Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: TPX2 is a direct target of miR-1294 in glioma cell lines. A. Predicted miR-1294 target sequence in the 3’-UTR of TPX2 mRNA. B. miR-1294 down-regulated the luciferase activity of the wild-type TPX2 3’UTR expression vector but did not reduce the expression of mutant TPX2. C. Expression of TPX2 in the CGGA public database. D. Expression of TPX2 in NBTs (n=5) and glioma specimens divided into LGG (n=8) and HGG (n=8). E. Expression of TPX2 in normal human astrocytes (NHAs) and four glioma cell lines (U87, U251, LN229, A172). F. Expression of TPX2 in U87 and U251 cells transfected with NC, miR-1294 mimic or miR-1294 inhibitor. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Sequencing, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Mutagenesis, Transfection

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: Down-regulation of TPX2 suppresses glioma cell proliferation, migration, and invasion and enhances chemosensitivity to temozolomide in vitro. A. qRT-PCR analysis of TPX2 expression in U87 and U251 cells transfected with NC or si-TPX2. B, C. The CCK-8 assay for U87 and U251 cells transfected with NC or si-TPX2. D. The migration assay for U87 and U251 cells transfected with NC or si-TPX2. E. The Transwell for U87 and U251 cells transfected with NC or si-TPX2. F, G. Cell viability was examined for U87 and U251 cells transfected with NC or si-TPX2 following TMZ treatments at various doses. H, I. Cell viability was examined every 24 hours in U87 and U251 cells transfected with NC or si-TPX2 following TMZ treatments at the indicated concentrations. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Migration, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: TPX2 overexpression reverses the suppressive effects of miR-1294 on glioma cells in vitro. A. qRT-PCR analysis of TPX2 expression in U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. B, C. The CCK-8 assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. D. The migration assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. E. The Transwell assay for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2. F, G. Cell viability was examined for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2 following TMZ treatments at various doses. H, I. Cell viability was examined every 24 hours for U87 and U251 cells transfected with NC, miR-1294 or miR-1294 together with TPX2 following TMZ treatments at the indicated concentrations. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Migration, Transwell Assay

Journal: American Journal of Cancer Research

Article Title: MicroRNA-1294 inhibits the proliferation and enhances the chemosensitivity of glioma to temozolomide via the direct targeting of TPX2

doi:

Figure Lengend Snippet: miR-1294 inhibits glioma growth in vivo. A. Tumor formation was assessed in nude mice, and the tumors of U87 xenografts were excised. B. The volume of excised tumors according to the formula: V (mm3)=0.5 * a* b2 (a represents the longest axis and b the shortest axis). C. The weight of excised tumors. D, E. Expression of TPX2 in tumor tissue. *P<0.05, **P<0.01.

Article Snippet: After incubation with specific primary antibodies against TPX2 (1:1000, Abnova, China) overnight at 4°C, membranes were further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies.

Techniques: In Vivo, Expressing

Journal: eBioMedicine

Article Title: Somatic structural variants drive upper tract urothelial carcinoma muscle invasiveness via activation of TPX2 transcription

doi: 10.1016/j.ebiom.2026.106182

Figure Lengend Snippet: SVs disrupt gene expression in UTUC. A. Variant Manhattan plot showing genes whose expression is significantly correlated with the proximity of SSV breakpoints. The upper panel displays the number of samples with SSV breakpoints near each gene (y-axis) across the genome (x-axis). Labelled genes indicate significant SSV-expression associations. The lower panel presents genome-wide CNV G-scores for comparison. B. Bar plots quantifying the number of genes with SSV-associated expression changes, before and after adjusting for CNV effects. The reduction in significant associations after adjustment highlights SSVs with independent effects on gene expression. C. Pie charts illustrating the distribution of SSV types among expression-altering breakpoints (left), and the genomic distribution of DEL breakpoints (right), emphasising the prevalence of intronic deletions in gene expression regulation. D. Pie chart showing the proportion of expression-altering SSV breakpoints that are associated with ecDNA. E. Bar plot showing 14 genes with significantly higher SSV frequency in MI-UTUC than in NMI-UTUC (Fisher's exact test). The number of samples with SSVs in each gene is shown for both MI and NMI groups. F. Upper panel: Receiver operating characteristic curves evaluating the predictive performance of TPX2 SSV status for UTUC. AUC values and corresponding p-values are indicated. Lower panel: Box plots showing expression levels of TPX2 in patients with and without SSVs. G. Expression levels of TPX2 in MI and NMI groups across UTUC transcriptomic datasets from published data (BCM, MAD, WCM). H. Representative images of IHC in UTUC samples with TPX2 related SSV (left panel), without TPX2 SSVs (middel panel). Scale bar, 500 μm (left), 100 μm (right). Right panel: A semi-quantitative modified IHC score was calculated in IHC of TPX2 as staining intensity (0–3) × staining extent (0–3), yielding a score range of 0–9. Each dot represents one case; boxes indicate the median and interquartile range.

