Journal: Molecular Medicine Reports

Article Title: Characteristics of chromosomal instability-related lncRNAs associated with progression and prognosis in breast cancer

doi: 10.3892/mmr.2026.13834

Figure Lengend Snippet: Upregulation of U62317.4 induces malignant phenotypes in BC. (A) Validation of the expression level of U62317.4 in BC tissues, normal tissues, high- and low-CIN groups, and high- and low-AS groups from TCGA dataset. (B) The expression of U62317.4 in patients with ER + and ER − BC in TCGA dataset. (C) Comparison of U62317.4 expression among BC subtypes from TCGA dataset, including basal-like, HER2-enriched, luminal A and B subtypes, which was conducted using the Kruskal-Wallis test followed by Dunn's post hoc test. (D) Validation of U62317.4 expression levels in BC tissues and paired adjacent normal tissues (n=60). The comparison was conducted using the Wilcoxon signed-rank test. (E) The expression level of U62317.4 in MCF-10A, MCF-7, MDA-MB-231 and SKBR3 cell lines. (F) The efficiency of U62317.4 overexpression and ASO-mediated knockdown was determined by reverse transcription-quantitative PCR analysis. MCF-7 and MDA-MB-231 cells were transfected with U62317.4 pcDNA3.1 overexpression plasmids and ASOs. (G) Representative images and quantification of wound healing assays showing the migration of MCF-7 and MDA-MB-231 cells transfected with U62317.4 ASOs and overexpression plasmids. The area between cell fronts on either side of the wound was measured and calculated as a percentage of the scratch area at 0 h. Scale bar, 100 µm. (H) Western blot assay demonstrated the protein levels of p53, BUB1B and CDC20 in MCF-7 and MDA-MB-231 cells transfected with U62317.4 ASOs and overexpression plasmids, as well as their relevant controls. (I) Ethynyldeoxyuridine staining analysis was performed to measure the viability of MCF-7 and MDA-MB-231 transfected with U62317.4 ASOs and overexpression plasmids, as well as their relevant controls. Scale bar, 100 µm. (J) Matrigel assays were used to determine the invasive abilities of MCF-7 and MDA-MB-231 cells transfected with U62317.4 ASOs and overexpression plasmids, as well as their relevant controls. Comparisons between the results of assays performed in MCF-7 and MDA-MB-231 cells were analyzed by one-way ANOVA, followed by Tukey's post hoc test. Data were represented as the mean ± SD from three independent experiments. Scale bar, 100 µm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. BC, breast cancer; ASO, antisense oligonucleotide; CON, control without transfection treatment; NC, antisense oligonucleotide negative control; TCGA, The Cancer Genome Atlas; ER + , estrogen receptor-positive; ER − , estrogen receptor-negative; BRCA, breast cancer type 1 susceptibility protein; ns, not significant; U62317 .4ov, U62317.4 overexpression plasmids; Vector, overexpression control; CIN, chromosomal instability; AS, aneuploidy score; HER2, tyrosine kinase-type cell surface receptor HER2; BUB1B, mitotic checkpoint serine/threonine-protein kinase BUB1β; CDC20, cell division cycle protein 20.

Article Snippet: Samples on the membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, and then incubated with primary antibodies, including GAPDH (1:1,000; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.), mitotic checkpoint serine/threonine-protein kinase BUB1β (BUB1B; 1:1,000; cat. no. 11504-2-AP; Proteintech Group, Inc.), cell division cycle protein 20 (CDC20; 1:2,000; cat. no. 10252-1-AP; Proteintech Group, Inc.) and p53 (1:5,000; cat. no. 10442-1-AP; Proteintech Group, Inc.), at 4°C overnight.

Techniques: Biomarker Discovery, Expressing, Comparison, Over Expression, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Migration, Western Blot, Staining, Control, Negative Control, Plasmid Preparation, Cell Surface Receptor Assay

