Exploring the Effect of Cyclophosphamide on Key Genes and Signaling Pathways in Germinal Center B-Cell Lymphoma Based on Bioinformatics

Background: The diffuse large B-cell lymphoma (DLBCL) is the most prevalent lymphoma worldwide. Cyclophosphamide (CP) is an important anticancer drug used in various cancer types, including DLBCL, but its use in clinical settings is primarily limited by its dose-related cardiotoxicity. This study aimed to explore prognostic biomarkers of DLBCL in germinal center B cell (GCB) subtype, intending to develop personalized treatment regimens, monitor and assess post-treatment outcome, and improve survival rate. Furthermore, this study aimed to explore the underlying mechanism of CP and devise a more appropriate dosage plan for achieving maximum clinical benefits while minimizing toxicity.Method: GSE87371 dataset was obtained from Gene Expression Omnibus (GEO) to screen prognostic key genes for DLBCL using univariate Cox analysis, Least Absolute Shrinkage and Selection Operation (LASSO), Kaplan-Meier survival analysis, multivariate Cox regression analysis and operating characteristic curve (ROC) with area under the curve (AUC). Differentially expressed genes (DEGs) between the high and low risk scores groups were screened and analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to obtain key pathways involved in progression of DLBCL. Furthermore, the GCB DLBCL cell lines OCI-Ly1 and VAL were cultured and treated with different concentrations of CP. Moreover, cell proliferation was determined using Cell Counting Kit (CCK)-8 and colony-forming assay, cell migration activity was assessed using cell scratch and transwell, and cell apoptosis was evaluated using flow cytometry. Additionally, RT-qPCR and Western blot analyses were used to determine the expression levels of key genes and pathways involved in DLBCL.Results: The expressions of leucine zipper motif (APPL) isoform 2 (APPL2), histone cell cycle regulator (HIRA), zinc finger protein 814 (ZNF814), ZYG11A, leucine-tyrosine-arginine motif-containing protein 1 (LYRM1) and valosin-containing protein-interacting protein 1 (VCPIP1) were found to be correlated with the prognosis of DLBCL and were used in constructing prognostic model. It was found that patients in the low-risk group survived longer as compared to those in the high-risk group (p < 0.05). However, the nuclear factor-kappaB (NF-kappa B) and interleukin (IL)-17 pathways were enriched through functional analysis. Furthermore, CP induced the apoptosis in GCB DLCBL cells (p < 0.05) and inhibited their proliferation (p < 0.05) and migration (p < 0.05). Moreover, CP regulated the expression of 6 key genes. It was observed that CP treatment elevated the expression levels of APPL2, HIRA, ZNF814, and ZYG11A and decreased LYRM1 and VCPIP1, at both mRNA (p < 0.05) and protein (p < 0.05) levels. Additionally, CP decreased the activity of NF-kappa B (phosphorylation of p65 (p-p65) protein; p < 0.05) and IL-17 (IL-17 mRNA, p < 0.05; IL-17 protein, p < 0.05) pathways.Conclusion: APPL2, HIRA, ZNF814, ZYG11A, LYRM1 and VCPIP1 were identified as key genes involved in the prognosis of DLBCL. Moreover, CP could up-regulate the expressions of APPL2, HIRA, ZNF814, ZYG11A, down-regulate the expressions of LYRM1 and VCPIP1, restrain both NF-kappa B and IL-17 pathways, suppress cell proliferation, migration while promoting cell apoptosis, thus inhibiting the progression of GCB DLBCL.