Accumulating evidence indicates that oxidative stress plays a critical role in initiating the progression of inflammatory and fibrotic liver diseases, including cholestatic hepatitis. Peroxiredoxin 4 (PRDX4) is a secretory antioxidase that protects against oxidative damage by scavenging reactive oxygen species (ROS) in both the intracellular compartments and extracellular space. In this study, we examined the in vivo net effects of PRDX4 overexpression in a murine model of cholestasis. To induce cholestatic liver injury, we subjected C57BL/6J wild-type (WT) or human PRDX4 (hPRDX4) transgenic (Tg) mice to sham or bile duct ligation (BDL) surgery for seven days. Our results showed that the liver necrosis area was significantly suppressed in Tg BDL mice with a reduction in the severity of liver injuries. Furthermore, PRDX4 overexpression markedly reduced local and systemic oxidative stress generated by BDL. In addition, suppression of inflammatory cell infiltration, reduced proliferation of hepatocytes and intrahepatic bile ducts, and less fibrosis were also found in the liver of Tg BDL mice, along with a reduced mortality rate after BDL surgery. Interestingly, the composition of the hepatic bile acids (BAs) was more beneficial for Tg BDL mice than for WT BDL mice, suggesting that PRDX4 overexpression may affect BA metabolism during cholestasis. These features indicate that PRDX4 plays an important role in protecting against liver injury following BDL and might be a promising therapeutic modality for cholestatic diseases.

Electroporation is an efficient method for nucleotide and protein transfer, and is used for clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9)-mediated genome editing. In this study, we investigated the effects of electroporation on platelet-derived growth factor receptor alpha (PDGFRA) and receptor tyrosine kinase (RTK) expression in U-251 and U-87 MG cells. PDGFRA mRNA and protein expression decreased 2 days after electroporation in both cell lines, with recovery observed after 13 days in U-87 MG cells. However, in U-251 MG cells, PDGFRα expression remained suppressed, despite mRNA recovery after 13 days. Similar expression profiles were observed for lipofection in the U-251 MG cells. Comprehensive RNA sequencing confirmed electroporation-induced up- and down-regulation of RTK mRNA in U-251 MG cells 2 days post-electroporation. In contrast, recombinant adeno-associated virus (rAAV) transfected with mNeonGreen fluorescent protein or Cas9 did not affect PDGFRA, RTKs, or inflammatory cytokine expression, suggesting fewer adverse effects of rAAV on U-251 MG cells. These findings emphasize the need for adequate recovery periods following electroporation or the adoption of alternative methods, such as rAAV transfection, to ensure the accurate assessment of CRISPR-mediated gene editing outcomes.

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.

Major advances have been made in cancer treatment, but the prognosis for elderly cancer patients with sarcopenia and frailty remains poor. Myokines, which are thought to exert preventive effects against sarcopenia, have been reported to be associated with the prognosis of various cancers, but their effect on head and neck squamous cell carcinoma (HNSCC) is unknown. The aim of this study was to clarify the influence of exercise on the control of HNSCC and to examine the underlying mechanism involved. Mice were injected with HSC-3-M3 cells, a human cell line of highly metastatic and poorly differentiated tongue cancer, at the beginning of the study. Just prior to transplantation, blood was collected from the mice, and the levels of myokines were measured by ELISA. Oncostatin M (OSM), a selected myokine, was added to HSC-3-M3 cells, after which the cell proliferation ability, cell cycle, and protein expression were analyzed in vitro. Tumor cell viability was lower (control: 100%, exercise: 75%), tumors were smaller (control: 26.2 mm³, exercise: 6.4 mm³), and survival was longer in the exercise group than in the control group in vivo. OSM inhibited HSC-3-M3 cell proliferation in a concentration-dependent manner in vitro. The addition of OSM increased the proportion of cells in the G0/G1 phase, decreased the proportion of cells in the G2/M phase, and increased the expression of the CDK inhibitors p21 and p27. These results indicate that exercise may directly inhibit the proliferation of HNSCC cell lines via OSM.

Evaluating cancer gene mutations is critical for effective therapeutic selection. Although massive parallel sequencing can efficiently detect gene mutations, most are variants of uncertain significance (VUS). Saturation genome editing (SGE) can facilitate VUS analysis by leveraging CRISPR-Cas9 and homology-directed repair to simultaneously introduce abundant gene mutations. Chronic myelogenous leukemia-derived HAP1 cells are widely used in SGE because of their clear genotype–phenotype relationship; however, the sole use of haploid cells limits SGE applicability in cancer research. Therefore, we developed an SGE-based system for evaluating KRAS mutations in diploid HCT 116 colon carcinoma cells. Single-nucleotide variants (SNVs) in KRAS codons A11–V14 were generated using Cas9-based SGE. Massive parallel sequencing revealed increased abundance of KRAS ^G12 and KRAS ^G13 SNVs and decreased abundance of the KRAS ^G12C SNV after KRAS ^G12C inhibitor treatment in SGE pooled cells. These results indicate that SGE is applicable to diploid HCT 116 cells and useful for evaluating SNV population changes and drug sensitivity. Thus, although haploid HAP1 cells are the primary models for SGE, the successful application of SGE to diploid HCT 116 colon carcinoma cells provides a practical framework for implementing SGE in KRAS-dependent carcinoma cells.