Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: Differential expression of BC200 RNA in GB cell lines. ( A ) The differential expression of BC200 RNA in GB cell lines and normal human astrocytes are shown. ( B ) The viability of GB cells was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( C ) Flow cytometry analysis of the ALDH1 + /CD133 + portion in GB cell lines and normal human astrocytes. ( D ) The level of BC200, BCRP1, MDR1, MRP1 and MGMT in GB cell lines was analyzed using RT-qPCR. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: Quantitative Proteomics, Sulforhodamine B Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: BC200 RNA promotes TMZ resistance in GB through sponge miR-218-5p. ( A ) The viability of shBC200 and OEBC200 GB cell lines was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( B ) The levels of MGMT, BCRP1, MDR1, and MRP1 following shBC200 and OEBC200 in GB cells were determined through western blot. ( C ) MicroRNA profiling analyses showed that shBC200 and OEBC200 contained high and low levels of miR-218-5p, respectively. ( D ) LncBase Predicted v.2 predicted that a high binding score of miR-218-5p with BC200. ( E ) BC200 directly interacts with multiple binding sites to hsa-miR-218-5p. *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: Sulforhodamine B Assay, Western Blot, Binding Assay

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: miR-218-5p regulated cell sphere formation, colony formation, and TMZ resistance in GB cells in vitro. ( A ) The differential expression of miR-218-5p in GB cell lines and normal human astrocytes. ( B ) Pearson’s correlation curve identified a negative correlation between BC200 and miR-218-5p in GB tissues. ( C ) Sphere formation assays showed that inhibition or mock transfection of miR-218-5p regulated GB cell stemness. ( D ) Colony formation assays showed that inhibition or mock transfection of miR-218-5p regulated GB cell survival. ( E ) The viability of GB cells with inhibition or mock transfection of miR-218-5p was analyzed through SRB assay 48 h after TMZ (0–1000 μM) treatment. ( F ) CCK-8 assay showed that inhibition or mock transfection of miR-218-5p had no effect on GB cell proliferation. ( G ) The protein levels of SOX2, Oct4, BRPC1, MRP1 and MDR1 with inhibition or mock transfection of miR-218-5p. ( H ) The protein levels of MGMT, MLH1, MSH2, MSH6 and PMS2 following inhibition or mock transfection of miR-218-5p in GB cells were determined through western blot. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques: In Vitro, Quantitative Proteomics, Inhibition, Transfection, Sulforhodamine B Assay, CCK-8 Assay, Western Blot

Journal: Cells

Article Title: Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

doi: 10.3390/cells9081859

Figure Lengend Snippet: The lncRNA BC200 RNA sponges miR-218-5p regulated MGMT and MMR system enhancing self-renewal and TMZ resistance of GBM cells.

Article Snippet: The human MGMT open reading frame (ORF) plasmid was purchased from OriGene (Cat# MGMT (RC229131, Taipei, Taiwan) and the cells transfected according to vendor’s instructions; MGMT-overexpressing U87MG cells were then allowed to grow at 37 °C in 5% CO 2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM).

Techniques:

Journal: Clinical Cancer Research

Article Title: Profound Prevention of Experimental Brain Metastases of Breast Cancer by Temozolomide in an MGMT-Dependent Manner

doi: 10.1158/1078-0432.ccr-13-2588

Figure Lengend Snippet: Figure 4. MGMT expression in matched sets of human breast primary tumors and resected brain metastases. A to D, sixty-two patient-matched sets were collected from tumor banks in Poland and Germany. TMAs of the specimens were stained for MGMT and evaluated for the percentage of positively staining tumor cells (nuclear staining only), dichotomized at 5%. The number and percentage of specimens in each category is given below each representative photomicrograph.

