Journal: Cancer Cell International

Article Title: Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer

doi: 10.1186/s12935-023-02904-y

Figure Lengend Snippet: NIK upregulation facilitated inherent tumor growth but not the lung metastatic potential of LM05 cells. A Cell growth curves of LM05-shGFP, shNIK no.1 and shNIK no.2 cells on planar culture (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). B Representative images (upper) and quantification data (lower) (n = 5, one-way ANOVA followed by Tukey’s multiple comparison test) of the soft agar colony formation assay in LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The scale bar is 5 mm. C Representative images of primary tumors (upper) and quantification data of the primary tumor weights (lower) (n = 4, one-way ANOVA followed by Tukey’s multiple comparison test) in LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The scale bar is 1 cm. D Representative in vivo bioluminescent images of LM05-shGFP and shNIK no.2 cells (upper). Tumor growth curves (lower) (n = 6 per group, two-way ANOVA followed by Tukey’s multiple comparison test) of NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. E Representative ex vivo bioluminescent images (upper) and quantification data of the lung metastasis tissue (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) derived from NOD-SCID mice orthotopically injected with LM05-shGFP (n = 5), shNIK no.1 (n = 4) and shNIK no.2 cells (n = 5). F Representative HE and IHC staining images (upper) and quantification data (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) of NIK protein production in lung metastasis tissue derived from NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The average CAM5.2-positive percentages were calculated from 5 fields of view from n = 4 individual lung metastasis slides. The scale bar is 100 μm. G Representative HE and IHC staining images (upper) and quantification data (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) from the TUNEL assay and α-SMA protein production in primary tumor tissues derived from NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The average number of TUNEL- and α-SMA-positive cells were calculated from 5 fields of view from n = 4 individual primary tumor slides. The scale bar is 100 μm. H Representative images (upper) and quantification data (lower) (n = 5, one-way ANOVA followed by Tukey’s multiple comparison test) of the Boyden chamber assay with TIG-3 cells that were co-cultured with LM05-shGFP, shNIK no.1, and shNIK no.2 cells. The scale bar is 500 μm. I Western blotting (upper) and immunofluorescence staining (lower) of α-SMA expression in TIG-3 cells that were co-cultured with LM05-shGFP, shNIK no.1 and shNIK no.2 cells or no cells (NC). The scale bar is 50 μm. All data are shown as the mean ± SEM. n.s. not significant. * P < 0.05

Article Snippet: Brest cancer cell lines and TIG-3 cells were fixed with 4% paraformaldehyde–PBS for 15 min and permeabilized with 0.1% Triton X-100-PBS (Fujifilm Wako Pure Chemical Corporation) for 15 min. After blocking with Blocking One regent for 1 h, cells were incubated with primary antibodies against α-SMA (#19245, Cell Signaling Technology, 1:500), Vimentin (V2258, Sigma-Aldrich Co., MO, USA, 1:200), NF-κB2 (#05–361, Merck, Darmstadt, Deutschland, 1:100), RelB (#4922, Cell Signaling Technology, 1:100) at 4 °C overnight.

Techniques: Comparison, Soft Agar Assay, In Vivo, Injection, Ex Vivo, Derivative Assay, Immunohistochemistry, TUNEL Assay, Boyden Chamber Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐mi R ‐134‐5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells

doi: 10.1111/jcmm.18523

Figure Lengend Snippet: Evaluation of signalling regulation of hsa‐miR‐134‐5p in human EPCs by enzyme‐linked immunosorbent assay (ELISA) and western blot analysis. In miR‐134‐5p‐overexpressed EPCs, (A) transforming growth factor Beta 1 (TGF‐β1) level in the supernatant normalized by cell number was respectively reduced in young and senescent cells, compared to miRNA mimic negative control (NC) group ( n = 4). Also note, the difference between young and senescent groups was indicated. The findings showed that, when performing a comparison between groups with or without overexpression of miR‐134‐5p, the expression level of TGF‐β1 was higher in senescent than young EPCs. Additionally, in senescent EPCs, (B) the TGF‐β1‐associated protein, transforming growth factor β‐activated kinase 1‐binding protein 1 (TAB1) ( n = 5) and (C) phosphorylated p38 mitogen‐activated protein kinase (p‐p38, n = 3) were down‐regulated compared to NC. (D) After senescent EPCs were treated with siRNA specific to TAB1 (TAB1si) for 24 h (h) and 48 h, TAB1 was suppressed ( n = 7) and, subsequently, (E) p‐p38 was down‐regulated at the time point of 48 h after TAB1si treatment compared to non‐sense siRNA (NS) ( n = 6). (F) and (G) When TAB1si or p38 mitogen‐activated protein kinase inhibitor SB203580 was added to senescent EPCs, TGF‐β1 level in the supernatant normalized by cell number showed minimal change between treatment group and its corresponding control group/( n = 4). Data are mean ± SEM and analysed using unpaired Student's t ‐test for comparison between groups. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus corresponding control.