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Enhanced CDRs correlated with prostate cancer prognosis. (A) Visualization of core genes' dependency in different prostate cancer cells with CRISPR-Cas9 and siRNA screening. (B, C) Survival analysis of CDRs in different CRPC patient cohorts. (D) The relative expression of CDRs in normal prostate tissues and prostate cancer tissues in the TCGA-PRAD cohort. Gleason score and tumor stage correlation analysis of CDRs in different prostate cancer patient cohorts. All prostate patient cohorts were indicated in the corresponding panels. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: CRISPR, Expressing, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs linked with neuroendocrine features in prostate cancer cohorts. (A) The Venn diagram illustrates the genes specifically enhanced in prostate cancer compared with normal prostate tissues and NEPC compared with adenocarcinoma. (B, C) The relative expression of CDRs in NEPC compared with adenocarcinoma in different prostate cancer cohorts. (D) Correlation analysis of CDRs with NE score. The red indicates the higher NE score. (E, F) Pearson correlation analysis of CDRs with NE score CRPC cohort. (G) The heatmap indicates the relative expression of CDRs and AR, as well as KLK3, a transcription activity indication of AR. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NEPC, neuroendocrine prostate cancer; NE, neuroendocrine; AR, androgen receptor; KLK3, kallikrein-related peptidase 3.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs ablation suppressed prostate cancer cell proliferation. (A) The circuit diagram illustrates the correlation among CDRs co-expressed gene sets. (B) Venn analysis of the overlap of CDRs co-expressed genes. (C) The heatmap shows the relative expression of CDRs overlapped co-expressed genes in patients with NE signature high and low groups. (D – F) The heatmap illustrates the correlation of CDRs co-expressed genes with CDRs in ADPC (D) and CRPC (E, F) patient cohorts. (G, H) KEGG pathway analysis (G) and GSEA analysis (H) of enriched biological processes of CDRs and CDRs co-expressed genes. (I, J) The diagram illustrates the working model of CRISPR-Cas13 for RNA silencing and knockdown efficiency of CDRs with the corresponding gRNAs. (K, L) Cell growth assays indicate the impact of CDRs knockdown on the viability of different prostate cancer cells. (M) Western blotting analysis of the expression of cell cycle-regulated genes, including CCND1, CDK1, and p-CDK1, after CDRs knockdown. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NE, neuroendocrine; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; CCND1, cyclin D1; CDK1, cyclin-dependent kinase 1.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, CRISPR, Knockdown, Western Blot, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs were transcriptionally regulated by the RB1/E2F1 axis in prostate cancer. (A) The diagrams show the canonical E2F1 motif location within the promoter of CDRs. (B) ChIP-sequencing E2F1 enrichment peak within the promoter of CDRs. (C) The relative enrichment of E2F1 within the promoter of CDRs was determined using standard ChIP-qPCR. (D) ChIP-sequencing peaks show the relative enrichment of E2F1 in CDRs' promoters after RB1 is known. (E – G) The relative expression of CDRs in CRPC patients with different RB1 deletion status. (H) CDRs expression in patients with RB1 deletion mutations from different prostate cancer cohorts. (I) qRT-PCR detected the relative expression of CDRs after RB1 or E2F1 knockdown with CRISPR-Cas13. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). RB1, retinoblastoma tumor suppressor 1; E2F1, early 2 factor 1; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: ChIP-sequencing, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, CRISPR, Ubiquitin Proteomics, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Virtual screening identified compounds that suppressed advanced prostate cancer. (A) The diagram illustrates structure-based virtual screening strategies for CDRs-targeted compounds. (B) The heatmap shows the binding affinity of compounds with CDRs. The red indicated a higher binding affinity of compounds with the corresponding compounds. (C, D) Cell viability assays were used to determine the tumor suppressive effect of compounds with high CDRs-binding affinity in different prostate cancer cell models. (E – G) The 2D structure of Q199, XDD60, and A79, which exhibits the most significant anti-tumor efficacy in prostate cancer cell models. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Compounds targeting CDRs exhibited superior anti-tumor efficacy compared with AR antagonists. (A – E) The tumor cell growth inhibition effects of different dosages of Q199, XDD60, and A79, as well as enzalutamide, were determined with CCK-8 assays (A–D), and the IC 50 of each agent was calculated with three independent experiments (E). (F, G) The histograms show the relative cell viability after being treated with 5 M of Q199, XDD60, or A79 alone, or a combination. (H) The Venn diagram shows the overlap of Q199, XDD60, and A79 potential targets predicted with SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Molecular docking shows the binding of CDRs with Q199, XDD60, and A79. (I) The lowest binding (LB) affinity of CDRs with Q199, XDD60, and A79. ns, not significant. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). AR, androgen receptor.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Inhibition, CCK-8 Assay, Binding Assay, Ubiquitin Proteomics