3.1 Identification of dysregulated pseudogenes in four common gynecological malignancies.
According to epidemiological statistics, cervical squamous cell carcinoma, ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma, and uterine carcinosarcoma remain lethal diseases in women.(1) To explore the potential role of pseudogenes in carcinogenesis and cancer prognosis of four gynecological malignancies, we used the public dreamBase database to identify differentially expressed pseudogenes. As shown in Figure 1A and Table 1, we identified 63 up-regulated and 0 down-regulated pseudogenes simultaneously in the four gynecological malignancies after preliminary screening. We then measured the expression levels of the 63 up-regulated pseudogenes in 32 types of human cancer (Figure 1B). After removal of pseudogenes that were not highly expressed in the 32 types of human cancer, 40 pseudogenes were identified as playing potential roles in gynecological malignancies.
3.2 Prognostic analysis of up-regulated pseudogenes in 32 types of human cancer.
We next explored the prognostic values of 40 the up-regulated pseudogenes in the 32 kinds of human cancer using GEPIA. As shown in Figure 2, KRT8P3, KRT8P45, and LDHAP5 predicted poor overall survival in ovarian serous cystadenocarcinoma (HR = 1.3, P = 0.046; HR = 1.3, P = 0.019; HR = 1.3, P = 0.03, respectively), FTLP14 predicted poor unfavorable prognosis in uterine corpus endometrioid carcinoma (HR = 2.6, P = 0.018) No other pseudogenes t were significantly correlated with poor prognosis in the four types of gynecological malignancies.
3.3 Investigation of pseudogene-miRNA-mRNA regulatory network.
By searching the starBase v2.0 database, only LDHAP5 had its corresponding miRNAs. The specific characteristics of the nine retrieved miRNAs are shown in additional file- 1 (Table-S1). In addition, as shown inTable-S2, only hsa-miR-181d-5p, hsa-miR-181c-5p, hsa-miR-7-5p, hsa-miR-543, hsa-miR-151a-5p, and hsa-miR-181b-5p had their own target genes. In total, 148 miRNA target genes, which were validated by at least one of three robust method (i.e., reporter assay, western blot, and quantitative-real-time polymerase chain reaction (qRT-PCR)), were identified via miRTarBase. The pseudogene-miRNA-mRNA network was constructed using Cytoscape v_3.7.2 (Figure 3A).
3.4 KEGG pathway and Gene Oncology (GO) enrichment analysis of miRNA target mRNAs
The 148 miRNA target genes were imported into STRING v-11.0, with GO and KEGG pathway enrichment analysis then performed under the operational guidance of the website. We selected the top five significantly enriched GO terms and KEGG pathways according to false discovery rate (FDR) values. The top five Biological Process (BO), Molecular Function (MO) and Cellular Component (CO) and their corresponding FDR values are shown in Figure 3B. The top five significantly enriched KEGG pathways were MicroRNAs in cancer (hsa05206, FDR = 4.32E-26), Pathway in cancer (hsa05200, FDR = 6.77E-18), PI3K-AKT signaling pathway (hsa04151, FDR = 9.95E-16), Endocrine resistance (hsa01522, FDR = 9.95E-16), and Foxo signaling pathway (hsa04068, FDR = 2.65E-15) (Figure 3C). These findings confirmed that the LDHAP5 pseudogene may mediate the occurrence and progression of ovarian serous cystadenocarcinoma.
3.5 EGFR as target mRNA of LDHAP5 in ovarian serous cystadenocarcinoma.
We used the Centiscape plugin of Cytoscape v-3.7.2 to visualize the regulatory protein-protein network constructed using STRING v-11.0 (Figure 4). The top 10 hub genes (i.e., TP53, MYC, EGFR, PTEN, HRAS, SIRT1, TNF, RELA, KRAS, and CREB1) were then identified based on Degree unDir values (Table 2). We further explored the sequence mutations and copy-number alterations of the 10 hub genes in ovarian serous cystadenocarcinoma using cBioportal. The group (TCGA, Nature 2011) which contained 489 patients was selected. However, only 361 patients (64.6%) were suitable for further analysis. The mutation frequencies of the 10 hub genes were TP53 (96%), MYC (34%), EGFR (9%), PTEN (14%), HRAS (9%), KRAS (24%), SIRT1 (10%), TNF (24%), RELA (11%) and CREB1 (10%), respectively (Figure 5). Pearson correlation analysis showed that EGFR (R = 0.16, P = 0.00072), PTEN (R = 0.098, P = 0.043), SIRT1 (R = 0.094, P = 0.013), RELA (R = 0.18, P = 0.00013) and CREB1 (R = 0.16, P = 0.00094) were significantly correlated with LDHAP5 expression in ovarian serous cystadenocarcinoma (Table 3). Using the Oncomine Main database, only EGFR (fold-change = 1.192, P = 0.001), PTEN (fold-change = 1.214, P = 0.007), and CREB1 (fold-change = 1.723, P = 1.66E-04) mRNAs were more highly expressed in TCGA ovarian patients (n = 594) than in normal patients (n = 8) (Figure 6A). We further analyzed the prognostic values (overall survival) of the five hub genes in ovarian serous cystadenocarcinoma using Kaplan–Meier plotter (Table 4, Figure 6B). Only EGFR was significantly correlated with poor outcome (HR = 1.51, 95% CI = 1.15-2, P = 0.0033) in ovarian serous cystadenocarcinoma, whereas SIRT1 predicted a good outcome (HR = 0.75, 95% CI = 0.57-1, P = 0.047). Thus, according to the pseudogene-miRNA-mRNA regulatory mechanism, we concluded that LDHAP5 may play potential roles in ovarian serous cystadenocarcinoma by targeting EGFR.