Jingjing Liu

Escherichia coli Nissle 1917 (EcN) is widely used as a probiotic and engineering chassis, but its carriage of the pks genomic island raises safety concerns related to the genotoxin colibactin. Here, we constructed a clbD deletion mutant (EcNΔ clbD ) and a chromosomally complemented strain (EcNΔ clbD :: clbD ) using a CRISPR/Cas9-based genome-editing strategy. Locus-specific PCR confirmed the expected clbD deletion and restoration patterns. DNA damage responses were evaluated by γH2AX immunofluorescence and alkaline comet assay after bacterial co-culture. Compared with EcN, EcNΔ clbD markedly reduced γH2AX positivity and comet-positive cells, whereas clbD complementation restored both phenotypes to levels comparable to EcN. In NCM460 colonic epithelial cells, EcNΔ clbD induced lower mRNA expression of pro-inflammatory cytokines and TLR4–MYD88–NFKB1-related transcripts than EcN. Additional CCK-8 and cell-associated bacterial load assays showed no significant differences between EcN- and EcNΔ clbD -treated groups, suggesting that the reduced inflammatory transcription was not primarily attributable to altered host cell CCK-8 readout or bacterial exposure. qPCR analysis of neighboring pks genes showed detectable local transcriptional changes after clbD deletion that were restored toward the parental EcN pattern by complementation. Transmission electron microscopy revealed no detectable morphological or ultrastructural disruption after clbD deletion. Exploratory untargeted metabolomics suggested a metabolic shift associated with clbD deletion, while gastrointestinal stress assays revealed condition-dependent fitness effects without a generalized growth defect in LB medium. Together, these findings indicate that clbD deletion attenuates EcN-induced DNA damage responses and epithelial inflammatory transcription in vitro . EcNΔ clbD may represent a candidate safety-optimized EcN chassis requiring further validation in additional epithelial models and in vivo systems.

Pancreatic cancer cell metastasis is a major factor influencing prognosis. A Ras homologue member I (ARHI) was reported to regulate proliferation and apoptosis in pancreatic cancer; however, its role in invasion remains unclear. This study aimed to explore the role and related mechanisms of ARHI in pancreatic cancer metastasis. We revealed that in pancreatic cancer ARHI expression levels were consistent with aggressive cellular phenotypes, and that changes in endogenous ARHI protein expression led to corresponding alterations in epithelial-mesenchymal transition (EMT) markers. Furthermore, ARHI accelerated tumor invasion in pancreatic cancer cells and in a mouse hepatic metastasis model. Notably, this unusual promoting effect of ARHI on EMT and invasion in pancreatic cancer was primarily exerted through the Notch-1 signaling pathway. Collectively, our findings provide insight into the function and molecular mechanisms of ARHI in pancreatic cancer metastasis.

Gastric cancer (GC) is a common malignant disease worldwide. Aberrant miRNAs expression contributes to malignant cells behaviour, and in preclinical research, miRNA targeting has shown potential for improving GC therapy. Our present study demonstrated that miR-632 promotes GC progression in a trefoil factor 1 (TFF1)-dependent manner. We collected GC tissues and serum samples to detect miR-632 expression using real-time PCR. A dual-luciferase reporter assay was used to identify whether miR-632 directly regulates TFF1 expression. Tube formation and endothelial cell recruitment assays were performed with or without miR-632 treatment. Western blot and in situ hybridization assays were performed to detect angiogenesis and endothelial recruitment markers that are affected by miR-632. Our results showed that miR-632 is highly expressed in GC tissue and serum and negatively associated with TFF1 in GC. miR-632 improves tube formation and endothelial cell recruitment by negatively regulating TFF1 in GC cells. Recombinant TFF1 reversed miR-632-mediated angiogenesis. TFF1 is a target gene of miR-632. Our study demonstrated that miR-632 promotes GC progression by accelerating angiogenesis in a TFF1-dependent manner. Targeting of miR-632 may be a potential therapeutic approach for GC patients.