Unraveling the impact of AXIN1 mutations on HCC development: Insights from CRISPR/Cas9 repaired AXIN1-mutant liver cancer cell lines.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. METHODS: In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. RESULTS: For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced ß-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored ß-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive ß-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. CONCLUSIONS: Our study provides insights into the effects of repairing AXIN1 mutations on ß-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.

Application of bacteriophage φPaP11-13 attenuates rat Cutibacterium acnes infection lesions by promoting keratinocytes apoptosis via inhibiting PI3K/Akt pathway.

Acne vulgaris caused by antibiotic-resistant Cutibacterium acnes (C. acnes) infection is difficult to treat conventionally. Phages have been suggested as a potential solution, but research on the mechanism of phage treatment is inadequate. This research investigates the underlying molecular mechanisms of phage φPaP11-13 attenuating C. acnes-induced inflammation in rat models. We found that rats infected with C. acnes had higher average ear thickness, greater enrichment of inflammatory cells as shown by hematoxylin-eosin (HE) staining, and fewer TUNEL (TdT-mediated dUTP Nick-End Labeling)-positive keratinocytes visualized by IF staining. Moreover, an increase of IGF-1 and IGF-1 receptor (IGF-1r) was detected using the immunohistochemical (IHC) staining method, Western blot (WB), and quantitative real-time PCR (qRT-PCR) when infected with C. acnes, which was decreased after the application of phage φPaP11-13. By applying the IGF-1 antibody, it was demonstrated that the severity of C. acnes-induced inflammation was relevant to the expression of IGF-1. Through WB and qRT-PCR, activation of the PI3K/Akt pathway and a down-regulation of the BAD-mediated apoptosis pathway were discovered after C. acnes infection. Subsequently, it was shown that the activation of the PI3K/Akt pathway against BAD-mediated apoptosis pathway was alleviated after applying phage φPaP11-13. Furthermore, applying the IGF-1r inhibitor, Pan-PI3K inhibitor, and Akt inhibitor reversed the changing trends of BAD induced by C. acnes and phage φPaP11-13. This study demonstrates that one of the critical mechanisms underlying the attenuation of acne vulgaris by phage φPaP11-13 is lysing C. acnes and regulating keratinocyte apoptosis via the PI3K/Akt signaling pathway.IMPORTANCECutibacterium acnes infection-induced acne vulgaris may cause severe physical and psychological prognosis. However, the overuse of antibiotics develops drug resistance, bringing challenges in treating Cutibacterium acnes. Bacteriophages are currently proven effective in MDR (multiple drug-resistant) Cutibacterium acnes, but there is a significant lack of understanding of phage therapy. This study demonstrated a novel way of curing acne vulgaris by using phages through promoting cell death of excessive keratinocytes in acne lesions by lysing Cutibacterium acnes. However, the regulation of this cell cycle has not been proven to be directly mediated by phages. The hint of ternary relation among "phage-bacteria-host" inspires huge interest in future phage therapy studies.