Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: The expression level of PBRM1 varies in different tumors and tumor stages. (A) The expression status of the PBRM1 gene in diverse cancer types were analyzed through the TIMER dataset. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Based on the CPTAC dataset, the expression level of PBRM1 total protein between normal tissues and primary tumor tissues of breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), and kidney renal clear cell carcinoma (KIRC, ccRCC) were analyzed. (C) Based on the TCGA data, the expression levels of the PBRM1 gene were analyzed by the main pathological stages (stage I, stage II, stage III, and stage IV) of BRCA, COAD, LUAD, ccRCC. Log2 (TPM+1) was applied for log-scale. (D) PBRM1 protein expression levels in Stage I and Stage IV of our ccRCC samples. The expression levels were detected using immunohistochemistry and analyzed by unpaired t-test.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Mutation frequency of PBRM1 in tumors reproduced from the Cancer Genome Atlas (TCGA) database. (A) Alternation frequency of PBRM1 in various tumors are shown. (B) Mutation types of PBRM1 in ccRCC are shown. (C) 3D structure of PBRM1 and the mutation cite is shown. (D) Protein-protein interaction network of PBRM1 are shown. The top 10 experimentally determined genes ranked by degree are shown.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: The mutation of PBRM1 affects the survival rates of patients. Overall survival (A) and disease-free survival (B) of PBRM1 high and PBRM1 low patients in different tumors. GEPIA2 tool was used to analyze the TCGA dataset. BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; LUAD, lung adenocarcinoma; KIRC, kidney renal clear cell carcinoma (ccRCC). (C) Overall survival of KIRC (ccRCC) patients based on the PBRM1 expression level and tumor grade. Kaplan-Meier curves with positive results are given.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Mutagenesis, Expressing

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Low expression of PBRM1 correlates with decreased CD4 cells in human ccRCC. (A) The correlation of PBRM1 expression with CD4, CD8 within tumors from 533 ccRCC patients were analyzed using the TIMER database. (B) Opal Multiplex Immunohistochemistry detection of PBRM1, CD4, CD8 in human ccRCC samples.180 ccRCC samples were tested and classified as High (>80%), Medium (50-60%), Low (<20%) according to the expression level of PBRM1 in the tumor tissues. (C) Represents the numbers of CD4, CD8 cells in (B) . PBRM1 High, PBRM1 Low groups were calculated by unpaired t-test, **P < 0.01; ***P < 0.001. Data indicates Mean+SD.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Multiplex Assay, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Low expression of PBRM1 causes reduction of tumor-infiltrating CD4 cells in ccRCC mouse model. (A) PBRM1 protein expression level in PBRM1 high and PBRM1 low Renca cells were detected by western blot. PBRM1 low Renca cells were obtained using shRNA to PBRM1. β-actin was used as an internal control. (B) Growth curves of PBRM1 High and PBRM1 Low tumors in ccRCC mouse model. (C) Flow cytometry image of tumor-infiltrating T cells. TIL cells were isolated from tumors and gated CD45 + CD3 + cells. Percentage (D) and absolute numbers (E) of CD4 and CD8 cells in tumors of PBRM1 High and PBRM1 Low mice model. CD4 andCD8 cells gated in CD45 + CD3 + cells. Data represents 3 independent experiments at least 6 mice per group. *P < 0.05; ns, no significance.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Expressing, Western Blot, shRNA, Control, Flow Cytometry, Isolation

Journal: Frontiers in Oncology

Article Title: Mutational Analysis of PBRM1 and Significance of PBRM1 Mutation in Anti-PD-1 Immunotherapy of Clear Cell Renal Cell Carcinoma

doi: 10.3389/fonc.2021.712765

Figure Lengend Snippet: Anti-PD-1 immunotherapy increased CD4 T cell infiltration in ccRCC mouse model. (A) Treatment scheme of anti-PD-1 immunotherapy in ccRCC mouse model. Anti-PD-1 antibody and control antibody were administrated at day 6, day 9, and day 12 after tumor inoculation. (B) Growth curves of PBRM1 High and PBRM1 Low tumors treated with anti-PD-1 antibody or control isotype antibody in ccRCC mouse model. Test was stopped when mice died or tumors reached 1000mm 3 in size. (C) Survival rates of mice in different groups. Data were analyzed with GraphPad Prism 8.0.2. Absolute cell numbers (D) and percentages (E) of CD4 and CD8 T cells in tumors of different groups were shown. All live mice were euthanized at day39 and tumor infiltrating T cells were detected by Flow Cytometry. Data are presented as mean ± SD with at least 6 mice for each group. *p < 0.05; ns, no significance.

