tumor weights are shown (right). (B) Representative images of H&E staining of xenografted tumors are shown (left, magnification×200; right, magnification×400).