Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 is aberrantly expressed in patients with BLCA, and this has an adverse prognostic implication. (A) Box plot of the tissue-wide differential gene expression profile across cancer experiments using the GPL570 platform (HG-U133_Plus_2)]. (B) Volcano plot of the hazard ratios of differentially expressed genes in the TGCA BLCA cohort. MED10 is indicated by the red circle. Volcano plot was plotted with log hazard ratio (HR) on the x-axis and p-value on the y-axis, using the Cox PH model. (C) Kaplan–Meier plot showing the effect of altered MED10 expression on the overall survival of patients in the BLCA cohort of the GPL570 platform. (D) Box and dot plots showing the association between MED10 expression and tumor stage in the GPL570 platform BLCA cohort (left) . Chart showing the 2-sample t-test results for (D) (right) . (E) Forest plot of hazard ratios showing the effect of MED10 on overall and recurrence-free survival on the GPL570 platform BLCA cohort. (F) Box and dot plots of the MED10 expression profile based on tumor grade in the GPL570 platform BLCA cohort.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Gene Expression, Expressing

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 expression is positively correlated with disease progression, immune suppression, and therapy failure. Box plots showing the association between MED10 mRNA expression and (A) AJCC metastasis stage, (B) recurrence/progression or disease-free status of patients, (C) AJCC tumor stage, or (D) primary therapy outcome success type, in the TCGA BLCA cohort. (E) Dot plot of the tumor purity-adjust correlation between MED10 expression and level of tumor immune infiltrates. (F) Dot plot of the effect of high amplification of MED10 on the diploid-normalized immune cell infiltration level. Kaplan–Meier plot showing the effect of altered MED10 expression with or without altered (G) CD8+ T, (H) MDSC, or (I) Treg cell level on the cumulative survival.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Biomarker Discovery, Amplification

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 is functionally co-expressed with hsa-miR-590 but is inversely associated with tumor-suppressor microRNAs. (A) Association plot of the relationship between the expression of MED10 and miRs in the TCGA BLCA cohort (left) . Chart showing the 10 topmost miRs associated with upregulated or downregulated MED10 expression (right) . Heatmaps showing (B) positively and (C) negatively correlated significant miRs in the TCGA BLCA cohort. (D) Line and dot plots showing the correlation between MED10 and hsa-miR-590-5p expression levels in the TCGA BLCA cohort. FPKM, fragmented per kilobase of transcript per million; RPM, reads per million total/mapped reads. (E) Box and whiskers plot showing the association between MED10 mRNA expression and hsa-miR-590-5p copy number alterations.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 interacts directly with hsa-miR-590, a modulator of immune infiltration and survival. (A) Depiction of mutual exclusivity test data showing tendency and likelihood of MED10 and hsa-miR-590 to co-occur in pool of 5 BLCA/UC studies. (B) Sequence-derived 3D structure of MED10. (C) The optimal secondary structure in dot-bracket notation with a minimum free energy (MFE) of −31.20 kcal/mol (upper) . The RNAfold-generated MFE secondary folding structure pattern of hsa-miR-590 (lower left) . The hsa-miR-590 3D structure generated based on the secondary structure in dot-bracket notation (lower right) . (D) Molecular docking showing the direct interaction between MED10 and hsa-miR-590. The 3D transformation data, consisting of three rotational angles (2.42°, 0.38°, 120.70°) and three translational parameters (−1.18, −53.27, −102.28) applied on the ligand molecule, hsa-miR-590. (E) Representative agarose gel electrophoresis image of the effect of altered MED10 protein expression on the expression level of hsa-miR-590 in BdEC and SW1738 cells. GAPDH served as loading control. WT, wild type. (F) Dot plot showing the effect of high amplification of hsa-miR-590 on the immune cell infiltration levels. (G) Kaplan–Meier plot of the effect of altered hsa-miR-590 expression in the TCGA BLCA cohort.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Sequencing, Derivative Assay, Generated, Transformation Assay, Agarose Gel Electrophoresis, Expressing, Control, Amplification

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: Targeting MED10 elicits downregulation of hsa-miR-590-5p expression preferentially in metastatic, transitional cell (urothelial) carcinoma cells. Box and whisker plots of the differential expression of (A) MED10 or (B) hsa-miR-590-5p in cancer and normal samples from the TCGA BLCA cohort. (C) Scatter-plot of the biological coefficient of variation (BCOV) against the average abundance of MED10 and MIR590 in the GSE81157 aggressive bladder cancer cohort. (D) Graphical representation of the effect of MED10 loss of function on hsa-miR-590 copy number in 29 metastatic or primary BLCA cell lines from the 21Q2 Public cohort (left) . Chart showing the spatial distribution of the BLCA cell lines according to metastasis status (right) . Visualization of the effect of MED10 knockout on hsa-miR-590-5p expression in (E) mixed pool of BLCA cell lines (left) , or purely bladder transitional/UC cell lines (right) .