Article Snippet: The blots were blocked with 0.1 g/mL BSA for 1 h at room temperature and subsequently incubated with primary antibodies specific to TAB1, phosphorylated p38 (Thr180/Tyr182) and total p38 (all from Cell Signalling) with the dilution 1:1000 at 4°C for 16 h. The blotting membranes were then incubated with HRP‐conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch) for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control, Comparison, Over Expression, Expressing, Binding Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐mi R ‐134‐5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells

doi: 10.1111/jcmm.18523

Figure Lengend Snippet: Angiogenic activities of senescent human EPCs after treatment with siRNA specific to TAB1 (TAB1si)/p38 inhibitor (SB203580, 10 μM) and transforming growth factor Beta 1 (TGF‐β1, 5 ng/mL). In TAB1si‐treated EPCs, improved cellular activities of (A) cell number detected by cell counting ( n = 5) and (C) tube formation using Matrigel ( n = 3) were noted but not found in (B) migration using Boyden Chamber assay ( n = 4). Similarly, after treatment with SB203580, (D) cell number ( n = 5) and (F) tube formation ( n = 4) were improved but (E) migration ( n = 6) was not improved. In senescent EPCs with improved angiogenic activities after overexpression of miR‐134‐5p, TGF‐β1 treatment did not affect (G) cell number ( n = 4) and (H) migration ( n = 5), but (I) tube formation ( n = 6) was inhibited, compared to corresponding miR‐134‐5p‐overexpressed EPC group without TGF‐β1 treatment. Representative micrographs are shown in (C), (F) and (I). Scale bar: 300 μm. Data are mean ± SEM. Statistical significance was analysed using unpaired Student's t ‐test for comparison between two groups in (A)–(F) or using ANOVA with Tukey's post hoc test for multiple group comparisons in (G)–(I). * p < 0.05 versus corresponding control. Abbreviations are the same as in Figure .

Article Snippet: The blots were blocked with 0.1 g/mL BSA for 1 h at room temperature and subsequently incubated with primary antibodies specific to TAB1, phosphorylated p38 (Thr180/Tyr182) and total p38 (all from Cell Signalling) with the dilution 1:1000 at 4°C for 16 h. The blotting membranes were then incubated with HRP‐conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch) for 1 h at room temperature.

Techniques: Cell Counting, Migration, Boyden Chamber Assay, Over Expression, Comparison, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐mi R ‐134‐5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells

doi: 10.1111/jcmm.18523

Figure Lengend Snippet: Signalling regulation of hsa‐miR‐134‐5p in human senescent EPCs and its clinical implication. In human EPCs cultured from PBMCs, miR‐134‐5p in the senescent cells, mainly targeting TAB1 gene, is up‐regulated and such a change results in improvement of angiogenetic activity via TAB1/p38 signalling. Moreover, up‐regulated miR‐134‐5p in senescent EPCs is linked to TGF‐β1 modulation to promote tube formation activity. In PBMCs, the expression level of hsa‐miR‐134‐5p has an initial increase after 31 years, and a subsequent decrease after 65 years. Moreover, along the increase of Framingham risk score, the expression level of hsa‐miR‐134‐5p decreases. Hsa‐miR‐134‐5p presents its potential anti‐ageing character in the human ageing process and acts as a pro‐angiogenesis modulator. Abbreviations are as in Figure .

Article Snippet: The blots were blocked with 0.1 g/mL BSA for 1 h at room temperature and subsequently incubated with primary antibodies specific to TAB1, phosphorylated p38 (Thr180/Tyr182) and total p38 (all from Cell Signalling) with the dilution 1:1000 at 4°C for 16 h. The blotting membranes were then incubated with HRP‐conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch) for 1 h at room temperature.