Article Snippet: Mouse ccRCC cell line Renca was purchased from ATCC and maintained in RPMI-1640 containing 10% fetal bovine serum, non-essential amino acids (NEAA), and sodium pyruvate according to the ATCC instruction at 37°C with 5% CO 2 .

Techniques: Control, Flow Cytometry

Journal: Cell Proliferation

Article Title: Non‐SMC condensin I complex subunit H participates in anti‐programmed cell death‐1 resistance of clear cell renal cell carcinomas

doi: 10.1111/cpr.13400

Figure Lengend Snippet: Elevated non‐SMC condensin I complex subunit H (NCAPH) promotes clear cell renal cell carcinoma (ccRCC) growth. (A) NCAPH expression in 87 paired ccRCC tissues and normal tissues was assessed by qRT‐PCR. Paired t ‐test been used, *** p < 0.001. (B) Kaplan–Meier plot showing the relationship between NCAPH levels and patient overall survival. (C) ROS curve showing the potential diagnostic value in ccRCC. (D) NCAPH protein expression in 786‐O and Caki‐1 cells with NCAPH overexpression or depletion was assessed by Western bolting. (E and F) Cell viability of 786‐O and Caki‐1 cells with NCAPH overexpression or depletion was assessed by CCK‐8 assays. *** p < 0.001 versus vector; ### p < 0.001 versus sh‐NC. (G) EDU staining shows the proliferative activity of 786‐O and Caki‐1 cells. *** p < 0.001 versus vector; ### p < 0.001 versus sh‐NC. (H) Clone formation assay showing the clonal formation ability of 786‐O and Caki‐1 cells. *** p < 0.001 versus vector; ### p < 0.001 versus sh‐NC. (I and J) Caki‐1 cells transfected with sh‐NC or sh‐NCAPH were injected into injected subcutaneously into Balb/c nude mice (five mice per group). The effect of NCAPH silencing on tumour volume curve and tumour weight was analysed. ### p < 0.001 versus sh‐NC. (K) Ki67 staining of xenografts. ### p < 0.001 versus sh‐NC. All experiments were repeated at least three times.

Article Snippet: Human Kidney‐2 (HK‐2) cells, human ccRCC cell lines (796‐P, 786‐O, ACHN and Caki‐1) and mouse Renca cells were bought from the ATCC and maintained in RPMI‐1640 medium supplemented with 10% FBS.

Techniques: Expressing, Quantitative RT-PCR, Diagnostic Assay, Over Expression, Western Blot, CCK-8 Assay, Plasmid Preparation, Staining, Activity Assay, Tube Formation Assay, Transfection, Injection

Journal: Cell Proliferation

Article Title: Non‐SMC condensin I complex subunit H participates in anti‐programmed cell death‐1 resistance of clear cell renal cell carcinomas

doi: 10.1111/cpr.13400

Figure Lengend Snippet: Non‐SMC condensin I complex subunit H (NCAPH) affects PD‐L1 expression by regulating the stability of β‐catenin protein. (A and B) The co‐IP assay was performed to detect the endogenous protein interaction between NCAPH and β‐catenin in 786‐O and Caki‐1 cells. (C) The treatment of MG132 blocked the change of β‐catenin level mediated by NCAPH overexpression or depletion. (D) Ubiquitination assay of β‐catenin in Caki‐1 cells co‐transfected with Myc‐Ub and Flag‐β‐catenin and treated with MG132. (E) PD‐L1 mRNA expression in clear cell renal cell carcinoma (ccRCC) cells was measured after NCAPH depletion or β‐catenin overexpression. *** p < 0.001 versus sh‐NC + vector; ### p < 0.001 versus sh‐NCAPH‐1. (F) The PD‐L1 protein level in ccRCC cells was measured after NCAPH depletion or β‐catenin overexpression. (G) Membrane PD‐L1 level in ccRCC cells was measured after NCAPH depletion or β‐catenin overexpression by flow cytometry. Each experiment was repeated at least three times.

