Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: (A) Predicted duplex formation between miR-BART9 and human E-cadherin 3′UTR (Wt). The seed sequence region is highlighted in bold. The putative target sequence of E-cadherin 3′UTR at nt 1795–1801. Mut indicates the mutated E-cadherin 3′UTR sequence used as a control in the reporter assay. Mutated bases are specified by underlining. (B) Luciferase activity of the wild type (Wt) or mutant (Mut) E-cadherin 3′UTR reporter in BM1, TW04 and HK1 cells expressing miR-BART9 or LacZ. (C) Luciferase activity of the wild type (Wt) or mutant (Mut) E-cadherin 3′UTR reporter in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). (D) Top panel: Immunoblotting analysis of E-cadherin in BM1, TW04 and HK1 cells expressing miR-BART9 or LacZ. (Left) or HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9) or scramble control (anti-Ctrl) (Right). GAPDH was used as a loading control. Bottom panel: E-cadherin protein levels were normalized to GAPDH levels, and then compared with the LacZ or anti-Ctrl cells whose normalized levels were expressed as 1.0. Bar graphs provide the means ± SEM of independent experiments and two-tailed Student's t-test were performed (*, P<0.05; **, P<0.01). (E) Representative immunofluorescence staining of E-cadherin and DAPI staining to detect the nucleus in BM1 and TW04 cells expressing miR-BART9 or LacZ. Arrows indicate cell-cell junctions. Scale bar = 20 µm. (F) Representative IHC staining of GFP, human Mac2BP and E-cadherin in sections of primary tumors formed by BM1 cells expressing miR-BART9 or LacZ. Scale bar = 500 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Sequencing, Control, Reporter Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Concentration Assay, Modification, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Immunohistochemistry

Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P<0.05; ***, P<0.001). Scale bar = 200 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Transwell Migration Assay, Invasion Assay, Expressing, Transfection, Negative Control, Western Blot, Control, Modification, Two Tailed Test

Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: Expression levels of matrix metalloproteases MMP1, MMP2, MMP9, MMP10 and MMP12 (A) and E-cadherin (CDH1), α-catenin (CTNNA1) and vimentin (B) in BM1 and TW04 cells infected with lentivirus expressing the miR-BART9 or control (LacZ) vector. mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. Bar graphs provide the means ± SEM of three independent experiments and two-tailed Student's t-test were performed (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Western blot analysis for the expression of indicated EMT markers. GAPDH protein was used as a protein loading control. (D) Representative immunofluorescence staining of vimentin and DAPI staining to detect the nucleus in BM1 and TW04 cells expressing miR-BART9 or LacZ. Scale bar = 20 µm. (E) Representative immunofluorescence staining of E-cadherin (CDH1) and vimentin and DAPI staining to detect the nucleus in sections of primary tumors formed by BM1 cells expressing miR-BART9 or LacZ. Scale bar = 20 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Two Tailed Test, Western Blot, Immunofluorescence, Staining