Techniques: Cell Culture, Activity Assay, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: MiR-944/CISH mediated inflammation via STAT3 is involved in oral cancer malignance by cigarette smoking

doi: 10.1016/j.neo.2020.08.005

Figure Lengend Snippet: Phenotypic effects of dysregulated CISH in OSCC cell lines. (A) Western blot analysis of the STAT3, 5 and their phosphorylation form after transfection of control vector (Vec) or CISH expression vector (CISH) for 48 h in SAS and SCC-4 cells. GAPDH was used as protein loading control. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH. (B) The effect of CISH on the transcriptional activity of the construct containing the STAT3 binding sequence. The relative luciferase activities are the ratios of Renilla luciferase normalized to the vector alone control. (C) Migration assay following CISH overexpression for 24 h using Matrigel non-coated Boyden chamber assay in SAS and SCC-4 cells. (D) Invasion assay following CISH overexpression for 24 h using Matrigel coated Boyden chamber assay in SAS and SCC-4 cells. (E) CCL3, CCL5 and IL-1β levels in cultured medium of CISH overexpression SAS and SCC-4 cells measured by ELISA (compared with Vec control). (F) Western blot analysis of the CISH, COX-2 and iNOS after transfection of CISH for 48 h in SAS and SCC-4 cells. GAPDH was used as protein loading control. All data are represented as mean ± SD; **, p < 0.01; ***, p < 0.001.

Article Snippet: The membranes were probed with specific antibodies against CISH (#8731, Cell signaling, Danvers, MA), STAT3 (#610189, BD Biosciences, NJ), phospho-STAT3 (p-STAT3, Tyr 705 ) (#9131, Cell signaling), STAT5 (#25656, Cell signaling), phospho-STAT3 (p-STAT5, Tyr 694 ) (#9359, Cell signaling), COX-2 (#12282, Cell signaling), and iNOS (ab204017, Abcam, Cambridge, MA).

Techniques: Western Blot, Phospho-proteomics, Transfection, Control, Plasmid Preparation, Expressing, Activity Assay, Construct, Binding Assay, Sequencing, Luciferase, Migration, Over Expression, Boyden Chamber Assay, Invasion Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: MiR-944/CISH mediated inflammation via STAT3 is involved in oral cancer malignance by cigarette smoking

doi: 10.1016/j.neo.2020.08.005

Figure Lengend Snippet: CISH is a direct target of miR-944. (A) Flow chat of predicted miRNA which targets CISH 3′-UTR using two independent algorithms (microRNA.org and TargetScan) combined with our patients’ miRNA array data (GSE45238). (B) Western blot analysis of CISH protein after transfection of miR-944 mimics (PM) with 30 nM for 48 h in OEC-M1 and SCC-25 cells. (C) Schematic representation of the putative miR-944 binding sequence in the 3′-UTR of CISH with wild-type form (CISH 3′UTR WT) and mutant form (CISH 3′UTR Mut). The mutated nucleotides are labeled with underline. (D) Luciferase activity assays were performed after OEC-M1 and SCC-25 cells transfected with luciferase vectors containing the wide-type or mutant CISH 3′-UTR, in response to miR-944 over-expression. The relative luciferase activity of each sample is measured at 48 h after transfection and normalized to Renilla luciferase activity. (E) Correlation analysis of miR-944 and CISH in human OSCC patients ( n = 33) by qRT-PCR analysis. Western blot analysis of the CISH, STAT3 and phosphor-STAT3 after transfection of miR-944 mimics (PM) (F) or miR-944 inhibitors (AM) (G) with indicated concentration for 48 h in OEC-M1 and SCC-25 cells. All data are presented as mean ± SD; ***, p < 0.001 versus scramble control (NC). GAPDH was used as protein loading control. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH.

Article Snippet: The membranes were probed with specific antibodies against CISH (#8731, Cell signaling, Danvers, MA), STAT3 (#610189, BD Biosciences, NJ), phospho-STAT3 (p-STAT3, Tyr 705 ) (#9131, Cell signaling), STAT5 (#25656, Cell signaling), phospho-STAT3 (p-STAT5, Tyr 694 ) (#9359, Cell signaling), COX-2 (#12282, Cell signaling), and iNOS (ab204017, Abcam, Cambridge, MA).

Techniques: Western Blot, Transfection, Binding Assay, Sequencing, Mutagenesis, Labeling, Luciferase, Activity Assay, Over Expression, Quantitative RT-PCR, Concentration Assay, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: MiR-944/CISH mediated inflammation via STAT3 is involved in oral cancer malignance by cigarette smoking

doi: 10.1016/j.neo.2020.08.005

Figure Lengend Snippet: miR-944 regulates OSCC cell functions through targeting CISH. OSCC cells were transfected with miR-944 mimics (PM) or scramble control (NC) for 24 h and then transfected with CISH expression vector without 3′-UTR (pCDNA3.1-CISH) or control vector (pCDNA3.1) for another 24 h. Under this condition, the level of CISH, STAT3 and phosphor-STAT3 were determined in OEC-M1 and SCC-25 cells by western blotting (A). The RNA level of COX-2 and iNOS (B), protein level of CCL3, CCL5 and IL-1β levels in cultured medium (C), migration ability (D), and invasion ability (E) were measured by qRT-PCR, ELISA and transwell assays in OEC-M1 cells. All data are presented as mean ± SD; **, p < 0.01; ***, p < 0.001 versus scramble control (NC). GAPDH was used as protein loading control. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH.

