Background: IGF2BP3 functions as an RNA-binding protein (RBP) and plays a role in the posttranscriptional control of mRNA localization, stability, and translation. Its dysregulation is frequently associated with tumorigenesis across various cancer types. Nonetheless, our understanding of how the expression of the IGF2BP3 gene is regulated remains limited. The specific functions and underlying mechanisms of IGF2BP3, as well as the potential benefits of targeting it for therapeutic purposes in bladder cancer, are not yet well comprehended. Methods: The mRNA and protein expression were examined by RT-qPCR and western blotting, respectively. The methylation level of CpG sites was detected by Bisulfite sequencing PCR (BSP). The regulation of IGF2BP3 expression by miR-320a-3p was analyzed by luciferase reporter assay. The functional role of IGF2BP3 was determined through proliferation, colony formation, wound healing, invasion assays, and xenograft mouse model. The regulation of HMGB1 by IGF2BP3 was investigated by RNA immunoprecipitation (RIP) and mRNA stability assays. Results: We observed a significant elevation in IGF2BP3 levels within bladder cancer samples, correlating with more advanced stages and grades, as well as an unfavorable prognosis. Subsequent investigations revealed that the upregulation of IGF2BP3 expression is triggered by copy number gain/amplification and promoter hypomethylation in various tumor types, including bladder cancer. Furthermore, miR-320a-3p was identified as another negative regulator in bladder cancer. Functionally, the upregulation of IGF2BP3 expression exacerbated bladder cancer progression, including the proliferation, migration, and invasion of bladder cancer. Conversely, IGF2BP3 silencing produced the opposite effects. Moreover, IGF2BP3 expression positively correlated with inflammation and immune infiltration in bladder cancer. Mechanistically, IGF2BP3 enhanced mRNA stability and promoted the expression of HMGB1 by binding to its mRNA, which is a factor that promotes inflammation and orchestrates tumorigenesis in many cancers. Importantly, pharmacological inhibition of HMGB1 with glycyrrhizin, a specific HMGB1 inhibitor, effectively reversed the cancer-promoting effects of IGF2BP3 overexpression in bladder cancer. Furthermore, the relationship between HMGB1 mRNA and IGF2PB3 is also observed in mammalian embryonic development, with the expression of both genes gradually decreasing as embryonic development progresses. Conclusions: Our present study sheds light on the genetic and epigenetic mechanisms governing IGF2BP3 expression, underscoring the critical involvement of the IGF2BP3-HMGB1 axis in driving bladder cancer progression. Additionally, it advocates for the investigation of inhibiting IGF2BP3-HMGB1 as a viable therapeutic approach for treating bladder cancer. Graphical

The repair of DNA double-strand breaks (DSBs) through alternative non-homologous end-joining (alt-NHEJ) pathway significantly contributes to genetic instability. However, the mechanism governing alt-NHEJ pathway choice, particularly its association with DSB complexity, remains elusive due to the absence of a suitable reporter system. In this study, we established a unique Escherichia coli reporter system for detecting complex DSB-initiated alternative end-joining (A-EJ), an alt-NHEJ-like pathway. By utilizing various types of ionizing radiation to generate DSBs with varying degrees of complexity, we discovered that high complexity of DSBs might be a determinant for A-EJ choice. To facilitate efficient repair of high-complexity DSBs, A-EJ employs distinct molecular patterns such as longer micro-homologous junctions and non-templated nucleotide addition. Furthermore, the A-EJ choice is modulated by the degree of homology near DSB loci, competing with homologous recombination machinery. These findings further enhance the understanding of A-EJ/alt-NHEJ pathway choice.

Esophageal cancer (ESCA) is the seventh most frequent and deadly neoplasm. Due to the lack of early diagnosis and high invasion/metastasis, the prognosis of ESCA remains very poor. Herein, we identify skin-related signatures as the most deficient signatures in invasive ESCA, which are regulated by the transcription factor ZNF750. Of note, we find that TRIM29 level strongly correlated with the expression of many genes in the skin-related signatures, including ZNF750. TRIM29 is significantly down-regulated due to hypermethylation of its promoter in both ESCA and precancerous lesions compared to normal tissues. Low TRIM29 expression and high methylation levels of its promoter are associated with malignant progression and poor clinical outcomes in ESCA patients. Functionally, TRIM29 overexpression markedly hinders proliferation, migration, invasion, and epithelial–mesenchymal transition of esophageal cancer cells, whereas opposing results are observed when TRIM29 is silenced in vitro. In addition, TRIM29 inhibits metastasis in vivo. Mechanistically, TRIM29 downregulation suppresses the expression of the tumor suppressor ZNF750 by activating the STAT3 signaling pathway. Overall, our study demonstrates that TRIM29 expression and its promoter methylation status could be potential early diagnostic and prognostic markers. It highlights the role of the TRIM29-ZNF750 signaling axis in modulating tumorigenesis and metastasis of esophageal cancer.