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Whisker Assay, Quantitative Proteomics, Knock-Out

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: shRNA-mediated targeting of MED10 in bladder urothelial carcinoma cells significantly attenuate their oncogenicity and metastatic phenotype. Photomicrographs and histograms showing the effect of knocking down MED10 on the (A) proliferation, ( B ) migration, (C) invasion, and (D) colony formation of SW1738 and JMSU1 cells. (E) Representative Western blot images showing the effect of shMED10 on the expression of MED10, BAX, BCL-xL, VIM, SNAI1, OCT4, or LIN28A protein expression level in SW1738 and JMSU1 cells. GAPDH serve as loading control. ns, not significant; **p < 0.01; ***p < 0.001.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: shRNA, Migration, Western Blot, Expressing, Control

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: shRNA-mediated targeting of MED10 in bladder urothelial carcinoma cells significantly attenuate their cancer stemness phenotype. (A) Heatmap showing the correlation between the expression of MED10, and stemness markers NANOG, SOX2, POU5F1/OCT4, CD44, PROM1/CD133, KLF4, and LIN28A in patients with urothelial recurrence or local recurrence from the AFFY_HG_U133_PLUS_2, GSE31684 cohort. Columns with similar annotations are collapsed by taking mean inside each group. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. 24 rows, 4 columns. (B) Immunohistochemistry photomicrographs of the differential expression of MED10, MKI67, OCT4, and LIN28A in benign, T1, T2, T3/4, or M1 tissue samples from the TMU-SHH UC cohort. Scale bar 200 μm. (C) Photomicrographs and histograms showing the effect of shMED10 on the formation of primary or secondary tumorspheres from SW1738 and JMSU1 cells. (D) Representative immunofluorescence images showing the effect of knocking down MED10 on the nuclear translocation and co-localization of OCT4A and LIN28A. DAPI served as nuclear marker. Scale bar 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Monoclonal antibodies against MED10 (C-2: #sc-393450), BAX (B-9: #sc-7480), BCL-xL (H-5: #sc-8392), Ki67 (#sc-23900), Vimentin (V9: #sc-6260), and SNAI1 (G-7: #sc-271977) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); OCT4A (#2840) and LIN28A (#8706) were purchased from Cell Signaling Technology, Inc. (CST, Beverly, MA, USA); and GAPDH (#sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: shRNA, Expressing, Immunohistochemistry, Quantitative Proteomics, Immunofluorescence, Translocation Assay, Marker

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 is aberrantly expressed in patients with BLCA, and this has an adverse prognostic implication. (A) Box plot of the tissue-wide differential gene expression profile across cancer experiments using the GPL570 platform (HG-U133_Plus_2)]. (B) Volcano plot of the hazard ratios of differentially expressed genes in the TGCA BLCA cohort. MED10 is indicated by the red circle. Volcano plot was plotted with log hazard ratio (HR) on the x-axis and p-value on the y-axis, using the Cox PH model. (C) Kaplan–Meier plot showing the effect of altered MED10 expression on the overall survival of patients in the BLCA cohort of the GPL570 platform. (D) Box and dot plots showing the association between MED10 expression and tumor stage in the GPL570 platform BLCA cohort (left) . Chart showing the 2-sample t-test results for (D) (right) . (E) Forest plot of hazard ratios showing the effect of MED10 on overall and recurrence-free survival on the GPL570 platform BLCA cohort. (F) Box and dot plots of the MED10 expression profile based on tumor grade in the GPL570 platform BLCA cohort.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 expression is positively correlated with disease progression, immune suppression, and therapy failure. Box plots showing the association between MED10 mRNA expression and (A) AJCC metastasis stage, (B) recurrence/progression or disease-free status of patients, (C) AJCC tumor stage, or (D) primary therapy outcome success type, in the TCGA BLCA cohort. (E) Dot plot of the tumor purity-adjust correlation between MED10 expression and level of tumor immune infiltrates. (F) Dot plot of the effect of high amplification of MED10 on the diploid-normalized immune cell infiltration level. Kaplan–Meier plot showing the effect of altered MED10 expression with or without altered (G) CD8+ T, (H) MDSC, or (I) Treg cell level on the cumulative survival.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: Expressing, Amplification