Article Snippet: The membranes were probed with specific antibodies against CISH (#8731, Cell signaling, Danvers, MA), STAT3 (#610189, BD Biosciences, NJ), phospho-STAT3 (p-STAT3, Tyr 705 ) (#9131, Cell signaling), STAT5 (#25656, Cell signaling), phospho-STAT3 (p-STAT5, Tyr 694 ) (#9359, Cell signaling), COX-2 (#12282, Cell signaling), and iNOS (ab204017, Abcam, Cambridge, MA).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Cell Culture, Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: MiR-944/CISH mediated inflammation via STAT3 is involved in oral cancer malignance by cigarette smoking

doi: 10.1016/j.neo.2020.08.005

Figure Lengend Snippet: NNK exposure induces miR-944 expression. (A) OEC-M1 and SCC-25 cells were treated with NNK (10–40 µM) for 72 h, and the miR-944 expression level was measured by qRT-PCR analysis. (B) OEC-M1 and SCC-25 cells were treated with 20 µM of NNK for indicated time (24–72 h), and the miR-944 expression level was measured by qRT-PCR analysis. (C) Luciferase activity assays were performed after OEC-M1 and SCC-25 cells transfected with luciferase vectors containing the wide-type or mutant CISH 3′-UTR, in response to DMSO control or 20 µM of NNK treatment. The relative luciferase activity of each sample is measured at 72 h after transfection and normalized to Renilla luciferase activity. OEC-M1 and SCC-25 cells were transfected with miR-944 inhibitors (AM) for 12 h, and followed by 20 µM of NNK treatment for another 72 h. Under this condition, the expression level of miR-944 was determined by qRT-PCR analysis (D), and the protein level of CISH, STAT3, phosphor-STAT3 and GAPDH were determined by western blot analysis (E). All data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Numerical values for protein band intensities are shown below the gels. The values were quantitated by densitometry and normalized to GAPDH.

Article Snippet: The membranes were probed with specific antibodies against CISH (#8731, Cell signaling, Danvers, MA), STAT3 (#610189, BD Biosciences, NJ), phospho-STAT3 (p-STAT3, Tyr 705 ) (#9131, Cell signaling), STAT5 (#25656, Cell signaling), phospho-STAT3 (p-STAT5, Tyr 694 ) (#9359, Cell signaling), COX-2 (#12282, Cell signaling), and iNOS (ab204017, Abcam, Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Mutagenesis, Control, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: MiR-944/CISH mediated inflammation via STAT3 is involved in oral cancer malignance by cigarette smoking

doi: 10.1016/j.neo.2020.08.005

Figure Lengend Snippet: The effects of NNK on the STAT3-mediated pro-inflammation genes. OEC-M1 and SCC-25 cells were transfected with miR-944 inhibitors (AM) for 12 h, and followed by 20 µM of NNK treatment for another 72 h. The RNA level of COX-2 and iNOS in cells (A), protein level of CCL3, CCL5 and IL-1β in cultured medium (B) were measured by qRT-PCR or ELISA. (C) Migration and invasion ability were measured by transwell assays in OEC-M1 and SCC-25 cells. (D) OEC-M1 and SCC-25 cells were treated with 20 µM of NNK for 72 h, and the TP63 expression level was measured by qRT-PCR analysis. (E) Correlation analysis of miR-944 and TP63 in human OSCC patients (n = 33) by qRT-PCR analysis. (F) Proposed model for comprehensive NNK-induced upregulation of miR-944 in stimulating STAT3 activation and pro-inflammatory genes expression through suppression of CISH. All data are represented as mean ± SD; **, p < 0.01; ***, p < 0.001.

Article Snippet: The membranes were probed with specific antibodies against CISH (#8731, Cell signaling, Danvers, MA), STAT3 (#610189, BD Biosciences, NJ), phospho-STAT3 (p-STAT3, Tyr 705 ) (#9131, Cell signaling), STAT5 (#25656, Cell signaling), phospho-STAT3 (p-STAT5, Tyr 694 ) (#9359, Cell signaling), COX-2 (#12282, Cell signaling), and iNOS (ab204017, Abcam, Cambridge, MA).

Techniques: Transfection, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Expressing, Activation Assay