Panoply™ Human NRP1 Knockdown Stable Cell Line

Gastric cancer has a high mortality rate, and its complex molecular pathogenesis leads to poor treatment outcomes. Autophagy plays a dual role in cancer, both promoting and inhibiting it. Here, researchers first obtained autophagy-related genes from a human autophagy database, and then applied consensus clustering analysis to perform molecular subtype analysis on gastric cancer samples from the TCGA database. The genes obtained after subtype analysis were used to construct a risk prognostic model. Subsequently, principal component analysis (PCA) and tSNE were used to assess the risk score, and the results showed that the model had good discriminative ability for gastric cancer samples. Cox regression analysis and time-dependent ROC curve analysis showed that the model had good risk prediction ability. Finally, combining pairwise expression profile comparison analysis, Kaplan-Meier survival curves, and external validation on the GEO dataset, NRP1 was ultimately selected as the research subject. In vitro experiments showed that NRP1 can regulate the proliferation and autophagy of gastric cancer cells by affecting the Wnt/β-catenin signaling pathway. Similarly, in vivo experiments also showed that NRP1 can affect tumor growth in vivo. Therefore, the researchers proposed that NRP1, by regulating autophagy, can serve as a prognostic factor and therapeutic target for gastric cancer.

Researchers used qPCR to detect differential expression of NRP1 (Figure 1A). Next, they constructed a stable NRP1 knockdown cell line and used Western blot to detect the expression levels of NRP1, the cell proliferation marker Ki67, and the autophagy-related marker LC3I/II. As shown in Figure 1B, after NRP1 knockdown, Ki67 expression decreased, while the LC3II/LC3I ratio increased, suggesting that NRP1 knockdown may lead to reduced cell proliferation and upregulated autophagy. Based on this, the researchers performed EdU assays and autophagy labeling experiments. In NRP1 knockdown cells, cell proliferation decreased by approximately 20%, and autophagy intensity increased (Figures 1C-E). To provide more reliable evidence that NRP1 knockdown leads to autophagy, they fixed the treated cells and used transmission electron microscopy to image the number and morphological changes of autophagic vesicles. In NRP1 knockdown cells, the number of intracellular autophagic vesicles was significantly increased (Figure 1F).

Figure 1. Validation of NRP1's regulation of tumor autophagy and proliferative capacity in vitro. (Yu Q Y, et al., 2023)