Functional genetic variants of GEN1 predict overall survival of Chinese epithelial ovarian cancer patients

Abstract

Background: Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored.

Methods: In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation.

Results: We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P < 0.001). And the impact of GEN1 rs56070363 C > T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment.

Conclusions: In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients.

Keywords: GEN1; DNA double strand break repair; Epithelial ovarian cancer; Genetic variant; Overall survival; Single nucleotide polymorphisms.

Fig. 1

The study analysis flowchart

Fig. 2

The GEN1 rs56070363 C > T contributed to the decreased binding affinity of miRNA-1287a-5p to GEN1 3′-UTR. A Graphic representation of the detailed location of rs56070363 in the 3′UTR of GEN1, which was also at the miRNA-binding site with the C allele. B Schematic drawing of the luciferase reporter system and sequencing results of the Psi-CHECK2 vector containing rs56070363 C or T allele. C, D Luciferase activity in the presence of the miRNA-1287a-5p transfected into SKOV3 and OVAC-433 cell lines. E Expression of GEN1 was detected by the qRT-PCR assay in SKOV3 and OVAC-433 cells overexpressing miRNA-1287a-5p and control cells. F Expression of GEN1 was detected by western blot assay in SKOV3 and OVAC-433 cells overexpressing miRNA-1287a-5p and control cells. *P < 0.05. **P < 0.01. Error bars, ± SEM from three biological replications

Fig. 3

Functional prediction of genetic variants GEN1 rs56070363. A The GEN1 rs56070363 polymorphism influenced the mRNA expression of GEN1 from GTEx database in whole blood. B The GEN1 rs56070363 polymorphism influenced the mRNA expression of GEN1 from GTEx database in ovary tissue. C GEN1 expression in normal ovarian tissues (n = 88, right) and human epithelial ovarian carcinoma (n = 419, left) from TCGA database. TPM, Transcripts Per Kilobase of exon model per Million mapped reads. D. GEN1 expression in Bordline Ovarian Surface Epithelial-Stromal Tumor (n = 18, left) and Ovarian Carcinoma (n = 171, right) from Tothil ovarian dataset. E GEN1 expression in Borderline Ovarian Serous Neoplasm (n = 20, left), Borderline Ovarian Serous Tumor, micropapillary variant (n = 10, middle) and Ovarian Serous Adenocarcinoma (n = 60, right) from Anglesio ovarian dataset. F GEN1 expression in LMP (low malignant potential, n = 18) tumor and malignant tumor (n = 277) from GSE9899 ovarian dataset. G The correlation between the mRNA expression of GEN1 and tumor stage in GSE9899 ovarian dataset. H The correlation between the mRNA expression of GEN1 and tumor grade in GSE9899 ovarian dataset. I Kaplan–Meier analyses with the log-rank test for overall survival stratified by GEN1 mRNA expression levels

Fig. 4

GEN1 promoted cell proliferation and metastasis. A, B Cell viability determined by CCK8 assay in SKOV3 and OVAC-433 cell lines. C, D Representative images and number of colonies in SKOV3 and OVAC-433 cell lines. E, F Detection of cell migration and invasion by transwell assay. G, H Quantitative analysis of migration and invasion cells. Error bars, ± SEM from three biological replications. WT, wild type. **P < 0.01. ***P < 0.001

Fig. 5

In silico analysis of GEN1 manifest its close correlation with TIME. A Significantly enriched biological processes (BP) correlated with GEN1. B Gene set enrichment analysis (GSEA). The most involved significant hallmark correlated with GEN1 in ovarian ancer. NES: normalized enrichment score. C Scatterplot showing stromal, immune and ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) scores for each sample in ovarian cancer. TCGA-OV, The Cancer Genome Atlas ovarian cancer- Ovarian Serous Cystadenocarcinoma. D Boxplot showing the comparison of antigen presentation, effector cells, suppressor cells and checkpoint scores between GEN1 expression high and low group. E Correlation between GEN1 expression and various tumor cells based on TIMER, QUANTISEQ and MCP method

Fig. 6

The relationship between GEN1 expression and neutrophil. A Kaplan–Meier analyses with the log-rank test for OS of neutrophil-to-lymphocyte ratio in EOC patients. B The forest plot of multivariate analysis concerning OS. C Expression of neutrophil markers was detected by the qRT-PCR assay in EOC patients. D Expression of N1/N2 and PMN-MDSCs related markers markers was detected by the qRT-PCR assay in EOC patients. OS, overall survival. EOC, epithelial ovarian cancer