| 摘要: | Introduction: Breast cancer is the most common cancer in women globally. Targeting and understanding various epigenetic pathway alterations is now a key avenue for cancer diagnostic, prognostic, and treatment response research to propel precision medicine. Purpose: Alterations in SRCIN1 and FGF13 genes in breast cancer were identified due to their methylation profile novelty in breast cancer. The biological and clinical significances of alterations of SRCIN1 and FGF13 genes in breast cancer were determined for their relevancy as early detection and or prognostic biomarkers. Methods: TCGA data was analyzed to assess methylation and expression data in Western cohorts, whereas we conducted qMSP and RT-qPCR to assess methylation and mRNA expression profiles in Taiwanese cohorts. TCGA methylation and mRNA expression correlation was assessed along with methylation and mRNA expression correlation with clinical parameters in both TCGA and Taiwanese cohorts. Cell viability of MDA-MB-231 and MCF-7 cell lines were conducted with MTT assay after over expression and knockdown of SRCIN1. RNA sequencing data of SRCIN1 silencing was also analyzed in MCF-7 cell line and MetaCore was used to analyze the biological pathways. Results: Methylation analysis using qMSP of tissue samples from Taiwanese breast cancer patients and data from The Cancer Genome Atlas (TCGA) were conducted for two candidate genes, SRC kinase signaling inhibitor 1 (SRCIN1) and Fibroblast growth factor 13 (FGF13). In both cohorts SRCIN1 showed increased methylation; Taiwanese cohort with 51/96 (53.1%, p<0.001). However, FGF13 which had some decreased methylated cg sites in tumor tissue of TCGA cohort showed increased methylation 41/96 (42.7% p<0.001) in tumor tissue of Taiwanese cohort. RT-qPCR was then conducted to analyze mRNA expression. TCGA mRNA expression data showed that SRCIN1 had 39.4% (30/76 p<0.001) overexpression of tumor tissue. Further analysis of TCGA cohorts showed that hypermethylated gene body cg sites were positively and significantly correlated with mRNA expression, while hypomethylated promoter cg sites were inversely and significantly correlated with mRNA expression (p<0.001). Additionally, evaluation of 5-year overall survival in TCGA showed that SRCIN1 gene body hypermethylation had worse survival in various cg sites. Correlation of SRCIN1 hypermethylation and mRNA expression with clinical data in TCGA cohort showed that SRCIN1 hypermethylation in various cg sites and upregulation of mRNA expression were able to significantly differentiate between ER, PR, TNBC and Luminal A patients, as well as age and histological type. Cell viability was decreased with SRCIN1 knockdown in MCF-7 cell lines (p<0.01) and decreased with SRCIN1 over-expression in MDA-MB-231 cell lines (p<0.001). Additionally, RNA-seq data revealed SRCIN1 knockdown in MCF-7 cells resulted in upregulation of pro-apoptotic proteins including p73 and p53, and their downstream proteins as well as upregulation in SMAD3 and its downstream proteins. Conclusion: FGF13 overexpression is seen in tumor tissue especially in TNBC. On the other hand, data showed SRCIN1 hypermethylation in gene body cg sites of breast tumor tissue and hypomethylation of SRCIN1 promoter sites. SRCIN1 promoter hypomethylation and gene body hypermethylation are inversely and positively correlated to mRNA expression in breast tumor tissue respectively. SRCIN1 gene body hypermethylation had worse overall survival. Silencing of SRCIN1 in MCF-7 cells and over-expression in MDA-MB-231 decreased cell viability. Preliminary data suggest, SRCIN1 hypermethylation in gene body and its consequent mRNA expression upregulation may represent an oncogenic pathway in Luminal A patients, however, downregulation of SRCIN1 in TNBC patients may signal the gene maneuvering in a tumor suppressive pathway, thereby maybe providing the first evidence delineating SRCIN1’s dual regulatory role in breast cancer. |