Article Snippet: Briefly, the human MGMT cDNA was purchased from Origene Technologies and cloned into the pCDH-CMVHygro lentiviral vector (Systems BioScience) for lentivirus production and subsequent infection of cells per the manufacturer’s recommended protocol.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: (A) Glioma cells were treated with TMZ (300µMol) for 24 hr. Cell viability was evaluated with an MTT assay. (B) Western blot analysis of MGMT expression in glioblastoma cells. (C) Real time PCR of miR-221 expression in glioblastoma cells. (D) RNA Hybrid prediction analyzes of miR-222, miR-221, and MGMT 3’ UTR. In bold are shown the mutated oligonucleotides. Luciferase activity of HEK-293 cells transiently co-transfected with the luciferase reporter containing wild-type MGMT-3’UTR or mutant MGMT-3’UTR in the presence of pre-miR-222, miR-221, or scrambled oligonucleotide. Representative of at least three independent experiments. *** p<0.001 versus control, ** p<0,0037 versus control.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: MTT Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Mutagenesis, Control

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: (A) Western blot analysis and real time PCR of MGMT protein and RNA after miR-221/222 transfection of T98G cells. (B) Western blot analysis and real time PCR of MGMT protein and RNA after anti-miR-221 and -222 transfection of U87MG cells. (C) Western blot of MGMT expression upon miR-221 transfection of LN428 cells. (D) Western blot analysis of MGMT expression in T98G cells, as a control, and the melanoma cell line A375 upon miR-221 transfection. (E) Analysis of methylation status of MGMT promoter in T98G and U87MG upon miR- or anti-miR-221/222 transfection. U is for the un-methylated form, M for methylated form, NL is for normal lymphocytes, used as control.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Expressing, Control, Methylation

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: ( A ) Cell viability of T98G, LN428, and A375 cells transfected with miR-221 and miR-222 upon TMZ treatment (300 µMol) for 24 hrs. **p value<0.0082 versus scr column, ***p value<0.005 versus scr column. ( B ) Growth curve of T98G and LN428 cells transfected or not with miR-221 after 24 hrs of treatment with TMZ. ( C ) Colony assay of T98G and LN428 cells transfected with miR-221 and then treated for 24 hrs with TMZ (300 µMol). Cells were left to grow for 6 days after treatment removal. ( D ) MGMT expression rescues cell viability after TMZ treatment in T98G and LN428 cells overexpressing miR-221 **p value<0.0082 versus untransfected MGMT column. ( E ) Correlation between miR-221 expression and TMZ sensitivity in nine primary glioblastoma cell lines and in six glioblastoma cell lines.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Transfection, Colony Assay, Expressing

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: (A) Apoptotic cell death assessed by FACS in T98G cells transfected with miR-221 or scrambled sequence and MGMT and treated with TMZ for 24 hrs. *** p value< 0.005 versus untrasfected MGMT column. (B) Active caspase-3 quantification in T98G cells as indicated and treated with TMZ for 24 hrs in the presence or absence of 3 hrs pre-treatment with ZVAD-fmk. (C) Upper panel Time course analysis of caspase-3 activation upon TMZ treatment in T98G cells transfected with miR-221 or with scrambled sequence. Lower panel Western blot analysis of caspase-3 activation after miR-221 and MGMT transfection. (D) Cell viability of T98G cells transfected with miR-221 or with scrambled sequence treated with TMZ for 24 hrs in the presence or absence of 3 hrs pre-treatment with ZVAD-fmk. ** p value< 0.0034 versus only treated TMZ column, Student’s t test.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Transfection, Sequencing, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: (A) Alkaline comet assay of T98G cells transfected with miR-221 and treated with TMZ for the indicated times. (B) Analysis of γH2AX in T98G cells transfected with scrambled control miR or miR-221, treated with TMZ in the presence or in the absence of MGMT cDNA, by immunocytofluorescence (upper and medium panel) or by Western blot (lower panel). (C) Western blot analysis of the indicated proteins upon transfection of T98G cells with miR-221 and MGMT cDNA and TMZ treatment for 24 hrs.

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: Alkaline Single Cell Gel Electrophoresis, Transfection, Control, Western Blot

Journal: PLoS ONE

Article Title: miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

doi: 10.1371/journal.pone.0074466

Figure Lengend Snippet: Mann–Whitney U test analysis was performed to evaluate the association between miR-221 and MGMT expression in long- and short -survival groups of patients. The expression of miR-221 (2^-Dct) ( A - B ) and MGMT (2^-Dct) are inversely correlated with patient survival ( p < 0.0490 and p = 0.043, respectively).

Article Snippet: For overexpression of MGMT, cells were transfected using Lipofectamine and Plus Reagent with 4 μg of MGMT cDNA (Origene, Rockville MD USA).

Techniques: MANN-WHITNEY, Expressing