Functional Characterization of ZFC3H1 and DYNLT3 in A375 Melanoma Cells Using CRISPR Knockout 2266763

ZNF695 Promotes Colorectal Cancer Progression Through Transcriptional Activation of CBX8 and Subsequent Wnt/β-Catenin Signaling Activation.

In this investigation, we examined the functional mechanism of the transcription factor zinc finger protein 695 (ZNF695) and its target gene chromobox protein homolog 8 (CBX8) in colorectal cancer (CRC) migration and invasion. HCT-116 and LOVO cell lines were used to establish cell models with knocked-down ZNF695 and knocked-down or over-expressed CBX8. To comprehensively evaluate the functional contributions of ZNF695 and CBX8 to cellular phenotypes, we employed CCK-8, wound-healing, and Transwell assays to evaluate cell proliferation, migration, and invasion, respectively. To assess the impact of ZNF695 on tumor progression, we generated a xenograft model utilizing nude mice. A FLAG-ZNF695 expression plasmid was constructed, and ChIP-seq experiments were performed. By integrating mRNA sequencing data following ZNF695 knockdown with highly expressed genes in CRC from the TCGA database, CBX8 was identified as a putative downstream target of ZNF695. We employed a dual-luciferase reporter assay to validate the specific binding affinity of ZNF695 toward the CBX8 promoter region. To elucidate the specific biological cascades modulated by ZNF695 and CBX8, we conducted a comprehensive pathway enrichment analysis. Rescue experiments were conducted to determine whether the ZNF695/CBX8 regulatory axis upregulates the expression of the Wnt signaling pathway downstream targets, AXIN2 and CCND1. Both in vitro assays and in vivo models confirmed that silencing ZNF695 dramatically suppresses CRC cell proliferation, migration, and invasion, while concurrently impeding tumor progression. ChIP-seq coupled with dual-luciferase reporter assays substantiated the direct binding of ZNF695 to the CBX8 promoter. Furthermore, CBX8 depletion significantly attenuated the migratory and invasive phenotypes of CRC cells. Restoring CBX8 expression effectively rescued the migratory and invasive deficits in CRC cells induced by ZNF695 silencing. Re-expression of CBX8 in ZNF695-silenced cells restored Wnt/β-catenin signaling activity, accompanied by increased expression of AXIN2 and CCND1. ZNF695 promotes CRC progression by transcriptionally activating CBX8 and subsequently enhancing Wnt/β-catenin signaling, thereby promoting tumor cell proliferation, migration, and invasion.

CRISPR/Cas9-Mediated Knockout of ZFP36L1 Impairs Cell Proliferation, Alters Cell-Cycle Progression, and Enhances DNA Damage Responses in MDA-MB-231 Triple-Negative Breast Cancer Cells

Background Zinc finger protein 36-like 1 (ZFP36L1) is an AU-rich element-binding RNA-binding protein that regulates post-transcriptional gene expression and has been implicated in tumor progression, cell-cycle regulation, and DNA damage responses. However, its functional role in triple-negative breast cancer (TNBC) remains poorly understood. This study investigated the effects of CRISPR/Cas9-mediated ZFP36L1 knockout on cell proliferation, doxorubicin (DOX) sensitivity, cell-cycle progression, and DNA damage responses in MDA-MB-231 TNBC cells. Methods Wild-type (WT) and CRISPR/Cas9-generated ZFP36L1 knockout (KO) MDA-MB-231 cells were cultured under standard conditions. Cellular proliferation was evaluated by cell counting over three weeks. Cell viability following DOX treatment was determined using the MTT assay, and half-maximal inhibitory concentration (IC50) values were calculated. Cell-cycle distribution was assessed by propidium iodide flow cytometry after 24 h of DOX exposure, while DNA damage was quantified by γ-H2AX flow cytometric analysis. Statistical significance was determined using Student’s t-test with P < 0.05 considered significant. Results ZFP36L1 knockout reduced the proliferative capacity of MDA-MB-231 cells compared with WT cells. Both cell lines exhibited dose-dependent decreases in viability following DOX treatment. KO cells demonstrated a higher mean IC50 than WT cells (9.64 vs. 8.40 μM), indicating a trend toward reduced DOX sensitivity; however, this difference was not statistically significant (P = 0.569). Flow cytometric analysis revealed enhanced accumulation of KO cells in the S and G2/M phases following DOX treatment, suggesting altered cell-cycle checkpoint regulation. Furthermore, KO cells exhibited elevated basal γ-H2AX expression and greater DOX-induced γ-H2AX accumulation than WT cells, indicating increased DNA damage and impaired maintenance of genomic stability. Conclusions CRISPR/Cas9-mediated loss of ZFP36L1 suppresses proliferation, alters cell-cycle checkpoint dynamics, and enhances DNA damage accumulation in MDA-MB-231 TNBC cells. These findings indicate that ZFP36L1 plays a context-dependent role in regulating genomic stability and cellular responses to genotoxic stress, highlighting its potential as a biomarker and therapeutic target in triple-negative breast cancer.

