A Ubiquitination-Related Gene Prognostic Signature and the Oncogenic Role of RNF149 in Nasopharyngeal Carcinoma: scRNAseq-Based Bioinformatics and Experimental Validation

Introduction: Nasopharyngeal carcinoma (NPC) is an aggressive malignancy with a poor prognosis. Ubiquitination is a complex post translational modification involved in cancer progression. However, ubiquitination related genes (URGs) in immunotherapy of NPC remains largely unexplored. Methods: Differentially expressed URGs were screened based on the single-cell RNA sequencing (scRNA-seq) dataset and a risk model of NPC was constructed and evaluated for prognostic significance. The oncogenic role of RNF149 in NPC was investigated through in vitro and in vivo experiments, including tumor cells, NPC-like organoids, and tumor-bearing mice. Results: scRNA-seq data showed that URGs scores were higher in cancer cells than in normal epithelial cells. We identified 216 differentially expressed URGs between cancer and normal epithelial cells, but only 33 differentially expressed URGs associated with prognosis. Based on 33 URGs, TCGA-HNSC samples were classified into two distinct subtypes with significant differences in the tumor immune microenvironment, immunotherapy effect, and survival-prognostic genes. Using LASSO algorithm, 13 URGs were selected to construct a risk model, which demonstrated high predictive performance. The expression profiles of these 13 URGs were analyzed in TCGA-HNSC tumor and adjacent non-cancerous samples, and six URGs (BSPRY, OTUB1, PJA1, RNF149, RNF181, USP10) exhibited consistent expression trends. Moreover, quantitative real-- time PCR revealed that RNF149 was up-regulated expression in NPC cell compared to the NP69 cells. RNF149 knockdown significantly impeded the proliferative, migratory, and invasive capabilities and exaggerated apoptosis of NPC cells. RNF149 knockdown cells exhibited a reduced capacity to form NPC organoids in a 3D culture system. shRNA- RNF149 diminished subcutaneous tumorigenic capacity of HK-1 cells compared to the control group. Discussion: The URGs-based prognostic risk model offers a robust tool for predicting immunotherapy efficacy in NPC and RNF9 promotes NPC progression. Conclusion: A URGs-related prognostic risk model capable of predicting clinical outcomes in NPC patients and RNF9 promotes NPC progression. Our findings are expected to provide new strategies to improve outcomes for NPC patients.