Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: TBX15/miR-152 pathway directly targets and inhibits KIF2C. A Western blotting analysis for protein expression of KIF2C in MCF7/ADR cells and T47D/ADR cells transfected with miR-152 mimics or miR-152 inhibitor, or TBX15-pCMV or siTBX15. All cell groups were treated with 1 µg/ml DOX. B Putative seed-matching sites or mutant sites (red) between miR-152 and the 3’-UTR of KIF2C were analyzed by TargetScan. The WT and mutant (mut) binding site sequences of miR-152 in the 3’ UTR of KIF2C were cloned into luciferase reporter vectors. HEK293T cells were co-transfected with the reporter vectors, renilla luciferase vector, and miR-152 mimics or miR-NC. After 24 h, the relative levels of luciferase activity were analyzed and normalized to values obtained from WT cells transfected with miR-NC. C Relative expression levels of KIF2C from normal tissue and breast tumors from human patients. KIF2C mRNA expression data were obtained from TCGA database containing 1114 breast tumors and 98 normal adjacent tissues, and were presented as the means ± SEM from three independent experiments. GSEA analysis to evaluate the correlation between expression levels of KIF2C and DOX resistance signatures in the GEO database. D MCF7/ADR cells were transfected with pCMV or KIF2C-pCMV plasmids (KIF2C), siNC or siKIF2C and were treated with DOX. After 48 h, drug sensitivity analysis was determined. E Flow cytometric analysis of MCF7/ADR cells which were transfected as in D and treated with 1 µg/ml DOX. F , G MCF7/ADR cells were transfected as above and were treated with 1 µg/ml DOX. After 48 h, the glucose consumption ( F , top), LA production ( F , bottom), and protein expression levels of KIF2C, p62, LC3, β-actin ( G ) were analyzed. All the data are presented as the means±SEM from three independent experiments and were normalized with corresponding control. *, ** P < 0.05 and P < 0.01, respectively

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: KIF2C overexpression reverses miR-152-induced DOX sensitivity in MCF7/ADR cells. A Levels of KIF2C in MCF7/ADR cells which were co-transfected with miR-NC, miR-152 mimics, or KIF2C. The data are presented as the means±SEM from three independent experiments. * Compared to miR-NC group at P < 0.05, ## compared to miR-152 at P < 0.01. B IC50 values of MCF7/ADR cells that were transfected as in A and were treated with DOX and cultured for 48 h. C Flow cytometric analysis of MCF7/ADR cells that were transfected as in (A) and were treated with 1 µg/ml DOX. D , E The glucose consumption ( D , left), LA production ( D , right), ECAR) ( E , left) and OCR ( E , right) were analyzed in MCF7/ADR cells treated as in A . F Levels of KIF2C, p62 and LC3 in MCF7/ADR cells treated as in ( A ). Graph represents the relative density of KIF2C, p62, or LC3-II/LC3-I expression. The data are presented as the means ± SEM from three independent experiments. ** Compared to miR-NC group at P < 0.01, ## compared to miR-152 at P < 0.01. G The glucose consumption ( G , top) and LA production ( G , bottom) were analyzed in MCF7/ADR cells treated with TBX15 or KIF2C overexpression. H Flow cytometric analysis of MCF7/ADR cells that were transfected as in G and were treated with 1 µg/ml DOX. I Levels of p62 and LC3 in MCF7/ADR cells treated as in G . Graph represents the relative density of KIF2C, p62, or LC3-II/LC3-I expression. The data are presented as the means ± SEM from three independent experiments. *, ** Compared to control groups at P < 0.05 and P < 0.01, # compared to TBX15 at P < 0.05

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Over Expression, Transfection, Cell Culture, Expressing, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: PKM2 is involved in DOX resistance in breast cancer, mediated by the miR-152/KIF2C signaling pathway. A Western blotting analysis of PKM2 levels in MCF7/ADR cells that were transfected with pCMV, TBX15, siNC or siTBX15, and were treated with 1 µg/ml DOX for 48 h. β-actin served as an internal control. B Western blotting analysis of PKM2 levels in MCF7/ADR cells transfected with KIF2C or siKIF2C, and were treated with 1 µg/ml DOX for 48 h. A , B Graph represents the relative density of PKM2 expression. β-actin was used as an internal control. The data are presented as the means ± SEM from three independent experiments.** Indicates significant difference compared to pCMV group at P < 0.01, # , ## compared to siNC at P < 0.05, P < 0.01, respectively. C Western blotting analysis of PKM2 levels in MCF7/ADR cells transfected with TBX15 and /or KIF2C, and were treated with 1 µg/ml DOX for 48 h. β-actin was used as an internal control. Graph represents the relative density of PKM2 expression. *,** Indicate significant difference compared to pCMV group at P < 0.05 and P < 0.01, respectively. # Compared to TBX15 at P < 0.05. D Western blotting analysis of PKM2 in MCF7/ADR cells that were co-transfected with miR-NC, miR-152 or KIF2C and were treated with 1 µg/ml DOX for 48 h. β-actin was used as an internal control. * Indicates significant difference compared to miR-NC group at P < 0.05, # compared to miR-152 at P < 0.05. E Western blotting analysis of KIF2C and PKM2 in MCF7/ADR cells that were co-transfected with siNC, siKIF2C or PKM2-pcDNA, and were treated with 1 µg/ml DOX for 48 h. Graph represents the relative density levels of PKM2, p62, or LC3-II/ I expression. β-actin was used as an internal control. ** Indicates significant difference compared to siNC group at P < 0.01, ## compared to siKIF2C at P < 0.01