HOXC9 transcriptionally activates NRP1 to promote cell malignant progression and immune evasion in colorectal cancer

NOP2-regulated m5C Methylation of FKBP4 in Promoting Lung Adenocarcinoma Progression.

BACKGROUND Nucleolar protein 2 (NOP2), an RNA methyltransferase that mediates m5C modification, has been implicated in cancer progression; however, its specific role in lung adenocarcinoma (LUAD) remains unclear. This study investigated the functional significance of NOP2 and its regulation of FK506-binding protein 4 (FKBP4) in LUAD pathogenesis. MATERIALS AND METHODS The human normal alveolar epithelial cell line HPAEpiC and the LUAD cell lines A549 and H1975 were utilized in this study. In vivo experiments were conducted using male Sprague-Dawley rats. The functional roles of NOP2 and FKBP4 were explored by transfecting LUAD cells with overexpression vectors or small interfering RNAs (siRNAs). Quantitative real-time PCR, western blotting, immunofluorescence, and immunohistochemical staining analyses were used to evaluate NOP2 and FKBP4 expression in cells and tissues. Cell viability and proliferation were assessed using Cell Counting Kit-8 assays and 5-ethynyl-2'-deoxyuridine (EdU) staining. Apoptosis was measured by flow cytometry and TUNEL staining. Transwell assays were conducted to determine cell migration and invasion. Co-immunoprecipitation and RNA pulldown assays were conducted to characterize the interaction between NOP2 and FKBP4. RESULTS Analysis of data from The Cancer Genome Atlas (TCGA) database revealed that elevated expression of NOP2 and FKBP4 correlated with poor prognosis in patients with LUAD. Both NOP2 and FKBP4 were significantly upregulated in LUAD cell lines compared to normal controls. Knockdown of NOP2 could inhibit LUAD cell proliferation, migration, and invasion, and enhance apoptosis. In vivo experiments confirmed that NOP2 depletion inhibited tumor growth and enhanced apoptosis. Mechanistically, NOP2 could directly bind to FKBP4 and regulate its expression. Importantly, FKBP4 knockdown reversed the oncogenic effects of NOP2 expression, establishing FKBP4 as a critical downstream effector of NOP2 in LUAD. CONCLUSION Our study demonstrates that NOP2 promotes LUAD progression by regulating FKBP4 expression, potentially through m5C methylation, thereby highlighting the NOP2/FKBP4 axis as a potential therapeutic target. Overall, these findings provide new insights into the epigenetic mechanisms driving LUAD aggressiveness.

HuR—SerpinB9 Axis Regulates Tumor T-Cell Fitness and Persistence via Post-Transcriptional Control 2267776

Findings highlight a HuR-SerpinB9 axis that regulates T-cell senescence and persistence, offering a potential therapeutic target in cancer and autoimmune diseases.

NTMT1 (METTL11A) promotes MYC-dependent proliferation and downstream HLA-A suppression in cervical cancer

Findings identify NTMT1 as a candidate regulatory node associated with MYC activation and downstream HLA-A suppression, warranting further mechanistic and in-vivo investigation, and highlighting NTMT1 as a promising candidate for future therapeutic investigation.