Identification and Validation of Prognostic Markers for Endometriosis-Associated Ovarian Cancer

Abstract

Background: Growing evidence suggests that endometriosis (EMs) is a risk factor for endometriosis-associated ovarian cancer (EAOC). The aim was to identify and validate gene signatures associated with EMs that may serve as potential biomarkers for evaluating the prognosis of patients with EAOC. Methods: The data of EMs and control samples was obtained from GEO database. The weighted gene co-expression network analysis (WGCNA) identified modular genes significantly associated with EMs. The KEGG pathway and GO functional enrichment analyses were also performed. Univariate Cox regression analysis was conducted to screen marker genes associated with the prognosis of EAOC patients. Finally, RT-qPCR and immunohistochemical verified the expression of ADAMTS19 and TUBB in normal ovarian and EAOC tissues, and the biological functions of ADAMTS19 and TUBB were preliminarily explored by CCK8 and Transwell assays. Results: The WGCNA identified 2 co-expression modules, which in total included 615 genes, and 7642 differentially expressed genes (DEGs) were detected thorough analysis of the EAOC dataset. After taking the intersection of 615 modular genes and 7642 DEGs, 214 shared genes were obtained, and univariate COX regression analysis pointed 10 genes associated with the prognosis of EAOC. Moreover, it was demonstrated by RT-qPCR and immunohistochemical staining experiments that ADAMTS19 expression was elevated, while TUBB expression was reduced in EAOC compared with normal ovarian cells and tissues. Finally, cell experiments revealed that ADAMTS19 promoted the proliferation and invasion in EAOC cells, while overexpression of TUBB inhibited these processes. Conclusions: The present study identified and validated new EMs-associated gene markers, which could serve as potential biomarkers for assessing the prognostic risk of EAOC patients. In addition, some of these genes may have significance as novel therapeutic targets and could be used to guide clinical applications.

Keywords: ADAMTS19; Endometriosis; Endometriosis-Associated Ovarian Cancer; Prognosis; TUBB.

Figure 1

Study flowchart.

Figure 2

Identification of the EMs-Associated Modules. (a) Analysis of the scale-free fit index for various soft-thresholding powers β. (b) Analysis of the mean connectivity for various soft-thresholding powers. (c) Heatmap of the correlation between module eigengenes and clinical traits of EMs. (d) Dendrogram based on a dissimilarity measure (1-TOM).

Figure 3

Functional Annotation and Analysis of EMs modular genes. (a) Gene enrichment in tumor-associated pathways. (b) Gene enrichment in biological processes. (c) Gene enrichment in molecular functions. (d) Gene enrichment in cellular components.

Figure 4

Identification of DEGs in EAOC. (a) Heatmap of the top 15 upregulated and top 15 downregulated genes. (b) Volcano map of DEGs.

Figure 5

Identification of genes associated with EAOC prognosis. (a) The Venn diagram shows that there are 214 intersecting genes between the modular signature genes of EMs and the differential genes of EAOC. (b) Univariate COX analysis of prognostic factors in patients with EAOC.

Figure 6

The expression of ADAMTS19 and TUBB in EAOC. (a) The expression levels of ADAMTS19 and TUBB were detected by RT-qPCR in the normal ovarian epithelial cell IOSE80 and the ovarian clear cell carcinoma cell ES-2, and the ovarian endometrioid carcinoma cell TOV-112D. (b) Representative images of ADAMTS19 and TUBB proteins immunohistochemical staining of ovarian tissue sections from patients in the Normal and Tumour groups, scale bar = 25 μm. (**: P<0.01, ***: P<0.001).

Figure 7

Cellular assays to evaluate the functions of ADAMTS19 and TUBB in EAOC. (a) CCK8 assay detected the cell viability of ES-2 and TOV-112D cells in Control, NC, and si-ADAMTS19 groups. (b) CCK8 assay detected the cell viability of ES-2 and TOV-112D cells in Control, NC, and oe-TUBB groups. (c) Transwell assay detected the effects of ADAMTS19 on the invasive ability of ES-2 and TOV-112D cells. (d) Transwell assay detected the effects of TUBB on the invasive ability of ES-2 and TOV-112D cells. (***: P<0.001).

Figure 8

Flowchart for management of ovarian atypical endometriosis. Abbreviations: MRI, magnetic resonance imaging; HC, hormonal contraceptive; US, ultrasound.