Prompt Description

2.2 Elucidate the key molecular mechanisms of polyphyllin III's action on different subtypes of breast cancer cells. ① Based on previous sensitivity difference results, select four cell lines: MCF-7 (Luminal A), BT-474 (Luminal B), SK-BR-3 (HER2-enriched), and MDA-MB-231 (TNBC). Use RNA-seq technology to systematically compare the transcriptome changes of each subtype before and after polyphyllin III treatment. Focus on the enrichment of apoptosis, oxidative stress, and subtype-specific signaling pathways (such as ER, HER2, STAT3), and screen for key differentially expressed genes. Combine Western blotting and qRT-PCR to verify the expression changes of candidate targets (such as Bcl-2 family members, NOXA, PUMA, EGFR, p-STAT3, etc.), and identify the core effector molecules with the most significant response in each subtype. ② Conduct functional intervention experiments targeting the dominant mechanisms of each subtype: In TNBC, set up a control group, a polyphyllin III group, an NAC (ROS scavenger) group, and a polyphyllin III + NAC group to detect apoptosis (Cleaved Caspase-3/PARP), ROS levels, mitochondrial membrane potential, and JNK/p38 phosphorylation status. In HER2⁺ cells, set up a control group, a polyphyllin III group, and a HER2 overexpression/knockdown group to evaluate HER2-AKT/ERK pathway activity and cell survival rate. In Luminal A cells, combine ER inhibitors (fulvestrant) or ERα siRNA to observe whether drug resistance is reversed. At the same time, evaluate functional phenotypic changes through Annexin V/PI flow cytometry, cell cycle analysis, and scratch/Transwell experiments. ③ Construct stable knockout or overexpression cell lines of key node genes (such as HIF-1α, STAT3, or HER2) to verify their necessity in polyphyllin III-induced cell death, signal inhibition, and phenotypic changes, and detect related protein ubiquitination or subcellular localization changes, thereby establishing the core molecular mechanism axis driving drug efficacy differences in each subtype.