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 is functionally co-expressed with hsa-miR-590 but is inversely associated with tumor-suppressor microRNAs. (A) Association plot of the relationship between the expression of MED10 and miRs in the TCGA BLCA cohort (left) . Chart showing the 10 topmost miRs associated with upregulated or downregulated MED10 expression (right) . Heatmaps showing (B) positively and (C) negatively correlated significant miRs in the TCGA BLCA cohort. (D) Line and dot plots showing the correlation between MED10 and hsa-miR-590-5p expression levels in the TCGA BLCA cohort. FPKM, fragmented per kilobase of transcript per million; RPM, reads per million total/mapped reads. (E) Box and whiskers plot showing the association between MED10 mRNA expression and hsa-miR-590-5p copy number alterations.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: MED10 interacts directly with hsa-miR-590, a modulator of immune infiltration and survival. (A) Depiction of mutual exclusivity test data showing tendency and likelihood of MED10 and hsa-miR-590 to co-occur in pool of 5 BLCA/UC studies. (B) Sequence-derived 3D structure of MED10. (C) The optimal secondary structure in dot-bracket notation with a minimum free energy (MFE) of −31.20 kcal/mol (upper) . The RNAfold-generated MFE secondary folding structure pattern of hsa-miR-590 (lower left) . The hsa-miR-590 3D structure generated based on the secondary structure in dot-bracket notation (lower right) . (D) Molecular docking showing the direct interaction between MED10 and hsa-miR-590. The 3D transformation data, consisting of three rotational angles (2.42°, 0.38°, 120.70°) and three translational parameters (−1.18, −53.27, −102.28) applied on the ligand molecule, hsa-miR-590. (E) Representative agarose gel electrophoresis image of the effect of altered MED10 protein expression on the expression level of hsa-miR-590 in BdEC and SW1738 cells. GAPDH served as loading control. WT, wild type. (F) Dot plot showing the effect of high amplification of hsa-miR-590 on the immune cell infiltration levels. (G) Kaplan–Meier plot of the effect of altered hsa-miR-590 expression in the TCGA BLCA cohort.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: Sequencing, Derivative Assay, Generated, Transformation Assay, Agarose Gel Electrophoresis, Expressing, Amplification

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: Targeting MED10 elicits downregulation of hsa-miR-590-5p expression preferentially in metastatic, transitional cell (urothelial) carcinoma cells. Box and whisker plots of the differential expression of (A) MED10 or (B) hsa-miR-590-5p in cancer and normal samples from the TCGA BLCA cohort. (C) Scatter-plot of the biological coefficient of variation (BCOV) against the average abundance of MED10 and MIR590 in the GSE81157 aggressive bladder cancer cohort. (D) Graphical representation of the effect of MED10 loss of function on hsa-miR-590 copy number in 29 metastatic or primary BLCA cell lines from the 21Q2 Public cohort (left) . Chart showing the spatial distribution of the BLCA cell lines according to metastasis status (right) . Visualization of the effect of MED10 knockout on hsa-miR-590-5p expression in (E) mixed pool of BLCA cell lines (left) , or purely bladder transitional/UC cell lines (right) .

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: Expressing, Whisker Assay, Knock-Out

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: shRNA-mediated targeting of MED10 in bladder urothelial carcinoma cells significantly attenuate their oncogenicity and metastatic phenotype. Photomicrographs and histograms showing the effect of knocking down MED10 on the (A) proliferation, ( B ) migration, (C) invasion, and (D) colony formation of SW1738 and JMSU1 cells. (E) Representative Western blot images showing the effect of shMED10 on the expression of MED10, BAX, BCL-xL, VIM, SNAI1, OCT4, or LIN28A protein expression level in SW1738 and JMSU1 cells. GAPDH serve as loading control. ns, not significant; **p < 0.01; ***p < 0.001.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: shRNA, Migration, Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

doi: 10.3389/fonc.2021.744937

Figure Lengend Snippet: shRNA-mediated targeting of MED10 in bladder urothelial carcinoma cells significantly attenuate their cancer stemness phenotype. (A) Heatmap showing the correlation between the expression of MED10, and stemness markers NANOG, SOX2, POU5F1/OCT4, CD44, PROM1/CD133, KLF4, and LIN28A in patients with urothelial recurrence or local recurrence from the AFFY_HG_U133_PLUS_2, GSE31684 cohort. Columns with similar annotations are collapsed by taking mean inside each group. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. 24 rows, 4 columns. (B) Immunohistochemistry photomicrographs of the differential expression of MED10, MKI67, OCT4, and LIN28A in benign, T1, T2, T3/4, or M1 tissue samples from the TMU-SHH UC cohort. Scale bar 200 μm. (C) Photomicrographs and histograms showing the effect of shMED10 on the formation of primary or secondary tumorspheres from SW1738 and JMSU1 cells. (D) Representative immunofluorescence images showing the effect of knocking down MED10 on the nuclear translocation and co-localization of OCT4A and LIN28A. DAPI served as nuclear marker. Scale bar 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: MED10-Human, 4 unique 29mer shRNA constructs in lentiviral GFP vector containing pGFP-C-shLenti (#TL303299; MED10 Human shRNA Plasmid Kit (Locus ID 84246; OriGene Technologies Inc., Rockville, MD, USA) were packaged and transfected into SW1738 or JMSU1 cells to silence MED10.

Techniques: shRNA, Expressing, Immunohistochemistry, Immunofluorescence, Translocation Assay, Marker