Article Snippet: For immunohistochemistry, TPX2 was detected using a rabbit polyclonal anti-TPX2 antibody (HUABIO, ER65201; RRID Tags AB_3731329), generated against a synthesised peptide derived from human TPX2 (aa 301–350), and recommended for IHC-P (1:100–1:300).

Techniques: Gene Expression, Variant Assay, Expressing, Genome Wide, Comparison, Modification, Staining

Journal: eBioMedicine

Article Title: Somatic structural variants drive upper tract urothelial carcinoma muscle invasiveness via activation of TPX2 transcription

doi: 10.1016/j.ebiom.2026.106182

Figure Lengend Snippet: An upstream structural variant interval enhances TPX2 expression and promotes invasive phenotypes in urothelial cancer cells. A. Schematic representation of somatic structural variants (SSVs) surrounding the TPX2 locus. A recurrent duplication event localised to the chr20:31000852–31001508 interval upstream of TPX2 was identified. This region contains eight tandem repeats of a 171-bp sequence. Two unique sgRNAs were designed to delete a 683-bp fragment spanning this interval. B. PCR and gel electrophoresis validation of CRISPR ribonucleoprotein (RNP)-mediated deletion of the TPX2 upstream interval in 5637 cells. The main band corresponding to the intact region (1025 bp) was markedly attenuated in TPX2-upstream knockout cells. (C, D). Quantitative RT-PCR (C) and Western blot (D) analyses showing that deletion of the chr20:31000852–31001508 interval significantly reduced TPX2 mRNA and protein expression in 5637 cells. (E, F) Quantitative RT-PCR (E) and Western blot (F) analyses demonstrating that transfection of a PCR-derived fragment corresponding to the TPX2 upstream interval increased TPX2 expression compared with control cells. G. Schematic of the dual-luciferase reporter construct in which the TPX2 promoter region (2331 bp, including exon 1) was cloned upstream of the Renilla luciferase (Rluc) gene in the psiCHECK-2 vector. H. Dual-luciferase reporter assay showing that co-transfection of the TPX2 promoter reporter with PCR-derived fragments from the chr20:31000852–31001508 interval significantly increased Rluc activity compared with co-transfection with a random 1-kb DNA fragment, indicating enhancer-like activity of this interval. (I, J) Western blot validation of TPX2 knockout (KO) (I) and TPX2 overexpression (OE) (J) in 5637 cells. (K, L) Wound-healing (K) and transwell migration and invasion assays (L) demonstrating that TPX2 knockout significantly impaired cell motility and invasive capacity. Scale bar = 1 mm in K, scale bar = 200 um in L. (M, N) Wound-healing (M) and transwell migration and invasion assays (N) showing that TPX2 overexpression markedly enhanced migratory and invasive properties of 5637 cells. Scale bar = 1 mm in M, scale bar = 200 um in N. Data are presented as mean ±SD (n = 3). Two-sided Mann-Whitney U test was used for comparisons between two groups. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). O. KEGG pathway enrichment analysis of genes downregulated upon TPX2 knockout (TPX2_KO vs. NC_KO). P. Gene set enrichment analysis (GSEA) of transcriptomic changes induced by amplification of the TPX2 upstream interval (Ups_OE vs. NC_OE). Pathways related to EMT, cell adhesion, and extracellular matrix remodelling were significantly enriched.

Article Snippet: For immunohistochemistry, TPX2 was detected using a rabbit polyclonal anti-TPX2 antibody (HUABIO, ER65201; RRID Tags AB_3731329), generated against a synthesised peptide derived from human TPX2 (aa 301–350), and recommended for IHC-P (1:100–1:300).

