فهرست نشریات علمی دسترسی آزاد ایران

Objective: The PI3K/Akt signaling pathway plays a central role in regulating cell growth, survival, and metabolism,and its dysregulation is a hallmark of many cancers. The PIK3CA gene, which encodes the alpha catalytic subunit ofPI3K, is altered in approximately 30% of breast cancers. Among its mutations, c.3140A>G (p.His1047Arg) in the kinasedomain is the most prevalent, producing a constitutively active enzyme with oncogenic potential. Here, we engineered apopulation of MCF7 cells carrying the PIK3CA c.3140A>G mutation using CRISPR-Cas9 with precise single-nucleotideediting, and evaluated its impact on cellular characteristics.Materials and Methods: In this experimental study, nearly homogeneous populations of PIK3CA H1047R mutant MCF7 cellswere generated using CRISPR-Cas9–mediated genome editing followed by hierarchical single-cell isolation. Editing efficiencywas validated through allele-specific polymerase chain reaction (PCR) and multiple rounds of Sanger sequencing. Cell cycledistribution and proliferation were analyzed using flow cytometry and cell count assays, respectively. Gene expression changeswere assessed by quantitative real-time PCR to evaluate the mutation’s impact on cell cycle-related genes.Results: Tracking of insertions, deletions, and recombination events (TIDER) analysis showed approximately 60%homology-directed repair (HDR) efficiency in the edited population. Flow cytometry revealed a 5% increase in theG2/M cell population in the edited clone compared with unedited controls (P<0.001). Proliferation assays demonstratedsignificantly accelerated growth (1.30 fold) under low fetal bovine serum (FBS) conditions (P=0.029). Quantitativereal-time PCR confirmed upregulation of cell cycle-promoting genes, with CCND1 and MYC expression increasing by1.62-fold (P<0.001) and 1.23-fold (P<0.001), respectively, relative to controls.Conclusion: The genetically edited cell lines represent robust and well-defined experimental models that enable directassessment of the functional consequences of oncogenic driver mutations on cellular behavior and signaling pathways. Ourfindings demonstrate that targeted genetic alterations induce measurable changes in proliferation, cell-cycle regulation, andgene expression, thereby providing mechanistic insight into tumorigenesis and the specific contribution of driver mutations tocancer-related cellular phenotypes.