Article Snippet: Human Kidney‐2 (HK‐2) cells, human ccRCC cell lines (796‐P, 786‐O, ACHN and Caki‐1) and mouse Renca cells were bought from the ATCC and maintained in RPMI‐1640 medium supplemented with 10% FBS.

Techniques: Expressing, Co-Immunoprecipitation Assay, Over Expression, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Membrane, Flow Cytometry

Journal: Cell Proliferation

Article Title: Non‐SMC condensin I complex subunit H participates in anti‐programmed cell death‐1 resistance of clear cell renal cell carcinomas

doi: 10.1111/cpr.13400

Figure Lengend Snippet: Non‐SMC condensin I complex subunit H (NCAPH) is related to aerobic glycolysis in clear cell renal cell carcinoma (ccRCC). (A and B) Glucose consumption and lactate production of ccRCC cells was measured. (C) The effect of NCAPH‐β‐catenin axis on the extracellular acidification rate (ECAR) in 786‐O and Caki‐1 cells. (D–G) The effect of NCAPH‐β‐catenin axis on the mRNA level of glycolysis‐related genes (HK2, PKM2, LDHA and GLUT1) in ccRCC cells. *** p < 0.001 vssh‐NC + vector; ### p < 0.001 versus sh‐NCAPH‐1. (H) Cell viability of ccRCC cells was measured by CCK‐8 assay after NCAPH overexpression or 2‐DG treatment. (I) EDU‐positive cells were counted in ccRCC cells after NCAPH overexpression or 2‐DG treatment. (J) The number of clones was counted in ccRCC cells with NCAPH overexpression or 2‐DG treatment. *** p < 0.001 versus vector + ddH2O; ### p < 0.001 versus NCAPH + ddH2O. Each experiment was repeated at least three times.

Article Snippet: Human Kidney‐2 (HK‐2) cells, human ccRCC cell lines (796‐P, 786‐O, ACHN and Caki‐1) and mouse Renca cells were bought from the ATCC and maintained in RPMI‐1640 medium supplemented with 10% FBS.

Techniques: Plasmid Preparation, CCK-8 Assay, Over Expression, Clone Assay

Journal: Cell Proliferation

Article Title: Non‐SMC condensin I complex subunit H participates in anti‐programmed cell death‐1 resistance of clear cell renal cell carcinomas

doi: 10.1111/cpr.13400

Figure Lengend Snippet: Non‐SMC condensin I complex subunit H (NCAPH) is related to the resistance to anti‐PD‐1 therapy in clear cell renal cell carcinoma (ccRCC). (A) Membrane PD‐1 expression in Renca cells with mouse NCAPH overexpression was measured by flow cytometry. (B) Schematic diagram showing the grouping and treatment plan of the in vivo study: C57BL/6 mice were inoculated with 10 6 control or mouse NCAPH overexpressed Renca cells and received PD‐1 mAb treatment or IgG2b control at the indicated time points (five mice per group). (C) Tumour growth curves of the xenograft. (D and E) The volumes and weight of captured xenograft. (F) Body weight of mouse in the process of the treatment. (G) Survival curves of mouse with indicated treatment. (H and I) HE and Ki67 staining of xenograft. (J) The Ki67‐positive cells in the slice were counted. * p < 0.05; *** p < 0.001 versus vector + IgG2b; ### p < 0.001 versus vector + PD‐1 mAb. (K) The infiltrated CD8 T cells were counted through CD8 IHC. ** p < 0.01. (L) Working model for NCAPH/β‐catenin axis in ccRCC. Three independent experiments were performed.

Article Snippet: Human Kidney‐2 (HK‐2) cells, human ccRCC cell lines (796‐P, 786‐O, ACHN and Caki‐1) and mouse Renca cells were bought from the ATCC and maintained in RPMI‐1640 medium supplemented with 10% FBS.

Techniques: Membrane, Expressing, Over Expression, Flow Cytometry, In Vivo, Control, Staining, Plasmid Preparation, Paraffin-embedded Immunohistochemistry