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: KIF2C directly binds to PKM2 and promotes PKM2 stability in breast cancer cells. A Co-IP analysis of MCF7/ADR cells that were transfected with pCMV, TBX15, siNC or siTBX15 and were treated with 1 µg /ml DOX for 48 h. B Co-IP analysis of MCF7/ADR cells that were transfected with pCMV, KIF2C, siNC or siKIF2C. Cell lysates were co-immunoprecipitated using an anti-KIF2C or anti-PKM2 antibody. C Western blotting analysis of MCF7/ADR cells transfected with pCMV or KIF2C and treated with 20 µg /ml CHX for 0, 6, 12 and 18 h. D Western blotting analysis of MCF7/ADR cells transfected with siNC or siKIF2C and were treated with CHX. The expression levels of KIF2C and PKM2 were analyzed. E Western blotting analysis of MCF7/ADR cells transfected with a pCMV or KIF2C and treated with 1 µM MG132 for 0, 12 and 24 h. F Western blotting analysis of MCF7/ADR cells transfected with siNC or siKIF2C and treated with MG132. The expression of KIF2C and PKM2 were analyzed. G Western blotting analysis of MCF7/ADR cells transfected with pCMV, KIF2C, siNC or siKIF2C and Ubiquitin-HA plasmid. Cell lysates were co-immunoprecipitated using anti-HA antibody and the expression levels of Ub-PKM2 were analyzed. H Western blotting analysis of MCF7/ADR cells transfected with a pCMV, KIF2C, and KIF2C domains (D1, D2, D3) in pCMV-flag plasmids. Cells were treated with 1 µg /ml DOX for 48 h. Cell lysates were co-immunoprecipitated using anti-flag antibody and the expression levels of PKM2 were analyzed. I Western blotting analysis of MCF7/ADR cells that were transfected as ( H ), and Ubiquitin-HA plasmid. Cell lysates were co-immunoprecipitated using an anti-HA antibody and the expression levels of Ub-PKM2 were analyzed. All the data above were presented as the means ± SEM from three independent experiments and were normalized to the percentage of corresponding control groups. *, ** P < 0.05, P < 0.01, respectively

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Ubiquitin Proteomics, Plasmid Preparation, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: TBX15/miR-152 overexpression reverses DOX resistance in vivo by decreasing KIF2C and PKM2 expression. A–C The MCF7/ADR cells expressing miR-152 or a control (miR-NC) were established using a lentivirus system and dispersed in 100 µl of serum-free DMEM medium. Cells were subcutaneously injected into female nude mice (n = 8). After subcutaneous injection of cancer cells for 24 days, mice were treated with DOX or PBS. Representative pictures of tumors ( A ), tumor size ( B ), and weight ( C ) were obtained at the indicated time point or following 45 days after initial tumor cell injection. Tumors were measured every two days and the tumor volumes were calculated. The tumors were excised and weighed after 45 days, and the representative pictures of trimmed tumors are displayed (Bar= 10 mm). Data are presented as the means ± SEM from all tumor samples. D , E Immunohistochemistry staining of tumor tissues with antibodies of KIF2C and PKM2 (bar = 100 μm). * P < 0.05, ** P < 0.01. F , J The MCF7/ADR cells overexpressing TBX15 or a Negative control (NC) were established using lentivirus system. Cells were subcutaneously injected into female nude mice (n = 8). 10 days after cancer cells subcutaneous injection, tumor xenografts were found in ADR-NC group. However, only 4 tumor samples were found 31 days after injection in TBX15 overexpression group ( F ). Tumors growth was measured as above. The tumors were excised and weighed, and the representative pictures of trimmed tumors were displayed (Bar= 10mm). Data were presented as the means ± SEM from all tumor samples ( G , H ). I qRT-PCR analysis of miR-152 gene expression in ADR-NC and ADR-TBX15 tumor tissues. Data were normalized to the ratio of miR-152 in ADR-NC group. Data are presented as the means± SEM from all tumor samples. ** P < 0.01. J Immunohistochemistry staining of tumor tissues with antibodies of KIF2C and PKM2 (Bar= 100 μm in Low Power view and Bar= 50 μm in High Power view)

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Over Expression, In Vivo, Expressing, Control, Injection, Immunohistochemistry, Staining, Negative Control, Quantitative RT-PCR, Gene Expression