Identification and Validation of DNA Methylation-driven Gene OSR1 as A Novel Tumor Suppressor for the Diagnosis and Prognosis of Breast Cancer

Jian Xu^1,2Biao Yang³Ling Cheng⁴Chen Gan²Runze Huang²Anlong Li²Han Ge²Longyu Hu⁵Meiwen Ling⁵Xinyi Zheng⁵Bao Zhao⁶Mingjun Zhang²Huaidong Cheng^2,5*

• ¹Anhui Medical University, Hefei, China
• ²Second Hospital of Anhui Medical University, Hefei, Anhui Province, China
• ³Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China
• ⁴First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei, Anhui Province, China
• ⁵Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong Province, China
• ⁶Institute of Health and Medicine, Hefei Comprehensive National Science Center, Heifei, China

Aberrant DNA methylation plays a critical role in the initiation and progression of cancer, yet its association with breast cancer remains inadequately defined. This study aims to clarify the link between methylation-driven genes and breast cancer pathogenesis. RNA sequencing and DNA methylation data for breast cancer were retrieved from The Cancer Genome Atlas (TCGA). By integrating the methylation R package with univariate Cox regression analysis, prognostically relevant methylation-driven genes were identified, with OSR1 emerging as the primary candidate. Gene expression profiles and corresponding clinical data were subsequently obtained from the TCGA. Differential expression analysis using the Wilcoxon rank-sum test revealed significantly reduced OSR1 expression in breast cancer tissues compared to normal counterparts. Kaplan-Meier survival curves and Cox regression models were applied to assess the prognostic significance of OSR1. Bioinformatic analyses indicated that OSR1 expression correlates negatively with breast cancer progression and is significantly associated with M stage, HER2 status, PAM50 subtypes, and histological classification. Low OSR1 expression was linked to poorer overall survival outcomes. Functional enrichment analysis implicated OSR1 in pathways related to peptide hormone secretion, peptide transport, metal ion response, and forebrain development. Moreover, elevated OSR1 expression was positively correlated with increased infiltration of immune cells, including NK cells, B cells, CD8 + T cells, and dendritic cells. Experimental validation was conducted by generating OSR1-overexpressing breast cancer cell lines to examine its effects on cell viability, migration, and proliferation via phenotypic assays and protein expression profiling. Both in vitro and in vivo studies demonstrated that OSR1 overexpression markedly suppressed breast cancer cell proliferation and migration. These findings confirm OSR1 as a methylation-regulated tumor suppressor gene and underscore its potential as a promising biomarker for individualized therapeutic strategies in breast cancer.