Background: ACAP1 plays a key role in endocytic recycling, which is essential for the normal function of lymphocytes. However, the expression and function of ACAP1 in lymphocytes have rarely been studied. Methods: Large-scale genomic data, including multiple bulk RNA-sequencing datasets, single-cell sequencing datasets, and immunotherapy cohorts, were exploited to comprehensively characterize ACAP1 expression, regulation, and function. Gene set enrichment analysis (GSEA) was used to uncover the pathways associated with ACAP1 expression. Eight algorithms, including TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, xCELL, MCPCOUNTER, EPIC, and TIDE, were applied to estimate the infiltrating level of immune cells. Western blotting, qPCR, and ChIP-PCR were used to validate the findings from bioinformatic analyses. A T-cell co-culture killing assay was used to investigate the function of ACAP1 in lymphocytes. Results: ACAP1 was highly expressed in immune-related tissues and cells and minimally in other tissues. Moreover, single-cell sequencing analysis in tumor samples revealed that ACAP1 is expressed primarily in tumor-infiltrating lymphocytes (TILs), including T, B, and NK cells. ACAP1 expression is negatively regulated by promoter DNA methylation, with its promoter hypo-methylated in immune cells but hyper-methylated in other cells. Furthermore, SPI1 binds to the ACAP1 promoter and positively regulates its expression in immune cells. ACAP1 levels positively correlate with the infiltrating levels of TILs, especially CD8+ T cells, across a broad range of solid cancer types. ACAP1 deficiency is associated with poor prognosis and immunotherapeutic response in multiple cancer types treated with checkpoint blockade therapy (ICT). Functionally, the depletion of ACAP1 by RNA interference significantly impairs the T cell-mediated killing of tumor cells. Conclusions: Our study demonstrates that ACAP1 is essential for the normal function of TILs, and its deficiency indicates an immunologically “cold” status of tumors that are resistant to ICT.

With increasing use of medical imaging (e.g., CT scans) and environmental radiation sources, over 2 % of pregnancies worldwide are inadvertently exposed to low-dose ionizing radiation (IR), raising urgent concerns about fetal neuroprotection. While prenatal IR is implicated in microcephaly and lifelong neuropsychiatric risks, prior studies have not resolved whether sirtuin-mediated pathways, particularly SIRT1/TPH2 signaling, drive these deficits or whether dietary phytochemicals like resveratrol can mitigate them. To determine (1) the role of SIRT1/TPH2 signaling in IR-induced neurodevelopmental and psychiatric impairments, and (2) the therapeutic potential of maternal resveratrol supplementation to counteract these effects-a strategy not previously explored in prenatal radiation models. Mouse cohorts received prenatal X-ray irradiation (0, 1.0 Gy, 2 Gy gestational day 8) with/without resveratrol supplementation, followed by longitudinal cortical and behavioral analyses. RNA sequencing/Western blotting quantified SIRT1, TPH2, BDNF, and senescence markers (P16, P21 and SA-β-gal). 5-HT levels were assessed by ELISA. Depression-like behaviors were tested via forced swim and tail suspension. IR-exposed fetuses exhibited progressive microcephaly with reduced cortical thickness, accompanied by SIRT1 downregulation, BDNF suppression, and elevated cellular senescence. Adult offspring displayed depression-like behaviors, linked to TPH2 downregulation and diminished 5-HT levels. Resveratrol supplementation normalized SIRT1/TPH2 signaling, restored cortical neurotrophic factors, and attenuated both microcephaly and depressive phenotypes. This study provides the first evidence that (1) SIRT1/TPH2 signaling is a central mediator of IR-induced neurodevelopmental and psychiatric impairments, and (2) maternal resveratrol supplementation prevents cortical damage and depression in offspring by rescuing this pathway. These findings position resveratrol as a novel, mechanism-driven intervention for fetal neuroprotection against environmental radiation.

The performance of CRISPR‐mediated genome editing near inverted repeats (IRs) potentially results in chromosomal translocations and other catastrophic rearrangements. However, the extent of this risk may be significantly underestimated because current reporter systems focus solely on site‐specific translocations. Here, trans‐acting reporter systems in Escherichia coli are developed to detect nontargeted translocations. Markedly increased frequency of translocations following CRISPR‐Cas9 activation is observed, with the magnitude determined primarily by the length of the IRs and the proximity between Cas9 target sites and IRs. These translocations arise through a combination of intramolecular single‐strand annealing and alternative end‐joining mechanisms. Furthermore, it is discovered that introducing segments homologous to IR loci can substantially mitigate nontargeted translocations without significantly compromising CRISPR‐Cas9‐mediated editing. The study provides valuable insights into the genetic risks associated with CRISPR technologies and suggests a viable strategy for developing genetically safer CRISPR systems.