Techniques: Variant Assay, Expressing, Sequencing, Nucleic Acid Electrophoresis, Biomarker Discovery, CRISPR, Knock-Out, Quantitative RT-PCR, Western Blot, Transfection, Derivative Assay, Control, Luciferase, Construct, Clone Assay, Plasmid Preparation, Reporter Assay, Cotransfection, Activity Assay, Over Expression, Migration, MANN-WHITNEY, Amplification

Journal: eBioMedicine

Article Title: Somatic structural variants drive upper tract urothelial carcinoma muscle invasiveness via activation of TPX2 transcription

doi: 10.1016/j.ebiom.2026.106182

Figure Lengend Snippet: scRNA-seq reveals TPX2 e xpression in proliferative tumour cell populations and associated pathways. A. UMAP visualisation of single-cell transcriptomic data from UTUC, identifying 20 distinct cell clusters. B. UMAP plot highlighting TPX2 expression, showing enrichment in cycling T cells, cycling epithelial cells, and cycling macrophages. Colour intensity corresponds to TPX2 expression levels. C. Dot plot displaying the percentage of cells expressing TPX2 (dot size) and the average TPX2 expression level (dot colour) across different cell types in patients with MI-UTUC and NMI-UTUC. Asterisks indicate statistically significant differences in expression between MI and NMI for specific cell types (two-sided Wilcoxon rank-sum test, Bonferroni-adjusted P values, ∗P < 0.05, ∗∗∗∗P < 0.001). D. KEGG pathway enrichment analysis of genes significantly upregulated in TPX2 -positive cycling epithelial cells compared with TPX2 -negative counterparts. Dot size represents the number of genes in each pathway; colour indicates the adjusted p-value of enrichment. E. Cell–cell communication heatmap showing the number of predicted ligand-receptor interactions among major cell types in the UTUC tumour microenvironment. Cell types are arranged on both axes, with senders (ligand-expressing cells) on the y-axis and receivers (receptor-expressing cells) on the x-axis. The intensity of red reflects the total number of interactions between each cell pair, as indicated by the colour scale. (F, G) Outgoing (F) and incoming (G) signalling patterns among cell types in the UTUC tumour microenvironment. Heatmaps show the relative strength of ligand-receptor signalling pathways based on CellChat analysis. Colour intensity indicates relative communication strength; bar plots on the top and right summarise overall pathway activity across cell types.

Article Snippet: For immunohistochemistry, TPX2 was detected using a rabbit polyclonal anti-TPX2 antibody (HUABIO, ER65201; RRID Tags AB_3731329), generated against a synthesised peptide derived from human TPX2 (aa 301–350), and recommended for IHC-P (1:100–1:300).

Techniques: Single Cell, Expressing, Activity Assay

Journal: eBioMedicine

Article Title: Somatic structural variants drive upper tract urothelial carcinoma muscle invasiveness via activation of TPX2 transcription

doi: 10.1016/j.ebiom.2026.106182

Figure Lengend Snippet: Spatial transcriptomic delineation of TPX2 positive epithelial cells and their tumour microenvironmental interactions in UTUC. A. Representative haematoxylin and eosin (H&E)-stained sections (left) and corresponding spatial transcriptomic cell-type annotations (right) from MI (upper) and NMI (lower)-UTUC biopsy, scale bar = 1 mm. Spatial spots were annotated by integrating spatial transcriptomics with single-cell reference datasets, revealing the spatial organisation of epithelial tumour populations and diverse TME components. B. Box plots showing TPX2 expression levels across major spatially defined cell types in MI-UTUC biopsy. TPX2 expression was significantly enriched in basal epithelial tumour cells and specific urothelial tumour subpopulations compared with immune and stromal compartments (∗∗∗∗, P < 0.0001; Wilcoxon rank-sum test). C. Bar plot comparing the proportion of TPX2 positive spots across spatial cell types between MI- and NMI-UTUC samples. TPX2 positive epithelial tumour populations were markedly enriched in MI-UTUC relative to NMI-UTUC. D. Box plots showing average epithelial mesenchymal transition (EMT) signature scores in spatial neighbours of TPX2 positive versus TPX2 negative epithelial tumour cells. Neighbourhoods surrounding TPX2 positive cells exhibited significantly higher EMT associated transcriptional activity (P = 3.33 × 10 −3 ; Wilcoxon rank-sum test), indicating localisation of TPX2 positive tumour cells within invasion-promoting microenvironments. E. Bar plot showing log2 fold changes of EGFR- and EPHA2-associated ligand expression in spatial neighbours of TPX2 positive compared with TPX2 negative epithelial tumour cells.

Article Snippet: For immunohistochemistry, TPX2 was detected using a rabbit polyclonal anti-TPX2 antibody (HUABIO, ER65201; RRID Tags AB_3731329), generated against a synthesised peptide derived from human TPX2 (aa 301–350), and recommended for IHC-P (1:100–1:300).

Techniques: Staining, Spatial Transcriptomics, Single Cell, Expressing, Activity Assay