Bioinformatics analysis and single-cell RNA sequencing: elucidating the ubiquitination pathways and key enzymes in lung adenocarcinoma.

BACKGROUND

Lung adenocarcinoma (LUAD) is a prevalent subtype of lung cancer associated with high mortality rates. We aimed to utilize single-cell multiomics analysis to identify the key molecules involved in ubiquitination modification, which plays a role in LUAD development and progression.

METHODS

We use a systematic approach to analyze LUAD-related single-cell and bulk transcriptome datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Single-cell RNA sequencing (scRNA-seq) data were normalized, clustered, and annotated with the Seurat package in R. InferCNV was used to distinguish malignant from epithelial cells, and AUCell evaluated the area under the curve (AUC) score of ubiquitination-related enzymes. Survival and differential analyses identified significant molecular markers associated with ubiquitination. expression was confirmed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assays, and its knockdown cell lines were assessed for effects on cellular processes and tumor formation in mice. 's interacting proteins were predicted, and its impact on AGR2 protein half-life and ubiquitination was evaluated. Rescue experiments involving overexpression and silencing assessed their impact on malignant behaviors.

RESULTS

By means of single-cell sequencing analysis, we probed the ubiquitination modification landscape in the LUAD microenvironment. Malignant cells had elevated scores for enzymes and ubiquitin-binding domains compared to normal epithelial cells, with 53 ubiquitination-related molecules showing prognostic disparities. , , and were identified as genes with prognostic significance, with showing higher expression in epithelial and malignant cells. Two missense mutation sites were identified in , which had a high copy number amplification ratio and positive correlation with messenger RNA (mRNA) expression. expression and tumor stage were found to be independent prognostic factors, and interfering with expression reduced the malignant behavior of LUAD cells. was found to bind to AGR2 protein and reduce its ubiquitination, leading to increased stability. Knockdown of inhibited the enhancement of cell viability, invasion, and migration resulting from overexpression.

CONCLUSIONS

This study examined ubiquitination modifications in LUAD using sequencing data, identifying 's critical role in malignancy regulation and its potential as a prognostic and therapeutic biomarker. These insights enhance understanding of LUAD mechanisms and treatment.